rna  (New England Biolabs)


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    Structured Review

    New England Biolabs rna
    Acetylation of H2A.Z is required for upregulation of Hes1 Notch target gene. ( A ) Schematic representation of the Flag-RBP-J/Tip60 fusion proteins used in Figure 4B and C and in Supplementary Figures S9 and S10 . Amino acid numbering is accordingly to accession NP_033061.3 for RBP-J and NP_874368.1 for Tip60. RBP-J domain: LAG1-DNAbind, LAG1 DNA binding (CDD:255260); Tip60 domain: MOZ/SAS, MOZ/SAS family (CDD:250916). ( B ) RBP-J/Tip60 wildtype (wt) but not its catalytic dead (cd) mutant upregulates Hes1 expression in MT cells. MT cells were infected with retroviral particles delivering plasmids encoding Flag-tagged RBP-J/Tip60-wt, cd mutant or empty vector (Control). Total <t>RNA</t> was reverse transcribed into <t>cDNA</t> and analysed by qPCR using primers specific for Tbp or Hes1 . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD ([**] P
    Rna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response"

    Article Title: Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky551

    Acetylation of H2A.Z is required for upregulation of Hes1 Notch target gene. ( A ) Schematic representation of the Flag-RBP-J/Tip60 fusion proteins used in Figure 4B and C and in Supplementary Figures S9 and S10 . Amino acid numbering is accordingly to accession NP_033061.3 for RBP-J and NP_874368.1 for Tip60. RBP-J domain: LAG1-DNAbind, LAG1 DNA binding (CDD:255260); Tip60 domain: MOZ/SAS, MOZ/SAS family (CDD:250916). ( B ) RBP-J/Tip60 wildtype (wt) but not its catalytic dead (cd) mutant upregulates Hes1 expression in MT cells. MT cells were infected with retroviral particles delivering plasmids encoding Flag-tagged RBP-J/Tip60-wt, cd mutant or empty vector (Control). Total RNA was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp or Hes1 . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD ([**] P
    Figure Legend Snippet: Acetylation of H2A.Z is required for upregulation of Hes1 Notch target gene. ( A ) Schematic representation of the Flag-RBP-J/Tip60 fusion proteins used in Figure 4B and C and in Supplementary Figures S9 and S10 . Amino acid numbering is accordingly to accession NP_033061.3 for RBP-J and NP_874368.1 for Tip60. RBP-J domain: LAG1-DNAbind, LAG1 DNA binding (CDD:255260); Tip60 domain: MOZ/SAS, MOZ/SAS family (CDD:250916). ( B ) RBP-J/Tip60 wildtype (wt) but not its catalytic dead (cd) mutant upregulates Hes1 expression in MT cells. MT cells were infected with retroviral particles delivering plasmids encoding Flag-tagged RBP-J/Tip60-wt, cd mutant or empty vector (Control). Total RNA was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp or Hes1 . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD ([**] P

    Techniques Used: Binding Assay, Mutagenesis, Expressing, Infection, Plasmid Preparation, Real-time Polymerase Chain Reaction

    H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. ( A ) Schematic representation of the NICD-inducible system established in MT cells. The NICD was fused to the estrogen receptor binding domain (NICD-ER) and retrovirally introduced into MT cells. The NICD-ER fusion protein is retained into the cytoplasm unless cells are treated with ( Z )-4-hydroxytamoxifen (4-OHT) that induces its nuclear translocation and activation of Notch target genes. ( B ) Hes1 and Il2ra Notch target genes are induced upon 4-OHT treatment of MT NICD-ER cells. Total RNA from MT NICD-ER cells, treated for 24 h with 4-OHT or EtOH as control, was reverse transcribed into cDNA and analyzed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of three independent experiments. ( C ) H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. MT NICD-ER cells were treated for 24 h with 4-OHT or EtOH as control and subjected to ChIP analysis using antibodies against H2A.Z, H2A.Zac, H3 or IgG as control. The qPCR analysis was focused at the Notch-dependent enhancers (red squares) represented on the left ( Hes1 +0.6 kb and Il2ra -26 kb ). Chrom X was used as negative control ( Control ). Data were normalized to the positive control ( GAPDH 0 kb ) and, in the case of H2A.Zac/H2A.Z, the H2A.Zac signals were further normalized to H2A.Z. Shown is the mean ± SD of two independent experiments.
    Figure Legend Snippet: H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. ( A ) Schematic representation of the NICD-inducible system established in MT cells. The NICD was fused to the estrogen receptor binding domain (NICD-ER) and retrovirally introduced into MT cells. The NICD-ER fusion protein is retained into the cytoplasm unless cells are treated with ( Z )-4-hydroxytamoxifen (4-OHT) that induces its nuclear translocation and activation of Notch target genes. ( B ) Hes1 and Il2ra Notch target genes are induced upon 4-OHT treatment of MT NICD-ER cells. Total RNA from MT NICD-ER cells, treated for 24 h with 4-OHT or EtOH as control, was reverse transcribed into cDNA and analyzed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of three independent experiments. ( C ) H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. MT NICD-ER cells were treated for 24 h with 4-OHT or EtOH as control and subjected to ChIP analysis using antibodies against H2A.Z, H2A.Zac, H3 or IgG as control. The qPCR analysis was focused at the Notch-dependent enhancers (red squares) represented on the left ( Hes1 +0.6 kb and Il2ra -26 kb ). Chrom X was used as negative control ( Control ). Data were normalized to the positive control ( GAPDH 0 kb ) and, in the case of H2A.Zac/H2A.Z, the H2A.Zac signals were further normalized to H2A.Z. Shown is the mean ± SD of two independent experiments.

    Techniques Used: Activation Assay, Binding Assay, Translocation Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Negative Control, Positive Control

    Histone variant H2A.Z has a negative impact on the expression of Notch target genes. ( A ) Histone Variant H2A.Z is efficiently depleted by CRISPR/Cas9 in MT cells. Whole Cell Extract (WCE) was prepared from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells and analysed by Western blotting. GAPDH was used as loading control. ( B ) Hes1 and Il2ra Notch target genes are upregulated upon depletion of H2A.Z. Total RNA from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of five independent experiments ([*] P
    Figure Legend Snippet: Histone variant H2A.Z has a negative impact on the expression of Notch target genes. ( A ) Histone Variant H2A.Z is efficiently depleted by CRISPR/Cas9 in MT cells. Whole Cell Extract (WCE) was prepared from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells and analysed by Western blotting. GAPDH was used as loading control. ( B ) Hes1 and Il2ra Notch target genes are upregulated upon depletion of H2A.Z. Total RNA from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of five independent experiments ([*] P

    Techniques Used: Variant Assay, Expressing, CRISPR, Western Blot, Real-time Polymerase Chain Reaction

    2) Product Images from "Capping-RACE: a simple, accurate, and sensitive 5′ RACE method for use in prokaryotes"

    Article Title: Capping-RACE: a simple, accurate, and sensitive 5′ RACE method for use in prokaryotes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky739

    Experimental verification of Capping-RACE with in vitro transcribed RNA. ( A ) Different treatments of in vitro transcribed RNA. ( B ) The cDNA products from the differently treated RNA described in (A). Lane 1, the negative control, performed without adding reverse transcriptase (RT). Lane 2, in vitro transcribed RNA that was not subjected to any treatment. Lane 3, in vitro transcribed RNA subjected to RppH treatment. Lane 4, in vitro transcribed RNA subjected to dual treatment, i.e. RppH treatment prior to VCE treatment. Lane 5, in vitro transcribed RNA subjected to vaccinia capping enzyme (VCE) treatment. The reaction products were analysed on a 12% non-denaturing polyacrylamide gel and detected by a fluorescence image analyser (FUJIFILM, FLA-5100).
    Figure Legend Snippet: Experimental verification of Capping-RACE with in vitro transcribed RNA. ( A ) Different treatments of in vitro transcribed RNA. ( B ) The cDNA products from the differently treated RNA described in (A). Lane 1, the negative control, performed without adding reverse transcriptase (RT). Lane 2, in vitro transcribed RNA that was not subjected to any treatment. Lane 3, in vitro transcribed RNA subjected to RppH treatment. Lane 4, in vitro transcribed RNA subjected to dual treatment, i.e. RppH treatment prior to VCE treatment. Lane 5, in vitro transcribed RNA subjected to vaccinia capping enzyme (VCE) treatment. The reaction products were analysed on a 12% non-denaturing polyacrylamide gel and detected by a fluorescence image analyser (FUJIFILM, FLA-5100).

    Techniques Used: In Vitro, Negative Control, Fluorescence

    3) Product Images from "Utility of the Trypanosoma cruzi Sequence Database for Identification of Potential Vaccine Candidates by In Silico and In Vitro Screening "

    Article Title: Utility of the Trypanosoma cruzi Sequence Database for Identification of Potential Vaccine Candidates by In Silico and In Vitro Screening

    Journal: Infection and Immunity

    doi: 10.1128/IAI.72.11.6245-6254.2004

    Expression of TcG1-TcG8 in different developmental stages of T. cruzi . Total RNA isolated from epimastigote (E), trypomastigote (T), and amastigote (A) stages of T. cruzi was reverse transcribed, and the cDNA was amplified by PCR amplification with the gene-specific forward and reverse primers. Amplicons were resolved on 1% agarose gel. The GPI8 gene, constitutively expressed in all three stages of T. cruzi , was amplified as a positive control. No amplification was obtained when template cDNAs were incubated in a PCR with gene-specific forward or reverse primers only.
    Figure Legend Snippet: Expression of TcG1-TcG8 in different developmental stages of T. cruzi . Total RNA isolated from epimastigote (E), trypomastigote (T), and amastigote (A) stages of T. cruzi was reverse transcribed, and the cDNA was amplified by PCR amplification with the gene-specific forward and reverse primers. Amplicons were resolved on 1% agarose gel. The GPI8 gene, constitutively expressed in all three stages of T. cruzi , was amplified as a positive control. No amplification was obtained when template cDNAs were incubated in a PCR with gene-specific forward or reverse primers only.

    Techniques Used: Expressing, Isolation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Positive Control, Incubation

    4) Product Images from "PRC2 binds to active promoters and contacts nascent RNAs in embryonic stem cells"

    Article Title: PRC2 binds to active promoters and contacts nascent RNAs in embryonic stem cells

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.2700

    Genome-wide analysis of EZH2 CLIP in mouse ESCs. ( a ) Distribution of RCSs identified with PARalyzer relative to genomic features and their distribution. The number of features in each set is indicated at the top. ( b ) EZH2 CLIP tags mapping to two representative protein-coding genes. An EZH2 ChIP-seq track is shown, with the scale indicated to the right in reads per 10 million mapped (RP10M). Red bars show RCSs called by PARalyzer. Rep1–4, biological replicate. ( c ) Density profile of RCSs over the 250 unique RefSeq transcripts with the most RCSs, each divided in 100 bins. TSS, transcription start site; TTS, transcription termination site. The 10 kb upstream and downstream are included, each divided in 50 200 nts bins. The distribution of RNA-seq reads (gray dashed line) is shown for comparison. ( d ) Same as c but for CLIP tags (after duplicate removal) and comparing tags containing the T > C transition (solid black line) with tags not containing the mutation (dashed gray line). ( e ) Distribution of CLIP tags and RNA-seq reads on the first 50 bp of first exons (E 1 ), first introns (i 1 ), last introns (i n ) and last exons (E n ) from RefSeq transcripts with at least two exons. The bars represent the mean percentage of total reads mapping to these features from 2 (RNA-seq) or 4 (CLIP-seq) biological replicates + numerical range. ( f ) PAR-CLIP as in Fig. 1d after chasing the 4-SU with uridine for the indicated time. Autoradiography (top) and HA blot (bottom) demonstrating equal protein loading.
    Figure Legend Snippet: Genome-wide analysis of EZH2 CLIP in mouse ESCs. ( a ) Distribution of RCSs identified with PARalyzer relative to genomic features and their distribution. The number of features in each set is indicated at the top. ( b ) EZH2 CLIP tags mapping to two representative protein-coding genes. An EZH2 ChIP-seq track is shown, with the scale indicated to the right in reads per 10 million mapped (RP10M). Red bars show RCSs called by PARalyzer. Rep1–4, biological replicate. ( c ) Density profile of RCSs over the 250 unique RefSeq transcripts with the most RCSs, each divided in 100 bins. TSS, transcription start site; TTS, transcription termination site. The 10 kb upstream and downstream are included, each divided in 50 200 nts bins. The distribution of RNA-seq reads (gray dashed line) is shown for comparison. ( d ) Same as c but for CLIP tags (after duplicate removal) and comparing tags containing the T > C transition (solid black line) with tags not containing the mutation (dashed gray line). ( e ) Distribution of CLIP tags and RNA-seq reads on the first 50 bp of first exons (E 1 ), first introns (i 1 ), last introns (i n ) and last exons (E n ) from RefSeq transcripts with at least two exons. The bars represent the mean percentage of total reads mapping to these features from 2 (RNA-seq) or 4 (CLIP-seq) biological replicates + numerical range. ( f ) PAR-CLIP as in Fig. 1d after chasing the 4-SU with uridine for the indicated time. Autoradiography (top) and HA blot (bottom) demonstrating equal protein loading.

    Techniques Used: Genome Wide, Cross-linking Immunoprecipitation, Chromatin Immunoprecipitation, RNA Sequencing Assay, Mutagenesis, Autoradiography

    EZH2 binds to RNA in mESCs. ( a ) CLIP blots for HA-tagged EZH2 in control cells and cells induced with doxycycline and before or after irradiation with UVC. The autoradiography is shown at the top and the approximate position of HA-EZH2 is indicated. The corresponding HA immunoblot is shown at the bottom. ( b ) PAR-CLIP autoradiography (top) and western blot (bottom). Different 4-SU concentrations, UV wavelength, and RNAse treatments were tested. ( c ) Scheme of the purification strategy used for PAR-CLIP-seq experiments. ( d ) Autoradiography of 3 biological replicates (rep1–3) utilized for PAR-CLIP-seq library construction. The dashed red boxes indicate the position of the excised bands. ( e ) Histogram plot for the mutation frequencies in PAR-CLIP-seq reads. The bars represent the average % of unique mapped CLIP tags containing the indicated mutation from the 4 biological replicates + s.d. ( f ) Genome browser view of EZH2 CLIP tags mapping to the Meg3 lncRNA (top) or Kcnq1ot1 antisense ncRNA (bottom). The 4 biological replicates are plotted separately. RCSs called by PARalyzer are shown as red bars. UCSC gene models are displayed. Rep1–4, biological replicate.
    Figure Legend Snippet: EZH2 binds to RNA in mESCs. ( a ) CLIP blots for HA-tagged EZH2 in control cells and cells induced with doxycycline and before or after irradiation with UVC. The autoradiography is shown at the top and the approximate position of HA-EZH2 is indicated. The corresponding HA immunoblot is shown at the bottom. ( b ) PAR-CLIP autoradiography (top) and western blot (bottom). Different 4-SU concentrations, UV wavelength, and RNAse treatments were tested. ( c ) Scheme of the purification strategy used for PAR-CLIP-seq experiments. ( d ) Autoradiography of 3 biological replicates (rep1–3) utilized for PAR-CLIP-seq library construction. The dashed red boxes indicate the position of the excised bands. ( e ) Histogram plot for the mutation frequencies in PAR-CLIP-seq reads. The bars represent the average % of unique mapped CLIP tags containing the indicated mutation from the 4 biological replicates + s.d. ( f ) Genome browser view of EZH2 CLIP tags mapping to the Meg3 lncRNA (top) or Kcnq1ot1 antisense ncRNA (bottom). The 4 biological replicates are plotted separately. RCSs called by PARalyzer are shown as red bars. UCSC gene models are displayed. Rep1–4, biological replicate.

    Techniques Used: Cross-linking Immunoprecipitation, Irradiation, Autoradiography, Western Blot, Purification, Mutagenesis

    5) Product Images from "Histidine-Rich Glycoprotein Can Prevent Development of Mouse Experimental Glioblastoma"

    Article Title: Histidine-Rich Glycoprotein Can Prevent Development of Mouse Experimental Glioblastoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0008536

    Presence and expression of viral transduced PDGF-B and HRG in tumors. ( A ) Insertion of the viral transduced human PDGF-B and HRG cDNA in genomic DNA prepared from PDGF-B+X (P+X) and PDGF-B+HRG (P+H) induced tumors. The tumor grade is given above each sample. Genomic DNA from U-706MG-a cells was used as positive control (+), and genomic DNA from an untreated mouse was used as negative control (−). ( B ) Expression of human PDGF-B and HRG mRNA in P+X and P+H tumors. RNA extracted from U-343MG and DF-1 RCAS-HRG cells were used as positive control for PDGF-B and HRG, respectively (+), and RNA from an untreated mouse brain was used as negative control (−).
    Figure Legend Snippet: Presence and expression of viral transduced PDGF-B and HRG in tumors. ( A ) Insertion of the viral transduced human PDGF-B and HRG cDNA in genomic DNA prepared from PDGF-B+X (P+X) and PDGF-B+HRG (P+H) induced tumors. The tumor grade is given above each sample. Genomic DNA from U-706MG-a cells was used as positive control (+), and genomic DNA from an untreated mouse was used as negative control (−). ( B ) Expression of human PDGF-B and HRG mRNA in P+X and P+H tumors. RNA extracted from U-343MG and DF-1 RCAS-HRG cells were used as positive control for PDGF-B and HRG, respectively (+), and RNA from an untreated mouse brain was used as negative control (−).

    Techniques Used: Expressing, Positive Control, Negative Control

    6) Product Images from "Polyadenylation Linked to Transcription Termination Directs the Processing of snoRNA Precursors in Yeast"

    Article Title: Polyadenylation Linked to Transcription Termination Directs the Processing of snoRNA Precursors in Yeast

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2008.10.003

    Polyadenylation and Processing of snoRNAs in TRAMP and Exosome Mutants (A–D and F–H) Northern analysis of polyadenylated pre-snoRNAs in total and poly(A) + fractions from various strains. U6 and SMX2 mRNA are loading controls for total and poly(A) + RNAs, respectively. Site I species characteristic for rrp6Δ are indicated with an arrow in (B). Poly(A) + fractions ([C], lanes 9 and 10, 19 and 20 and [H], lanes 19 and 20) or total RNA (D) deadenylated by RNase H treatment in the presence of oligo(dT). An arrow in (D) points at deadenylated pre-snR65 from terminator II; RNA size marker is on the right. To deplete Trf5 (C) or Mtr4 (F), trf4Δ/GAL1::TRF5 or GAL1::MTR4 cells were pregrown on YPGal and transferred to YPD; trf4Δ and nrd1-102 cells were grown at 23°C or transferred to 37°C (F); Dis3 was depleted by growth in the presence of doxycycline (H). (E) CR-RT-PCR of polyadenylated pre-snR65 terminated in region II in the trf4Δ strain. The number of added A residues is in subscript.
    Figure Legend Snippet: Polyadenylation and Processing of snoRNAs in TRAMP and Exosome Mutants (A–D and F–H) Northern analysis of polyadenylated pre-snoRNAs in total and poly(A) + fractions from various strains. U6 and SMX2 mRNA are loading controls for total and poly(A) + RNAs, respectively. Site I species characteristic for rrp6Δ are indicated with an arrow in (B). Poly(A) + fractions ([C], lanes 9 and 10, 19 and 20 and [H], lanes 19 and 20) or total RNA (D) deadenylated by RNase H treatment in the presence of oligo(dT). An arrow in (D) points at deadenylated pre-snR65 from terminator II; RNA size marker is on the right. To deplete Trf5 (C) or Mtr4 (F), trf4Δ/GAL1::TRF5 or GAL1::MTR4 cells were pregrown on YPGal and transferred to YPD; trf4Δ and nrd1-102 cells were grown at 23°C or transferred to 37°C (F); Dis3 was depleted by growth in the presence of doxycycline (H). (E) CR-RT-PCR of polyadenylated pre-snR65 terminated in region II in the trf4Δ strain. The number of added A residues is in subscript.

    Techniques Used: Northern Blot, Marker, Reverse Transcription Polymerase Chain Reaction

    Polyadenylated snoRNA Species in the rrp6Δ Strain Originate from Transcription Terminators (A, B, and G) SnoRNAs are oligoadenylated in the absence of Rrp6. Northern hybridization and CR-RT-PCR analysis of different box C/D and box H/ACA snoRNAs in wild-type and rrp6Δ strains (A and B). CR-RT-PCR for snR65 in the rrp6Δ/trf4Δ strain (G). Only the last few residues of mature ends are shown; undigested nucleotides and adenines are in bold. (C) Inactivation of Nop1 in a temperature-sensitive nop1-2 strain leads to accumulation of adenylated boxC/D snR13 and snR65 (marked with asterisks). (D) Northern hybridization of total and poly(A) + RNA from the rrp6Δ strain for snR65 and snR13. U6 and SMX2 mRNA are loading controls for total and poly(A) + RNAs, respectively. Two poly(A) + populations are indicated by vertical lines. Arrows show major oligoadenylated precursor terminated at site I. Sequencing of RT-PCR products on polyadenylated precursors from the rrp6Δ strain is shown on the right. An arrowhead indicates the cleavage site in terminator I of snR13. (E) RNase H treatment of total RNA from the rrp6Δ strain in the presence of oligo(dT) for snR65. Arrows point at deadenylated species. RNA size marker is on the right. (F) CR-RT-PCR of polyadenylated pre-snR65 terminated in region II in the rrp6Δ strain. The number of added A residues is in subscript, and numerals in parentheses denote number of clones.
    Figure Legend Snippet: Polyadenylated snoRNA Species in the rrp6Δ Strain Originate from Transcription Terminators (A, B, and G) SnoRNAs are oligoadenylated in the absence of Rrp6. Northern hybridization and CR-RT-PCR analysis of different box C/D and box H/ACA snoRNAs in wild-type and rrp6Δ strains (A and B). CR-RT-PCR for snR65 in the rrp6Δ/trf4Δ strain (G). Only the last few residues of mature ends are shown; undigested nucleotides and adenines are in bold. (C) Inactivation of Nop1 in a temperature-sensitive nop1-2 strain leads to accumulation of adenylated boxC/D snR13 and snR65 (marked with asterisks). (D) Northern hybridization of total and poly(A) + RNA from the rrp6Δ strain for snR65 and snR13. U6 and SMX2 mRNA are loading controls for total and poly(A) + RNAs, respectively. Two poly(A) + populations are indicated by vertical lines. Arrows show major oligoadenylated precursor terminated at site I. Sequencing of RT-PCR products on polyadenylated precursors from the rrp6Δ strain is shown on the right. An arrowhead indicates the cleavage site in terminator I of snR13. (E) RNase H treatment of total RNA from the rrp6Δ strain in the presence of oligo(dT) for snR65. Arrows point at deadenylated species. RNA size marker is on the right. (F) CR-RT-PCR of polyadenylated pre-snR65 terminated in region II in the rrp6Δ strain. The number of added A residues is in subscript, and numerals in parentheses denote number of clones.

    Techniques Used: Northern Blot, Hybridization, Reverse Transcription Polymerase Chain Reaction, Sequencing, Marker, Clone Assay

    7) Product Images from "Novel rapidly evolving hominid RNAs bind nuclear factor 90 and display tissue-restricted distribution"

    Article Title: Novel rapidly evolving hominid RNAs bind nuclear factor 90 and display tissue-restricted distribution

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm668

    snaR-A is a rapidly synthesized PolIII transcript. ( A ) In vitro transcription of linearized DNA containing the CMV promoter, adenovirus VA RNA II (pVA II ), and snaR-A from pR3 with HeLa cell nuclear extract in the presence of 0, 20 or 200 μg/ml α-amanitin. M: 3′-end labeled cytoplasmic RNA. ( B ) In vitro transcription in HeLa cell nuclear extract of snaR-A from pR1 and pR2, and by T7 RNA polymerase from linearized pT7-snaR-A. A schematic of pR1-R3 constructs (boxed) gives upstream sequence (numbered) with respect to the snaR-A transcriptional start site (bent arrow). ( C ) Northern blot of total RNA from HeLa S3 cells after exposure to 1 μg/ml actinomycin D for 15, 30, 60 or 180 min or to 100 μg/ml cycloheximide (ChX), 10 μg/ml α-amanitin, 10 μM DRB or DMSO for 180 min. Blot was probed with Probe-A (upper) and 5S rRNA antisense oligonucleotide (lower).
    Figure Legend Snippet: snaR-A is a rapidly synthesized PolIII transcript. ( A ) In vitro transcription of linearized DNA containing the CMV promoter, adenovirus VA RNA II (pVA II ), and snaR-A from pR3 with HeLa cell nuclear extract in the presence of 0, 20 or 200 μg/ml α-amanitin. M: 3′-end labeled cytoplasmic RNA. ( B ) In vitro transcription in HeLa cell nuclear extract of snaR-A from pR1 and pR2, and by T7 RNA polymerase from linearized pT7-snaR-A. A schematic of pR1-R3 constructs (boxed) gives upstream sequence (numbered) with respect to the snaR-A transcriptional start site (bent arrow). ( C ) Northern blot of total RNA from HeLa S3 cells after exposure to 1 μg/ml actinomycin D for 15, 30, 60 or 180 min or to 100 μg/ml cycloheximide (ChX), 10 μg/ml α-amanitin, 10 μM DRB or DMSO for 180 min. Blot was probed with Probe-A (upper) and 5S rRNA antisense oligonucleotide (lower).

    Techniques Used: Synthesized, In Vitro, Labeling, Construct, Sequencing, Northern Blot

    snaR is highly expressed in human testis and cell lines. ( A ) Northern blots of total RNA extracted from normal adult human tissue (left) or testis tumor (Tum) and normal adjacent tissue (NAT, right), probed with Probe-A (upper) or for 5.8S rRNA (lower). Tissues tested were (from left): brain, thyroid, thymus, heart, adipose, trachea, lung, skeletal muscle, esophagus, liver, spleen, small intestine, colon, prostate, kidney, testes, ovary, cervix and placenta. ( B ) Northern blot of cell lines with radiolabeled RNA complementary to full-length snaR-A (upper) or 5.8S rRNA antisense oligonucleotide (lower). Asterisks denote residual snaR-A bands. ( C ) Northern blot of 293 cell, brain and testis RNA, probed with Probe-A or oligonucleotides specific for piR-36011 or piR-36189. 3′-end labeled cytoplasmic RNA and MspI-digested pBR322 DNA (M) served as size markers. The blot was cut into strips before hybridization as shown by dashed lines. The lower panel was exposed 36-fold longer than the upper panel. Asterisk marks the low molecular weight snaR-related bands.
    Figure Legend Snippet: snaR is highly expressed in human testis and cell lines. ( A ) Northern blots of total RNA extracted from normal adult human tissue (left) or testis tumor (Tum) and normal adjacent tissue (NAT, right), probed with Probe-A (upper) or for 5.8S rRNA (lower). Tissues tested were (from left): brain, thyroid, thymus, heart, adipose, trachea, lung, skeletal muscle, esophagus, liver, spleen, small intestine, colon, prostate, kidney, testes, ovary, cervix and placenta. ( B ) Northern blot of cell lines with radiolabeled RNA complementary to full-length snaR-A (upper) or 5.8S rRNA antisense oligonucleotide (lower). Asterisks denote residual snaR-A bands. ( C ) Northern blot of 293 cell, brain and testis RNA, probed with Probe-A or oligonucleotides specific for piR-36011 or piR-36189. 3′-end labeled cytoplasmic RNA and MspI-digested pBR322 DNA (M) served as size markers. The blot was cut into strips before hybridization as shown by dashed lines. The lower panel was exposed 36-fold longer than the upper panel. Asterisk marks the low molecular weight snaR-related bands.

    Techniques Used: Northern Blot, Labeling, Hybridization, Molecular Weight

    8) Product Images from "Transient Reversal of Episome Silencing Precedes VP16-Dependent Transcription during Reactivation of Latent HSV-1 in Neurons"

    Article Title: Transient Reversal of Episome Silencing Precedes VP16-Dependent Transcription during Reactivation of Latent HSV-1 in Neurons

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002540

    Elevated Phase II viral transcript levels in neurons expressing human Oct-1. (A) Schematic of Oct-1 showing the location of the POU DNA-binding domain near the middle of the protein and an alignment of the human, mouse and rat POU homeo (POU H ) subdomain sequences. The four variable positions are located in helix-1 and helix-2 and are numbered according to their position within the POU H sequence. A rodent-like Oct-1 derivative (Oct-1 E30D/M33L ) was constructed by changing glutamic acid-30 and methionine-33 to the aspartic acid and leucine of the mouse/rat sequence. (B) SCG neuron cultures were infected with HSV-1 GFP-Us11, maintained for 5 days in the presence of ACV and then infected with lentiviral vectors encoding GFP or human Oct-1. After a further 5 days, ACV was removed and reactivation induced with 20 µM LY294002. RNA was collected at intervals and analyzed by qRT-PCR using human Oct-1 specific primers. For each time point, values for each transcript were compared to those from the GFP-expressing neurons (set to 1.0). (C) Relative levels of viral transcripts (ICP27, UL5, UL30, VP16 and UL36) after reactivation of HSV-1 GFP-Us11 in neurons expressing GFP, wild type human Oct-1 (WT) and human Oct-1 E30D/M33L (MUT). (D) Wild type (WT) and E30D/M33L (MUT) versions of human Oct-1, VP16 (residues 5–412, VP16ΔC) and the β-propeller domain of human HCF-1 (residues 1–380, HCF-1 N380 ) were synthesized by in vitro translation in the presence of 35 S-methionine and visualized by 10% SDS-PAGE followed by autoradiography. (E) Assembly of the VP16-induced complex (VIC) by rodent-like (E30D/M33L) Oct-1 is greatly reduced compared to wild type human Oct-1. Recombinant Oct-1, VP16 and HCF-1 proteins were assayed for VIC formation by gel shift assay using a 32 P-labeled probe containing an (OCTA + )TAATGARAT element from the HSV-1 ICP0 promoter [23] . The first three lanes are controls showing probe alone (lane 1), un-programmed rabbit reticulocyte lysate (lane 2), and a mix of lysates containing recombinant VP16 and HCF-1 (lane 3). The shift formed by the rabbit Oct-1 present in the lysate is greatly enhanced by the presence of either wild type or mutant human Oct-1 (lanes 4 and 10). A slower migrating complex (VIC) is formed by addition of VP16 and HCF-1 in the presence of wild type Oct-1 (lane 5) and only weakly by the mutant (lane 11). Reducing the amount of wild type Oct-1 by 5, 10, 50 and 100-fold respectively (lanes 6–9) reduces but does not eliminate this complex. No VIC is detected over a similar range of using mutant Oct-1.
    Figure Legend Snippet: Elevated Phase II viral transcript levels in neurons expressing human Oct-1. (A) Schematic of Oct-1 showing the location of the POU DNA-binding domain near the middle of the protein and an alignment of the human, mouse and rat POU homeo (POU H ) subdomain sequences. The four variable positions are located in helix-1 and helix-2 and are numbered according to their position within the POU H sequence. A rodent-like Oct-1 derivative (Oct-1 E30D/M33L ) was constructed by changing glutamic acid-30 and methionine-33 to the aspartic acid and leucine of the mouse/rat sequence. (B) SCG neuron cultures were infected with HSV-1 GFP-Us11, maintained for 5 days in the presence of ACV and then infected with lentiviral vectors encoding GFP or human Oct-1. After a further 5 days, ACV was removed and reactivation induced with 20 µM LY294002. RNA was collected at intervals and analyzed by qRT-PCR using human Oct-1 specific primers. For each time point, values for each transcript were compared to those from the GFP-expressing neurons (set to 1.0). (C) Relative levels of viral transcripts (ICP27, UL5, UL30, VP16 and UL36) after reactivation of HSV-1 GFP-Us11 in neurons expressing GFP, wild type human Oct-1 (WT) and human Oct-1 E30D/M33L (MUT). (D) Wild type (WT) and E30D/M33L (MUT) versions of human Oct-1, VP16 (residues 5–412, VP16ΔC) and the β-propeller domain of human HCF-1 (residues 1–380, HCF-1 N380 ) were synthesized by in vitro translation in the presence of 35 S-methionine and visualized by 10% SDS-PAGE followed by autoradiography. (E) Assembly of the VP16-induced complex (VIC) by rodent-like (E30D/M33L) Oct-1 is greatly reduced compared to wild type human Oct-1. Recombinant Oct-1, VP16 and HCF-1 proteins were assayed for VIC formation by gel shift assay using a 32 P-labeled probe containing an (OCTA + )TAATGARAT element from the HSV-1 ICP0 promoter [23] . The first three lanes are controls showing probe alone (lane 1), un-programmed rabbit reticulocyte lysate (lane 2), and a mix of lysates containing recombinant VP16 and HCF-1 (lane 3). The shift formed by the rabbit Oct-1 present in the lysate is greatly enhanced by the presence of either wild type or mutant human Oct-1 (lanes 4 and 10). A slower migrating complex (VIC) is formed by addition of VP16 and HCF-1 in the presence of wild type Oct-1 (lane 5) and only weakly by the mutant (lane 11). Reducing the amount of wild type Oct-1 by 5, 10, 50 and 100-fold respectively (lanes 6–9) reduces but does not eliminate this complex. No VIC is detected over a similar range of using mutant Oct-1.

    Techniques Used: Expressing, Binding Assay, Sequencing, Construct, Infection, Quantitative RT-PCR, Synthesized, In Vitro, SDS Page, Autoradiography, Recombinant, Electrophoretic Mobility Shift Assay, Labeling, Mutagenesis

    During reactivation, HSV-1 exhibits a biphasic profile of viral transcripts in SCG neurons. (A) Scheme showing a typical reactivation experiment. Neuron cultures were established and then infected with HSV-1 GFP-Us11 (MOI = 1) in the presence of 100 µM acyclovir (ACV). Latency was established over a 7-day period before re-feeding with fresh media lacking ACV. The next day, reactivation was induced with 20 µM LY294002. (B) Profile of viral mRNA accumulation in response to LY294002. RNA was collected at the indicated times and analyzed by qRT-PCR. Values are normalized against the 0 h sample [ICP27, 171 copies/sample; UL5, 135 copies/sample; UL30, 94 copies/sample; VP16, 347 copies/sample and UL36 130 copies/sample]. Data is derived from three or more independent cultures and reactivation experiments. (C) Reactivation profiling in the presence of the viral DNA encapsidation inhibitor WAY150138 (20 µg/ml). (D) Transcript levels at 20 h post induction in the absence (−) or presence (+) of protein synthesis inhibitor cyclohexamide (CHX, 10 µg/ml). To ensure cell viability, CHX was added 10 h after LY294002, prior to the appearance of new viral transcripts.
    Figure Legend Snippet: During reactivation, HSV-1 exhibits a biphasic profile of viral transcripts in SCG neurons. (A) Scheme showing a typical reactivation experiment. Neuron cultures were established and then infected with HSV-1 GFP-Us11 (MOI = 1) in the presence of 100 µM acyclovir (ACV). Latency was established over a 7-day period before re-feeding with fresh media lacking ACV. The next day, reactivation was induced with 20 µM LY294002. (B) Profile of viral mRNA accumulation in response to LY294002. RNA was collected at the indicated times and analyzed by qRT-PCR. Values are normalized against the 0 h sample [ICP27, 171 copies/sample; UL5, 135 copies/sample; UL30, 94 copies/sample; VP16, 347 copies/sample and UL36 130 copies/sample]. Data is derived from three or more independent cultures and reactivation experiments. (C) Reactivation profiling in the presence of the viral DNA encapsidation inhibitor WAY150138 (20 µg/ml). (D) Transcript levels at 20 h post induction in the absence (−) or presence (+) of protein synthesis inhibitor cyclohexamide (CHX, 10 µg/ml). To ensure cell viability, CHX was added 10 h after LY294002, prior to the appearance of new viral transcripts.

    Techniques Used: Infection, Quantitative RT-PCR, Derivative Assay

    Transactivation function of VP16 is required during Phase II. (A) Structure of VP16 showing the 12-bp insertion ( in 1814) between the structured N-terminal domain and the C-terminal activation domain (AD) that disrupts VP16-induced complex assembly [32] . (B) SCG neurons were infected with mutant ( in 1814) or marker rescue ( in 1814R) viruses (MOI = 1) in the presence of 100 µM acyclovir and maintained for 7 days before measuring the relative amounts of viral genomic DNA by qPCR. (C) Reactivation was induced with 20 µM LY294002 in media lacking ACV and maintained for 7 days before harvest and plaque assay to detect infectious virus. (D) Comparison of viral transcript levels during reactivation by in 1814 (‘M’) and in 1814R (‘R’) at 15 and 20 h post-induction (Phase I) and at 72 h post-induction (Phase II). For each time point, transcript levels from the in 1814 (‘M’) sample were set to 1 and the value for the corresponding transcript from in 1814R (‘R’) plotted as the fold difference. (E) Depletion of VP16 using RNA interference. Latently infected neuron cultures were infected with a lentivirus expressing a VP16-specific short-hairpin RNA [shRNA] (KD) or with a control lentivirus (Con). ShRNAs were allowed to accumulate for 5 days before reactivation was induced with LY294002 and allowed to proceed for 5 days in media lacking ACV. Lysates were prepared and probed by immunoblotting to detect VP16 and the loading control, Rho-GDI. (F) Quantitation of infectious virus by plaque assay. (G) Comparison of viral transcript levels in the absence of VP16. Values from the control culture are plotted relative to the corresponding value from the VP16 shRNA (KD) culture.
    Figure Legend Snippet: Transactivation function of VP16 is required during Phase II. (A) Structure of VP16 showing the 12-bp insertion ( in 1814) between the structured N-terminal domain and the C-terminal activation domain (AD) that disrupts VP16-induced complex assembly [32] . (B) SCG neurons were infected with mutant ( in 1814) or marker rescue ( in 1814R) viruses (MOI = 1) in the presence of 100 µM acyclovir and maintained for 7 days before measuring the relative amounts of viral genomic DNA by qPCR. (C) Reactivation was induced with 20 µM LY294002 in media lacking ACV and maintained for 7 days before harvest and plaque assay to detect infectious virus. (D) Comparison of viral transcript levels during reactivation by in 1814 (‘M’) and in 1814R (‘R’) at 15 and 20 h post-induction (Phase I) and at 72 h post-induction (Phase II). For each time point, transcript levels from the in 1814 (‘M’) sample were set to 1 and the value for the corresponding transcript from in 1814R (‘R’) plotted as the fold difference. (E) Depletion of VP16 using RNA interference. Latently infected neuron cultures were infected with a lentivirus expressing a VP16-specific short-hairpin RNA [shRNA] (KD) or with a control lentivirus (Con). ShRNAs were allowed to accumulate for 5 days before reactivation was induced with LY294002 and allowed to proceed for 5 days in media lacking ACV. Lysates were prepared and probed by immunoblotting to detect VP16 and the loading control, Rho-GDI. (F) Quantitation of infectious virus by plaque assay. (G) Comparison of viral transcript levels in the absence of VP16. Values from the control culture are plotted relative to the corresponding value from the VP16 shRNA (KD) culture.

    Techniques Used: Activation Assay, Infection, Mutagenesis, Marker, Real-time Polymerase Chain Reaction, Plaque Assay, Expressing, shRNA, Quantitation Assay

    Acute replication of HSV-1 in SCG neurons follows the canonical ordered cascade of mRNA accumulation. (A) Primary neurons were isolated from superior cervical ganglia (SCG) of E21 rats, cultured for 7 days in the presence of 5 µM aphidicolin and 20 µM 5-fluorouracil to eliminate proliferating cells, and then infected with HSV-1 GFP-Us11 at a multiplicity of 3 plaque forming units per neuron (MOI = 3). RNA was collected at 0, 3, 6, 9, and 12 h post-infection (p.i.) and analyzed by quantitative reverse transcription PCR (qRT-PCR) to determine the relative levels of viral immediate-early (ICP27), early (UL30), γ1 leaky-late (VP16) and γ2 true-late (UL36) transcripts. Values represent the average and standard error from the mean from three independent infection experiments. (B) Neuron cultures were treated with the viral DNA polymerase inhibitor phosphonoacetic acid (PAA, 300 µg/ml) for 1 h (hatched bars) or mock treated (filled bars) and then infected with HSV-1 GFP-Us11. Total DNA was prepared at the indicated times and the relative levels of viral genomic DNA determined by quantitative (qPCR) using primers complementary to the HSV-1 UL30 gene. Input DNA was normalized by qPCR detection of the rat RPL19 gene. (C, D) Analysis of γ1 leaky-late (VP16) and γ2 true-late (UL36) transcript levels in the presence or absence of PAA.
    Figure Legend Snippet: Acute replication of HSV-1 in SCG neurons follows the canonical ordered cascade of mRNA accumulation. (A) Primary neurons were isolated from superior cervical ganglia (SCG) of E21 rats, cultured for 7 days in the presence of 5 µM aphidicolin and 20 µM 5-fluorouracil to eliminate proliferating cells, and then infected with HSV-1 GFP-Us11 at a multiplicity of 3 plaque forming units per neuron (MOI = 3). RNA was collected at 0, 3, 6, 9, and 12 h post-infection (p.i.) and analyzed by quantitative reverse transcription PCR (qRT-PCR) to determine the relative levels of viral immediate-early (ICP27), early (UL30), γ1 leaky-late (VP16) and γ2 true-late (UL36) transcripts. Values represent the average and standard error from the mean from three independent infection experiments. (B) Neuron cultures were treated with the viral DNA polymerase inhibitor phosphonoacetic acid (PAA, 300 µg/ml) for 1 h (hatched bars) or mock treated (filled bars) and then infected with HSV-1 GFP-Us11. Total DNA was prepared at the indicated times and the relative levels of viral genomic DNA determined by quantitative (qPCR) using primers complementary to the HSV-1 UL30 gene. Input DNA was normalized by qPCR detection of the rat RPL19 gene. (C, D) Analysis of γ1 leaky-late (VP16) and γ2 true-late (UL36) transcript levels in the presence or absence of PAA.

    Techniques Used: Isolation, Cell Culture, Infection, Polymerase Chain Reaction, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    9) Product Images from "Apoptosis-like programmed cell death induces antisense ribosomal RNA (rRNA) fragmentation and rRNA degradation in Leishmania"

    Article Title: Apoptosis-like programmed cell death induces antisense ribosomal RNA (rRNA) fragmentation and rRNA degradation in Leishmania

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2012.85

    Fragmentation of asLSU- γ RNA upon induction of apoptosis is correlated with extensive degradation of the sLSU- γ LSU- γ rRNA in L. infantum axenic amastigotes. ( a , upper panel) Northern blot analysis of RNA samples extracted from L. infantum amastigotes treated with various concentrations of MF (0–20 μ M) for 24 h. ( a , bottom panel) Ethidium bromide (EtBr)-stained RNA gel is shown here. ( b ) Primer extension analysis of L. infantum axenic amastigotes treated for 24 h with either G418 (25 μ g/ml) or paramomycin sulphate (150 μ g/ml) or hygromycin-B (80 μ g/ml) or MF (20 μ M) and/or Sb III (25 μ M). A primer complementary to nucleotides 96–213 of sLSU- γ rRNA was used for the analysis. Arrows indicate the degradation pattern of sLSU- γ rRNA in MF- and SbIII-treated samples. ( c ) Lysates from Leishmania promastigotes treated with MF (25 μ M) were fractionated by a 15–45% sucrose gradient. RNA samples were isolated from the respective fractions as indicated in Figure 1d , resolved on a 10% urea acrylamide gel and hybridized with the 173 nt ss-DNA probe to detect asLSU- γ RNA. The asLSU- γ RNA fragments enriched in the 80S and polysome fractions are indicated
    Figure Legend Snippet: Fragmentation of asLSU- γ RNA upon induction of apoptosis is correlated with extensive degradation of the sLSU- γ LSU- γ rRNA in L. infantum axenic amastigotes. ( a , upper panel) Northern blot analysis of RNA samples extracted from L. infantum amastigotes treated with various concentrations of MF (0–20 μ M) for 24 h. ( a , bottom panel) Ethidium bromide (EtBr)-stained RNA gel is shown here. ( b ) Primer extension analysis of L. infantum axenic amastigotes treated for 24 h with either G418 (25 μ g/ml) or paramomycin sulphate (150 μ g/ml) or hygromycin-B (80 μ g/ml) or MF (20 μ M) and/or Sb III (25 μ M). A primer complementary to nucleotides 96–213 of sLSU- γ rRNA was used for the analysis. Arrows indicate the degradation pattern of sLSU- γ rRNA in MF- and SbIII-treated samples. ( c ) Lysates from Leishmania promastigotes treated with MF (25 μ M) were fractionated by a 15–45% sucrose gradient. RNA samples were isolated from the respective fractions as indicated in Figure 1d , resolved on a 10% urea acrylamide gel and hybridized with the 173 nt ss-DNA probe to detect asLSU- γ RNA. The asLSU- γ RNA fragments enriched in the 80S and polysome fractions are indicated

    Techniques Used: Northern Blot, Staining, Isolation, Acrylamide Gel Assay

    Fragmentation of asLSU- γ RNA upon induction of apoptosis is correlated with extensive degradation of the sLSU- γ LSU- γ rRNA in L. infantum macrophage-derived amastigotes. ( a ) Primer extension analysis to detect fragmentation of asLSU- γ rRNA on total RNA extracted from THP-1 human macrophage-derived amastigotes treated with MF (25 μM) for various time points post infection (24, 48 and 72 h) and compared with the untreated control. ( b ) Primer extension analysis of macrophage-derived amastigotes treated with MF as in ( a ) to visualize degradation of sLSU- γ rRNA upon MF treatment
    Figure Legend Snippet: Fragmentation of asLSU- γ RNA upon induction of apoptosis is correlated with extensive degradation of the sLSU- γ LSU- γ rRNA in L. infantum macrophage-derived amastigotes. ( a ) Primer extension analysis to detect fragmentation of asLSU- γ rRNA on total RNA extracted from THP-1 human macrophage-derived amastigotes treated with MF (25 μM) for various time points post infection (24, 48 and 72 h) and compared with the untreated control. ( b ) Primer extension analysis of macrophage-derived amastigotes treated with MF as in ( a ) to visualize degradation of sLSU- γ rRNA upon MF treatment

    Techniques Used: Derivative Assay, Infection

    Leishmania promastigote to amastigote differentiation and exposure to various stresses induce fragmentation of the asLSU- γ RNA. ( a ) Size-fractionated RNA (≤200 nt) was isolated from L. infantum unstressed and heat–stress promastigotes (Pro) and subjected to primer extension analysis using a forward primer corresponding to nucleotides 101–118 of the sLSU- γ rRNA. ( b ) L. infantum parasites were treated with 1 mM and 2 mM of H 2 O 2 for 8 h and RNA was used for primer extension analysis as in A . ( c ) Primer extension analysis of L. infantum promastigotes and amastigotes (Ama) as indicated in ( a ). M, the end-labeled DNA ladder (Promega) was used as a reference for molecular size
    Figure Legend Snippet: Leishmania promastigote to amastigote differentiation and exposure to various stresses induce fragmentation of the asLSU- γ RNA. ( a ) Size-fractionated RNA (≤200 nt) was isolated from L. infantum unstressed and heat–stress promastigotes (Pro) and subjected to primer extension analysis using a forward primer corresponding to nucleotides 101–118 of the sLSU- γ rRNA. ( b ) L. infantum parasites were treated with 1 mM and 2 mM of H 2 O 2 for 8 h and RNA was used for primer extension analysis as in A . ( c ) Primer extension analysis of L. infantum promastigotes and amastigotes (Ama) as indicated in ( a ). M, the end-labeled DNA ladder (Promega) was used as a reference for molecular size

    Techniques Used: Isolation, Labeling

    The ATP-dependent DEAD-box RNA helicase HEL67 interacts with both the sLSU- γ and asLSU- γ rRNAs to prevent asrRNA fragmentation. ( a ) A modified UV-crosslinking method was used to identify protein factors bound to in vitro -transcribed sLSU- γ and asLSU- γ rRNAs. The 67 kDa and 30 kDa proteins bound to both sLSU- γ and asLSU- γ rRNAs are indicated. ( b , left panel) Strategy to generate a L. infantum null mutant strain ( Lin HEL (−/−) ) for the LinJ.32.0410 gene encoding an ATP-dependent RNA helicase of 67 kDa (HEL67). Both alleles of the HEL67 gene were replaced by the hygromycin phosphotransferase gene ( HYG ) and neomycin phosphotransferase gene ( NEO ) cassettes, respectively, through homologous recombination. An add-back mutant ( Lin HEL (−/−) REV, b , bottom panel) was generated by overexpressing HEL67 as part of the pSP α ZEO α -HEL67 vector in the Lin HEL67 (−/−) mutant background. ( b , right panel) Southern blot hybridization of genomic DNA digested with Xba I and Blp I using the HEL67 3‘-flank sequence as a probe. In Lin HEL67 (−/−) , two hybridizing bands of 2.3 kb (for the HYG gene replacement) and 2.1 kb (for the NEO gene replacement) were detected but not the 3.1 kb HEL67 endogenous band. ( c ) Primer extension analysis was performed to detect asLSU- γ RNA fragmentation using the end-labeled forward primer corresponding to nucleotides 101–118 of the LSU- γ rRNA. ( c , left panel) RNA was extracted from wild-type (WT), Lin HEL67 (−/−) and Lin HEL67 (−/−) REV L. infantum promastigotes subjected to O/N temperature (37 °C) and pH (5.5) stress. MF (15 μ M)-treated L. infantum axenic amastigotes were used as a positive control for the induction of apoptosis and asLSU- γ RNA fragmentation. ( c , right panel) SS-qRT-PCR to detect asLSU- γ RNA levels in WT, Lin HEL67 (−/−) and Lin HEL67 (−/−) REV. A primer corresponding to nucleotides 1–18 of sLSU- γ rRNA was used for cDNA synthesis. ( d ) Primer extension analysis using a reverse primer complementary to nucleotides 196–213 of the sLSU- γ rRNA
    Figure Legend Snippet: The ATP-dependent DEAD-box RNA helicase HEL67 interacts with both the sLSU- γ and asLSU- γ rRNAs to prevent asrRNA fragmentation. ( a ) A modified UV-crosslinking method was used to identify protein factors bound to in vitro -transcribed sLSU- γ and asLSU- γ rRNAs. The 67 kDa and 30 kDa proteins bound to both sLSU- γ and asLSU- γ rRNAs are indicated. ( b , left panel) Strategy to generate a L. infantum null mutant strain ( Lin HEL (−/−) ) for the LinJ.32.0410 gene encoding an ATP-dependent RNA helicase of 67 kDa (HEL67). Both alleles of the HEL67 gene were replaced by the hygromycin phosphotransferase gene ( HYG ) and neomycin phosphotransferase gene ( NEO ) cassettes, respectively, through homologous recombination. An add-back mutant ( Lin HEL (−/−) REV, b , bottom panel) was generated by overexpressing HEL67 as part of the pSP α ZEO α -HEL67 vector in the Lin HEL67 (−/−) mutant background. ( b , right panel) Southern blot hybridization of genomic DNA digested with Xba I and Blp I using the HEL67 3‘-flank sequence as a probe. In Lin HEL67 (−/−) , two hybridizing bands of 2.3 kb (for the HYG gene replacement) and 2.1 kb (for the NEO gene replacement) were detected but not the 3.1 kb HEL67 endogenous band. ( c ) Primer extension analysis was performed to detect asLSU- γ RNA fragmentation using the end-labeled forward primer corresponding to nucleotides 101–118 of the LSU- γ rRNA. ( c , left panel) RNA was extracted from wild-type (WT), Lin HEL67 (−/−) and Lin HEL67 (−/−) REV L. infantum promastigotes subjected to O/N temperature (37 °C) and pH (5.5) stress. MF (15 μ M)-treated L. infantum axenic amastigotes were used as a positive control for the induction of apoptosis and asLSU- γ RNA fragmentation. ( c , right panel) SS-qRT-PCR to detect asLSU- γ RNA levels in WT, Lin HEL67 (−/−) and Lin HEL67 (−/−) REV. A primer corresponding to nucleotides 1–18 of sLSU- γ rRNA was used for cDNA synthesis. ( d ) Primer extension analysis using a reverse primer complementary to nucleotides 196–213 of the sLSU- γ rRNA

    Techniques Used: Modification, In Vitro, Mutagenesis, Homologous Recombination, Generated, Plasmid Preparation, Southern Blot, Hybridization, Sequencing, Labeling, Positive Control, Quantitative RT-PCR

    The asrRNA fragmentation process is evolutionary conserved. Single-stranded (SS) RT-PCR was performed to detect asRNA complementary to the LSU- γ rRNA of Trypanosoma brucei ( a ) and to the 28S rRNA in the THP-1 human acute monocytic leukemia cell line ( b ) using specific forward primers (see Supplementary Table 1 ). ( c ) Primer extension analysis using a forward primer complementary to nucleotides 101–118 of the T. brucei LSU- γ rRNA to detect asLSU- γ RNA fragmentation in T. brucei exposed to H 2 O 2 (0–400 μ M). ( d ) Primer extension to detect asRNA complementary to the human 28S rRNA in THP-1 cell line treated with H 2 O 2 (0–20 mM). A forward primer complementary to nucleotides 1–18 of the human 28S rRNA was used
    Figure Legend Snippet: The asrRNA fragmentation process is evolutionary conserved. Single-stranded (SS) RT-PCR was performed to detect asRNA complementary to the LSU- γ rRNA of Trypanosoma brucei ( a ) and to the 28S rRNA in the THP-1 human acute monocytic leukemia cell line ( b ) using specific forward primers (see Supplementary Table 1 ). ( c ) Primer extension analysis using a forward primer complementary to nucleotides 101–118 of the T. brucei LSU- γ rRNA to detect asLSU- γ RNA fragmentation in T. brucei exposed to H 2 O 2 (0–400 μ M). ( d ) Primer extension to detect asRNA complementary to the human 28S rRNA in THP-1 cell line treated with H 2 O 2 (0–20 mM). A forward primer complementary to nucleotides 1–18 of the human 28S rRNA was used

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    asLSU- γ RNA fragmentation is dramatically increased upon drug-induced ALPCD. ( a ) Primer extension analysis (same primer as in Figure 2a ) of total RNA isolated from L. infantum amastigotes treated with either G418 (25 μ g/ml) or paramomycin sulphate (150 μ g/ml) or hygromycin-B (80 μ g/ml) or MF (20 μ M) and or antimony (SbIII) (25 μ M) for 24 h. Arrows indicate the asLSU- γ RNA-derived fragments. End-labeled Decade marker (Ambion) was used as a ladder. ( b ) Primer extension carried out on total RNA extracted from axenic amastigotes treated with increasing concentrations of MF (0–20 μ M) for 24 h. Primer extension was performed with different primers that generated in all cases an induced fragmentation pattern for asLSU- γ RNA upon MF treatment (data not shown). ( c , upper panel) Northern blot analysis of RNA from 0 to 20 μ M MF-treated amastigotes for 24 h using the 173 nt ss-DNA probe. ( c , bottom panel) Overexposed RNA blot to detect asLSU- γ RNA cleavage products accumulated upon increased concentrations of MF
    Figure Legend Snippet: asLSU- γ RNA fragmentation is dramatically increased upon drug-induced ALPCD. ( a ) Primer extension analysis (same primer as in Figure 2a ) of total RNA isolated from L. infantum amastigotes treated with either G418 (25 μ g/ml) or paramomycin sulphate (150 μ g/ml) or hygromycin-B (80 μ g/ml) or MF (20 μ M) and or antimony (SbIII) (25 μ M) for 24 h. Arrows indicate the asLSU- γ RNA-derived fragments. End-labeled Decade marker (Ambion) was used as a ladder. ( b ) Primer extension carried out on total RNA extracted from axenic amastigotes treated with increasing concentrations of MF (0–20 μ M) for 24 h. Primer extension was performed with different primers that generated in all cases an induced fragmentation pattern for asLSU- γ RNA upon MF treatment (data not shown). ( c , upper panel) Northern blot analysis of RNA from 0 to 20 μ M MF-treated amastigotes for 24 h using the 173 nt ss-DNA probe. ( c , bottom panel) Overexposed RNA blot to detect asLSU- γ RNA cleavage products accumulated upon increased concentrations of MF

    Techniques Used: Isolation, Derivative Assay, Labeling, Marker, Generated, Northern Blot, Northern blot

    Natural asRNA complementary to the rRNA is produced in Leishmania in the context of translating ribosomes. ( a ) asRNA complementary to the LSU- γ rRNA was detected by strand-specific (ss) RT-PCR. The +RT−AS is to detect the asRNA. The −RT reaction is to check for DNA contamination. PCR with genomic DNA (+) was included as a positive control. ( b and c ) RNA blots to detect the mature and precursor bands of antisense (as) and sense (s) LSU- γ rRNAs in Leishmania promastigote (Pro) and amastigote (Ama) life stages. Total RNA resolved on 1% agarose gel was hybridized with the 173 nt ss-DNA probe corresponding to nucleotides 41–213 of sLSU- γ rRNA and recognizing the asLSU- γ RNA ( b ) or with a 5'-labeled 42 nt oligonucleotide complementary to nucleotides 172–213 of the sLSU- γ rRNA ( c ). ( d ) Leishmania promastigote lysates were loaded onto linear 15–45% (w/w) sucrose gradient and fractionated by ultracentrifugation by continuously recording absorbance (A) at 254 nm to separate the 40S and 60S ribosomal subunits from the 80S monosome and polysome fractions (upper panel). Fractionated RNA material corresponding to the small ribonuclear protein complexes (RNPs) (F1 to F2), ribosomal subunits 40S (F3) and 60S (F4), 80S monosome (F5 and F6) and polysomes (F7–F12) was resolved on 10% urea acrylamide gel and analyzed by northern blot hybridization with the 173 nt ss-DNA probe (24 h exposure) (bottom panel). The mature ∼200 nt asLSU- γ rRNA is indicated. Smaller asLSU- γ RNA-derived hybridizing fragments are indicated within a bracket. ( e ) The same membrane as in d was used for northern blot hybridization after stripping to detect sLSU- γ rRNA using the 42 nt oligonucleotide probe (2 h exposure). sLSU- γ rRNA-derived hybridizing fragments are indicated within a bracket. Plain arrows indicate sLSU- γ and asLSU- γ rRNA-derived fragments of a similar length and open arrows indicate fragments of a different length
    Figure Legend Snippet: Natural asRNA complementary to the rRNA is produced in Leishmania in the context of translating ribosomes. ( a ) asRNA complementary to the LSU- γ rRNA was detected by strand-specific (ss) RT-PCR. The +RT−AS is to detect the asRNA. The −RT reaction is to check for DNA contamination. PCR with genomic DNA (+) was included as a positive control. ( b and c ) RNA blots to detect the mature and precursor bands of antisense (as) and sense (s) LSU- γ rRNAs in Leishmania promastigote (Pro) and amastigote (Ama) life stages. Total RNA resolved on 1% agarose gel was hybridized with the 173 nt ss-DNA probe corresponding to nucleotides 41–213 of sLSU- γ rRNA and recognizing the asLSU- γ RNA ( b ) or with a 5'-labeled 42 nt oligonucleotide complementary to nucleotides 172–213 of the sLSU- γ rRNA ( c ). ( d ) Leishmania promastigote lysates were loaded onto linear 15–45% (w/w) sucrose gradient and fractionated by ultracentrifugation by continuously recording absorbance (A) at 254 nm to separate the 40S and 60S ribosomal subunits from the 80S monosome and polysome fractions (upper panel). Fractionated RNA material corresponding to the small ribonuclear protein complexes (RNPs) (F1 to F2), ribosomal subunits 40S (F3) and 60S (F4), 80S monosome (F5 and F6) and polysomes (F7–F12) was resolved on 10% urea acrylamide gel and analyzed by northern blot hybridization with the 173 nt ss-DNA probe (24 h exposure) (bottom panel). The mature ∼200 nt asLSU- γ rRNA is indicated. Smaller asLSU- γ RNA-derived hybridizing fragments are indicated within a bracket. ( e ) The same membrane as in d was used for northern blot hybridization after stripping to detect sLSU- γ rRNA using the 42 nt oligonucleotide probe (2 h exposure). sLSU- γ rRNA-derived hybridizing fragments are indicated within a bracket. Plain arrows indicate sLSU- γ and asLSU- γ rRNA-derived fragments of a similar length and open arrows indicate fragments of a different length

    Techniques Used: Produced, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Positive Control, Agarose Gel Electrophoresis, Labeling, Acrylamide Gel Assay, Northern Blot, Hybridization, Derivative Assay, Stripping Membranes

    Overexpression of asLSU- γ rRNA stimulates srRNA degradation upon induction of apoptosis. ( a ) Schematic diagram of Leishmania expression vectors harboring the full-length LSU- γ (213 bp) and part of the LSU- α and LSU- β in the sense (s) and antisense (as) orientation. ( b ) qRT-PCR to validate overexpression of the asLSU- γ RNA in the asLSU1.2 overexpressor in comparison with the sLSU1.2 overexpressor. ( c , upper panel) EtBr-stained RNA gel of MF-treated parasites overexpressing either the sLSU1.2 (0–20 μ M) or the asLSU1.2 (0–15 μ M) rRNA. ( c , bottom panel) RNA blot with the 173 nt ss-DNA probe recognizing asLSU- γ RNA showing more accumulation of the mature asLSU- γ RNA in the untreated asLSU1.2 overexpressor but increased degradation of this RNA upon MF treatment. ( d ) Primer extension analysis to detect sLSU- γ rRNA and its degradation products in both sLSU1.2- and asLSU1.2-overexpressed strains using a reverse primer complementary to nucleotides 196–213 of sLSU- γ
    Figure Legend Snippet: Overexpression of asLSU- γ rRNA stimulates srRNA degradation upon induction of apoptosis. ( a ) Schematic diagram of Leishmania expression vectors harboring the full-length LSU- γ (213 bp) and part of the LSU- α and LSU- β in the sense (s) and antisense (as) orientation. ( b ) qRT-PCR to validate overexpression of the asLSU- γ RNA in the asLSU1.2 overexpressor in comparison with the sLSU1.2 overexpressor. ( c , upper panel) EtBr-stained RNA gel of MF-treated parasites overexpressing either the sLSU1.2 (0–20 μ M) or the asLSU1.2 (0–15 μ M) rRNA. ( c , bottom panel) RNA blot with the 173 nt ss-DNA probe recognizing asLSU- γ RNA showing more accumulation of the mature asLSU- γ RNA in the untreated asLSU1.2 overexpressor but increased degradation of this RNA upon MF treatment. ( d ) Primer extension analysis to detect sLSU- γ rRNA and its degradation products in both sLSU1.2- and asLSU1.2-overexpressed strains using a reverse primer complementary to nucleotides 196–213 of sLSU- γ

    Techniques Used: Over Expression, Expressing, Quantitative RT-PCR, Staining, Northern blot

    10) Product Images from "PIWIL1 Promotes Gastric Cancer via a piRNA-Independent Mechanism"

    Article Title: PIWIL1 Promotes Gastric Cancer via a piRNA-Independent Mechanism

    Journal: bioRxiv

    doi: 10.1101/2020.05.03.075390

    PIWIL1 target RNAs and transcriptomic changes in PIWIL1-KO SNU-1 cells. (A) WGCNA analysis of RNA co-expression modules regulated by PIWIL1. Topological overlap dissimilarity measure is clustered by average linkage hierarchical clustering. Module assignments (using a dynamic hybrid algorithm) are denoted in the color bar (bottom). (B) Heatmap of the correlation between module eigengenes and the trait of with or without PIWIL1 expression. Red color represents a positive correlation between a module and the trait, and blue color represents a negative correlation. Each cell contained the corresponding correlation and P-value . (C) KEGG pathway analysis of hub genes of the blue or turquoise module. Any gene with KME ≥0.9 was assigned as a hub gene. GSEA/MSigDB gene sets tool was used for the KEGG pathway analysis. (D) Venn diagram of RNAs positively or negatively regulated by PIWIL1 or bound by PIWIL1. PIWIL1-positively or negatively regulated RNAs are identified by DESeq2 analysis with P
    Figure Legend Snippet: PIWIL1 target RNAs and transcriptomic changes in PIWIL1-KO SNU-1 cells. (A) WGCNA analysis of RNA co-expression modules regulated by PIWIL1. Topological overlap dissimilarity measure is clustered by average linkage hierarchical clustering. Module assignments (using a dynamic hybrid algorithm) are denoted in the color bar (bottom). (B) Heatmap of the correlation between module eigengenes and the trait of with or without PIWIL1 expression. Red color represents a positive correlation between a module and the trait, and blue color represents a negative correlation. Each cell contained the corresponding correlation and P-value . (C) KEGG pathway analysis of hub genes of the blue or turquoise module. Any gene with KME ≥0.9 was assigned as a hub gene. GSEA/MSigDB gene sets tool was used for the KEGG pathway analysis. (D) Venn diagram of RNAs positively or negatively regulated by PIWIL1 or bound by PIWIL1. PIWIL1-positively or negatively regulated RNAs are identified by DESeq2 analysis with P

    Techniques Used: Expressing

    11) Product Images from "Identification of m6A residues at single-nucleotide resolution using eCLIP and an accessible custom analysis pipeline"

    Article Title: Identification of m6A residues at single-nucleotide resolution using eCLIP and an accessible custom analysis pipeline

    Journal: bioRxiv

    doi: 10.1101/2020.03.11.986174

    Experimental validation of select m 6 A residues. RNA immunoprecipitation using anti-m 6 A antibody (meRIP) followed by RT-qPCR was used (protocol outlined in top flowchart) to confirm the presence of m 6 A within the transcripts. Residues were chosen based on their location within the gene in order to gauge the ability of our method to identify m 6 A sites over a diverse profile of positions. Enrichment is measured as the percent of input recovered from the immunoprecipitation with anti-m 6 A compared to amount of input recovered using anti-IgG control. ‘Positive Control’ is a known m 6 A site within the EEF1A1 gene.
    Figure Legend Snippet: Experimental validation of select m 6 A residues. RNA immunoprecipitation using anti-m 6 A antibody (meRIP) followed by RT-qPCR was used (protocol outlined in top flowchart) to confirm the presence of m 6 A within the transcripts. Residues were chosen based on their location within the gene in order to gauge the ability of our method to identify m 6 A sites over a diverse profile of positions. Enrichment is measured as the percent of input recovered from the immunoprecipitation with anti-m 6 A compared to amount of input recovered using anti-IgG control. ‘Positive Control’ is a known m 6 A site within the EEF1A1 gene.

    Techniques Used: Immunoprecipitation, Quantitative RT-PCR, Positive Control

    Overview of the meCLIP strategy, including summary of library preparation and the subsequent algorithm to identify m 6 A residues from the sequencing reads. A) Following isolation of mRNA from total RNA samples, the transcripts are fragmented and UV crosslinked to anti-m 6 A antibody (top). Following immunoprecipitation (bottom right), the antibody is removed and the RNA is reverse transcribed. Residual amino acid adducts resulting from the RNA:antibody crosslinking cause C-to-T mutations that are detectable in the resulting sequencing reads (bottom middle). These mutations are used as input for a custom algorithm that identifies sites of elevated C-to-T conversion frequency that occur within the m 6 A consensus motif (bottom left). B) Following sequencing, the resulting reads are used for a custom algorithm that uses the ‘mpileup’ command of SAMtools ( Li et al. 2009 ) to identify sites of elevated C-to-T mutations. These positions are then filtered based on the frequency of the conversion ( > =2.5% and
    Figure Legend Snippet: Overview of the meCLIP strategy, including summary of library preparation and the subsequent algorithm to identify m 6 A residues from the sequencing reads. A) Following isolation of mRNA from total RNA samples, the transcripts are fragmented and UV crosslinked to anti-m 6 A antibody (top). Following immunoprecipitation (bottom right), the antibody is removed and the RNA is reverse transcribed. Residual amino acid adducts resulting from the RNA:antibody crosslinking cause C-to-T mutations that are detectable in the resulting sequencing reads (bottom middle). These mutations are used as input for a custom algorithm that identifies sites of elevated C-to-T conversion frequency that occur within the m 6 A consensus motif (bottom left). B) Following sequencing, the resulting reads are used for a custom algorithm that uses the ‘mpileup’ command of SAMtools ( Li et al. 2009 ) to identify sites of elevated C-to-T mutations. These positions are then filtered based on the frequency of the conversion ( > =2.5% and

    Techniques Used: Sequencing, Isolation, Immunoprecipitation

    12) Product Images from "Transposase assisted tagmentation of RNA/DNA hybrid duplexes"

    Article Title: Transposase assisted tagmentation of RNA/DNA hybrid duplexes

    Journal: bioRxiv

    doi: 10.1101/2020.01.29.926105

    Tagmentation activity of Tn5 transposome on RNA/DNA hybrids. (a) Denaturing (8 M urea) polyacrylamide gel analysis of reverse transcription products of an in vitro transcribed mRNA (IRF9). Lane 1: ssRNA marker. Lane 2: in vitro transcribed mRNA (IRF9). Lane 3 4: reverse transcription products of an in vitro transcribed mRNA (IRF9). Lane 5: reverse transcription product treated with DNase I. Lane 6: reverse transcription product treated with RNase H. ssRNA and ssDNA is marked with a red asterisk and a blue pound sign, respectively. (b) Gel picture showing size distribution of RNA/DNA hybrids products of 50 μl reaction systems without Tn5 transposome, and with 5 μl, 10 μl, and 15 μl Tn5 transposome, respectively. The blue and orange patches denote small and large fragments, respectively. (c) qPCR amplification curve of tagmentation products without Tn5 treatment or with Tn5 treatment in three different buffers (see methods). Average Ct values are 26.41, 18.39, 18.33 and 18.34, respectively. (d) Sanger sequencing chromatograms of PCR products following RNA/DNA hybrid tagmentation and strand extension. Adaptor A and B sequences are highlighted with blue background color and insert sequences are highlighted with yellow background.
    Figure Legend Snippet: Tagmentation activity of Tn5 transposome on RNA/DNA hybrids. (a) Denaturing (8 M urea) polyacrylamide gel analysis of reverse transcription products of an in vitro transcribed mRNA (IRF9). Lane 1: ssRNA marker. Lane 2: in vitro transcribed mRNA (IRF9). Lane 3 4: reverse transcription products of an in vitro transcribed mRNA (IRF9). Lane 5: reverse transcription product treated with DNase I. Lane 6: reverse transcription product treated with RNase H. ssRNA and ssDNA is marked with a red asterisk and a blue pound sign, respectively. (b) Gel picture showing size distribution of RNA/DNA hybrids products of 50 μl reaction systems without Tn5 transposome, and with 5 μl, 10 μl, and 15 μl Tn5 transposome, respectively. The blue and orange patches denote small and large fragments, respectively. (c) qPCR amplification curve of tagmentation products without Tn5 treatment or with Tn5 treatment in three different buffers (see methods). Average Ct values are 26.41, 18.39, 18.33 and 18.34, respectively. (d) Sanger sequencing chromatograms of PCR products following RNA/DNA hybrid tagmentation and strand extension. Adaptor A and B sequences are highlighted with blue background color and insert sequences are highlighted with yellow background.

    Techniques Used: Activity Assay, In Vitro, Marker, Real-time Polymerase Chain Reaction, Amplification, Sequencing, Polymerase Chain Reaction

    13) Product Images from "Ribonucleoprotein particles of bacterial small non-coding RNA IsrA (IS61 or McaS) and its interaction with RNA polymerase core may link transcription to mRNA fate"

    Article Title: Ribonucleoprotein particles of bacterial small non-coding RNA IsrA (IS61 or McaS) and its interaction with RNA polymerase core may link transcription to mRNA fate

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv1302

    Only non-specific binding of IsrA to RNAP can inhibit transcription. ( A ) Transcription on a linear template containing the T7A1 promoter. The order in which RNAP was allowed to associate with the promoter DNA or competitor RNA is given (+* added first; + second). Note the template-independent labeling of IsrA at the 3′ end by RNAP, which suggests non-specific binding near the active center of RNAP (see text). ( B ) Pre-formed 11-mer elongation complexes were chased in the presence or absence of sRNAs. ( C ) Abortive synthesis of CpApU on T7A1 promoter. Mutant IsrA RNAs or heparin were added before promoter DNA. Note that the IsrA variant Δ2, which cannot bind RNAP specifically, still inhibits initiation and is labelled at the 3′ end (see text for details).
    Figure Legend Snippet: Only non-specific binding of IsrA to RNAP can inhibit transcription. ( A ) Transcription on a linear template containing the T7A1 promoter. The order in which RNAP was allowed to associate with the promoter DNA or competitor RNA is given (+* added first; + second). Note the template-independent labeling of IsrA at the 3′ end by RNAP, which suggests non-specific binding near the active center of RNAP (see text). ( B ) Pre-formed 11-mer elongation complexes were chased in the presence or absence of sRNAs. ( C ) Abortive synthesis of CpApU on T7A1 promoter. Mutant IsrA RNAs or heparin were added before promoter DNA. Note that the IsrA variant Δ2, which cannot bind RNAP specifically, still inhibits initiation and is labelled at the 3′ end (see text for details).

    Techniques Used: Binding Assay, Labeling, Mutagenesis, Variant Assay

    Stem 2 of IsrA is required for specific binding to RNAP. ( A ) Mutant versions of IsrA (see also Supplementary Figure S1F) shown as a black line-diagram with deleted regions indicated in white. In the i2loop mutant the loop closing stem 2 has been replaced with GAAG (gray; see also Supplementary Figure S6A). ( B ) EMSA of RNAP and mutant labeled IsrA RNAs with heparin added before (+*) or after (+) complex formation. All reactions contained total yeast RNA as a non-specific competitor prior to complex formation. ( C ) Competition of unlabeled mutant IsrA RNAs (15-fold molar excess) with labeled wild-type variant. Unlabeled competitor RNA was always added before complex formation of RNAP with labeled RNA. All reactions contained total yeast RNA as a non-specific competitor prior to complex formation. Heparin was added in all reactions after formation of complexes. Only IsrA mutants that are able to bind RNAP in the presence of heparin (see panel B) compete against association of labeled IsrA. ( D ) Northern blot analysis of mutant IsrA RNAs that co-purified with RNAP (for protein gel see Supplementary Figure S6B. Plasmids from which the mutant IsrA RNAs (see panel A) were expressed, along with an empty vector (pGemT), were transformed into strain RL rpoC BCCP Δ isrA with a disrupted IsrA gene and carrying biotinylated RNAP. A strain expressing RNAP without the biotinylated tag (RLΔ isrA- p isrA (*)) was used as a negative control (input and pull-down are overloaded). sRNAs were released from beads with 3C, and the blots were probed against IsrA (bottom) or 6S RNA (top). Below the blot, quantitation of the bands corresponding to IsrA mutants (encircled in the blot), normalized to the corresponding bands of 6S RNA.
    Figure Legend Snippet: Stem 2 of IsrA is required for specific binding to RNAP. ( A ) Mutant versions of IsrA (see also Supplementary Figure S1F) shown as a black line-diagram with deleted regions indicated in white. In the i2loop mutant the loop closing stem 2 has been replaced with GAAG (gray; see also Supplementary Figure S6A). ( B ) EMSA of RNAP and mutant labeled IsrA RNAs with heparin added before (+*) or after (+) complex formation. All reactions contained total yeast RNA as a non-specific competitor prior to complex formation. ( C ) Competition of unlabeled mutant IsrA RNAs (15-fold molar excess) with labeled wild-type variant. Unlabeled competitor RNA was always added before complex formation of RNAP with labeled RNA. All reactions contained total yeast RNA as a non-specific competitor prior to complex formation. Heparin was added in all reactions after formation of complexes. Only IsrA mutants that are able to bind RNAP in the presence of heparin (see panel B) compete against association of labeled IsrA. ( D ) Northern blot analysis of mutant IsrA RNAs that co-purified with RNAP (for protein gel see Supplementary Figure S6B. Plasmids from which the mutant IsrA RNAs (see panel A) were expressed, along with an empty vector (pGemT), were transformed into strain RL rpoC BCCP Δ isrA with a disrupted IsrA gene and carrying biotinylated RNAP. A strain expressing RNAP without the biotinylated tag (RLΔ isrA- p isrA (*)) was used as a negative control (input and pull-down are overloaded). sRNAs were released from beads with 3C, and the blots were probed against IsrA (bottom) or 6S RNA (top). Below the blot, quantitation of the bands corresponding to IsrA mutants (encircled in the blot), normalized to the corresponding bands of 6S RNA.

    Techniques Used: Binding Assay, Mutagenesis, Labeling, Variant Assay, Northern Blot, Purification, Plasmid Preparation, Transformation Assay, Expressing, Negative Control, Quantitation Assay

    IsrA specifically binds core enzyme of RNAP. ( A ) Electrophoretic mobity shift assay (EMSA) with radioactively labeled, gel-purified T7 synthesized 32 P-labelled RNAs (tRNA Ala , 6S RNA or IsrA) which were mixed with equimolar amounts of purified core or holo (σ 70 ) enzymes of RNAP. The labeled RNA and a competitor (T7A1 promoter DNA or heparin) were added to RNAP first (+*) or second (+). ( B ) Competition of various unlabeled RNAs with 32 P-labeled 6S RNA and IsrA. 15-fold excess of unlabeled competitor RNA (Supplementary Figure S5) was added to RNAP before addition of labeled RNAs. All reactions contained total yeast RNA as a non-specific competitor prior to complex formation. Heparin was added to all reactions before loading on the gel to dissolve non-specific complexes.
    Figure Legend Snippet: IsrA specifically binds core enzyme of RNAP. ( A ) Electrophoretic mobity shift assay (EMSA) with radioactively labeled, gel-purified T7 synthesized 32 P-labelled RNAs (tRNA Ala , 6S RNA or IsrA) which were mixed with equimolar amounts of purified core or holo (σ 70 ) enzymes of RNAP. The labeled RNA and a competitor (T7A1 promoter DNA or heparin) were added to RNAP first (+*) or second (+). ( B ) Competition of various unlabeled RNAs with 32 P-labeled 6S RNA and IsrA. 15-fold excess of unlabeled competitor RNA (Supplementary Figure S5) was added to RNAP before addition of labeled RNAs. All reactions contained total yeast RNA as a non-specific competitor prior to complex formation. Heparin was added to all reactions before loading on the gel to dissolve non-specific complexes.

    Techniques Used: Shift Assay, Labeling, Purification, Synthesized

    RNAP and a defined set of proteins co-purify with IsrA carrying a streptotag. ( A ) Streptotagged IsrA-3tag (see Figure 1B ) and proteins associated with it were isolated on di-streptomycin beads from strain in which the endogenous gene for IsrA was disrupted (strain RL rpoC BCCP Δ isrA -p isrA3tag , see Supplementary table). Co-purifying proteins (Strepto lanes) were analyzed by SDS-PAGE on 4–20% gradient gels and stained with Coomassie or silver as described in the Materials and Methods. Mock isolation with the di-streptomycin beads using the parental strain with untagged IsrA (RL; lane 4) was used as a negative control. The presence and absence of affinity tags on IsrA is indicated by + and –, respectively. RNAP purified on a heparin column from strain BW-62 was used as size control (RC; lane 3). ( B ) RNA was analyzed by Northern blotting with an IsrA-specific probe. Note that the blot is overexposed to make the IsrA band in the input visible. The presence and absence of affinity tags on IsrA is indicated by + and –, respectively.
    Figure Legend Snippet: RNAP and a defined set of proteins co-purify with IsrA carrying a streptotag. ( A ) Streptotagged IsrA-3tag (see Figure 1B ) and proteins associated with it were isolated on di-streptomycin beads from strain in which the endogenous gene for IsrA was disrupted (strain RL rpoC BCCP Δ isrA -p isrA3tag , see Supplementary table). Co-purifying proteins (Strepto lanes) were analyzed by SDS-PAGE on 4–20% gradient gels and stained with Coomassie or silver as described in the Materials and Methods. Mock isolation with the di-streptomycin beads using the parental strain with untagged IsrA (RL; lane 4) was used as a negative control. The presence and absence of affinity tags on IsrA is indicated by + and –, respectively. RNAP purified on a heparin column from strain BW-62 was used as size control (RC; lane 3). ( B ) RNA was analyzed by Northern blotting with an IsrA-specific probe. Note that the blot is overexposed to make the IsrA band in the input visible. The presence and absence of affinity tags on IsrA is indicated by + and –, respectively.

    Techniques Used: Isolation, SDS Page, Staining, Negative Control, Purification, Northern Blot

    A small non-coding RNA, IsrA, co-purifies with RNA polymerase. ( A ) Pull-down of RNAs with 6xHis-tagged (strain RL rpoC HIS ) or tag-less RNAP on Ni 2+ -NTA sepharose. Isolated RNAs were 5′- or 3′ end labeled and analyzed as described in the Materials and Methods. ( B ) Secondary structure model of the purified RNA, identified as IsrA (( 23 ); a.k.a. IS61 ( 20 ) or McaS ( 21 , 22 )) which was based on phylogenetic analysis (see Supplementary Figure S2). An extension that substitutes the outer portion of stem 3 and provides affinity to streptomycin (streptotag ( 32 ); see Figure 2 ) is boxed. ( C ) Summary of IsrA structure in sequence logo representation; height of nucleotides mark the degree of conservation. Base pairs that form stems are indicated by brackets; rectangles indicate regions implicated in protein binding (CsrA), or translational regulation of flhDC , or csgD mRNAs ( 21 , 22 , 43 ). ( D ) Purification of RNAP on streptavidin sepharose through a biotinylated tag at the β’ subunit that can be cleaved off using HRV3C (3C) protease. RNAPs were isolated from strains; lane 1: RLΔ isrA -p isrA (tag-less RNAP, IsrA under mutant promoter on multi-copy plasmid); lane 2: RL (tag-less RNAP, genomic IsrA under mutant promoter); lane 3: MG1655 rpoC BCCP (BCCP-tagged RNAP, genomic IsrA under wild-type promoter); lane 4: JC7623 rpoC BCCP (BCCP-tagged RNAP, genomic IsrA under wild-type promoter); lane 5: RL rpoC BCCP (BCCP-tagged RNAP, genomic IsrA under mutant promoter); lane 6: RL rpoC BCCP hfq65 (BCCP-tagged RNAP, genomic IsrA under mutant promoter, Hfq lacks C-terminus) (see also Supplementary Table). Relative expression levels of IsrA are shown above the gels (expressed from wild-type promoter in chromosomal locus (+), expressed from a mutant promoter in the chromosomal locus (++) or from mutant promoter from a high-copy plasmid (+++)). RNAPs were released by HRV3C (3C) cleavage and analyzed by SDS/PAGE on a 4–20% gradient gel. Strains that have RNAP without a biotinylated tag were used as a control. Lanes 7 and 8 are controls for non-specific binding to and efficiency of release from beads of RNAP in lanes 1 and 5, respectively. ( E ) Northern blot analysis of RNAs that co-purified with biotinylated RNAP in panel (D). The blots were probed against IsrA (bottom) or 6S RNA (top). Indicated are the presence of the biotinylated affinity tag on RNAP β’ (+ or – tag), the expression levels of IsrA in the input extract (as in panel (D)), and whether Hfq in the cells was truncated to its core of 65 amino acids (ΔC) or was wild type (+).
    Figure Legend Snippet: A small non-coding RNA, IsrA, co-purifies with RNA polymerase. ( A ) Pull-down of RNAs with 6xHis-tagged (strain RL rpoC HIS ) or tag-less RNAP on Ni 2+ -NTA sepharose. Isolated RNAs were 5′- or 3′ end labeled and analyzed as described in the Materials and Methods. ( B ) Secondary structure model of the purified RNA, identified as IsrA (( 23 ); a.k.a. IS61 ( 20 ) or McaS ( 21 , 22 )) which was based on phylogenetic analysis (see Supplementary Figure S2). An extension that substitutes the outer portion of stem 3 and provides affinity to streptomycin (streptotag ( 32 ); see Figure 2 ) is boxed. ( C ) Summary of IsrA structure in sequence logo representation; height of nucleotides mark the degree of conservation. Base pairs that form stems are indicated by brackets; rectangles indicate regions implicated in protein binding (CsrA), or translational regulation of flhDC , or csgD mRNAs ( 21 , 22 , 43 ). ( D ) Purification of RNAP on streptavidin sepharose through a biotinylated tag at the β’ subunit that can be cleaved off using HRV3C (3C) protease. RNAPs were isolated from strains; lane 1: RLΔ isrA -p isrA (tag-less RNAP, IsrA under mutant promoter on multi-copy plasmid); lane 2: RL (tag-less RNAP, genomic IsrA under mutant promoter); lane 3: MG1655 rpoC BCCP (BCCP-tagged RNAP, genomic IsrA under wild-type promoter); lane 4: JC7623 rpoC BCCP (BCCP-tagged RNAP, genomic IsrA under wild-type promoter); lane 5: RL rpoC BCCP (BCCP-tagged RNAP, genomic IsrA under mutant promoter); lane 6: RL rpoC BCCP hfq65 (BCCP-tagged RNAP, genomic IsrA under mutant promoter, Hfq lacks C-terminus) (see also Supplementary Table). Relative expression levels of IsrA are shown above the gels (expressed from wild-type promoter in chromosomal locus (+), expressed from a mutant promoter in the chromosomal locus (++) or from mutant promoter from a high-copy plasmid (+++)). RNAPs were released by HRV3C (3C) cleavage and analyzed by SDS/PAGE on a 4–20% gradient gel. Strains that have RNAP without a biotinylated tag were used as a control. Lanes 7 and 8 are controls for non-specific binding to and efficiency of release from beads of RNAP in lanes 1 and 5, respectively. ( E ) Northern blot analysis of RNAs that co-purified with biotinylated RNAP in panel (D). The blots were probed against IsrA (bottom) or 6S RNA (top). Indicated are the presence of the biotinylated affinity tag on RNAP β’ (+ or – tag), the expression levels of IsrA in the input extract (as in panel (D)), and whether Hfq in the cells was truncated to its core of 65 amino acids (ΔC) or was wild type (+).

    Techniques Used: Isolation, Labeling, Purification, Sequencing, Protein Binding, Mutagenesis, Plasmid Preparation, Expressing, SDS Page, Binding Assay, Northern Blot

    14) Product Images from "Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine †"

    Article Title: Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine †

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1049

    DNA sequence of the RT-PCR products of 155 bp ( A ), 305 bp ( B ) and 188 bp ( C ) obtained by 3′5′ RACE from 16S rRNA m , p1 and p2 , respectively. The DNA sequences shown here are those found in the 16S ribosomal RNA. Sequences derived from the m 16S rRNA are highlighted in a grey background. Arrows indicate that complete DNA sequences extend until to the sequence of oligonucleotide FA, and to the reverse complement of oligonucleotide RA. In each panel, the two nucleotides shown in bold and underlined correspond to the junction, generated by the RNA ligation, of the 5′ and 3′ ends of the 16S RNAs.
    Figure Legend Snippet: DNA sequence of the RT-PCR products of 155 bp ( A ), 305 bp ( B ) and 188 bp ( C ) obtained by 3′5′ RACE from 16S rRNA m , p1 and p2 , respectively. The DNA sequences shown here are those found in the 16S ribosomal RNA. Sequences derived from the m 16S rRNA are highlighted in a grey background. Arrows indicate that complete DNA sequences extend until to the sequence of oligonucleotide FA, and to the reverse complement of oligonucleotide RA. In each panel, the two nucleotides shown in bold and underlined correspond to the junction, generated by the RNA ligation, of the 5′ and 3′ ends of the 16S RNAs.

    Techniques Used: Sequencing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Generated, Ligation

    ( A ) Schematic processing of the p1 16S rRNA. The extra-sequences of 115 nt and 33 nt, flanking the m 16S rRNA at its 5′ and 3′ ends, respectively, are shown on a grey background. RA and FA are the primers used for 3′5′ RACE analysis. The site of annealing of RA to m 16S rRNA, and that of FA to the reverse complement of the m 16S rRNA, are indicated by arrows. Figure not drawn to scale. ( B ) Expected sizes in bp of the RT-PCR products (amplicons) obtained from the different species of 16S rRNA ( p1 , p2 , p3 and m ) by 3′5′ RACE. ( C–F ) Agarose gel electrophoresis of RT-PCR products obtained by 3′5′ RACE from total RNA isolated from MC4100 bacteria grown at 30°C (C), or 44°C (D), or 45°C (E) or 46°C (F). Each RNA sample was thermo-denatured (lanes b), or not (lanes a) prior to the 3′5′ ligation. The sizes (in bp) of the molecular weight markers are indicated to the left of each gel (M). ( G ) The thermodenaturation step dissociates the complementary sequences present at the 3′ and 5′ends of the p1 16S rRNA, and therefore offers to all the 16S rRNA species an equal chance to access to the T4 RNA ligase.
    Figure Legend Snippet: ( A ) Schematic processing of the p1 16S rRNA. The extra-sequences of 115 nt and 33 nt, flanking the m 16S rRNA at its 5′ and 3′ ends, respectively, are shown on a grey background. RA and FA are the primers used for 3′5′ RACE analysis. The site of annealing of RA to m 16S rRNA, and that of FA to the reverse complement of the m 16S rRNA, are indicated by arrows. Figure not drawn to scale. ( B ) Expected sizes in bp of the RT-PCR products (amplicons) obtained from the different species of 16S rRNA ( p1 , p2 , p3 and m ) by 3′5′ RACE. ( C–F ) Agarose gel electrophoresis of RT-PCR products obtained by 3′5′ RACE from total RNA isolated from MC4100 bacteria grown at 30°C (C), or 44°C (D), or 45°C (E) or 46°C (F). Each RNA sample was thermo-denatured (lanes b), or not (lanes a) prior to the 3′5′ ligation. The sizes (in bp) of the molecular weight markers are indicated to the left of each gel (M). ( G ) The thermodenaturation step dissociates the complementary sequences present at the 3′ and 5′ends of the p1 16S rRNA, and therefore offers to all the 16S rRNA species an equal chance to access to the T4 RNA ligase.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Isolation, Ligation, Molecular Weight

    ( A and B ) Sedimentation profiles of ribosomal subunits prepared from strains MC4100 pWKS30 (A) and MC4100 pDM39 (B) labelled with [ 3 H]-uridine at 45°C. Sedimentation is from right to left. A 260 /ml, open circles. [ 3 H] c.p.m. × 10 3 , filled circles. ( C ) Total RNAs phenol-extracted from the same two bacterial batches were subjected to 3′5′ RACE analysis without prior thermodenaturation of RNA (lane a = MC4100 pWSK30, lane b = MC4100 pDM39). Relative DNA concentrations in the bands of the agarose gel were estimated using a Typhoon Trio phosphorImager and the ImageQuant software.
    Figure Legend Snippet: ( A and B ) Sedimentation profiles of ribosomal subunits prepared from strains MC4100 pWKS30 (A) and MC4100 pDM39 (B) labelled with [ 3 H]-uridine at 45°C. Sedimentation is from right to left. A 260 /ml, open circles. [ 3 H] c.p.m. × 10 3 , filled circles. ( C ) Total RNAs phenol-extracted from the same two bacterial batches were subjected to 3′5′ RACE analysis without prior thermodenaturation of RNA (lane a = MC4100 pWSK30, lane b = MC4100 pDM39). Relative DNA concentrations in the bands of the agarose gel were estimated using a Typhoon Trio phosphorImager and the ImageQuant software.

    Techniques Used: Sedimentation, Agarose Gel Electrophoresis, Software

    ( A ) Preparative sucrose gradient sedimentation of ribosomal particles from strain MC4100 labelled with [ 3 H]-uridine at 45°C. Fractions 18–21 were pooled, and 21S particles were concentrated by ultracentrifugation. ( B ) An aliquot of the 21S particles isolated from ( A ) was mixed with unlabelled 50S and 30S subunits from a wt strain and rerun on a new sucrose gradient under the same conditions. Sedimentation is from right to left. A 260 /ml, open circles. [ 3 H] c.p.m. × 10 3 , filled circles. ( C ) 16S rRNA phenol-extracted from the 21S particles isolated from ( A ) were subjected to 3′5′ RACE analysis, with (lane b) or without (lane a) a thermodenaturation step prior to the 3′5′ RNA ligation. The RT-PCR products of 305 bp and 188 bp were purified by preparative agarose gel electrophoresis (from lanes a and b, respectively) and sequenced. In lane c, an aliquot of the 16S rRNA was thermodenatured, but instead of rapid freezing leading to irreversible RNA denaturation (as shown in lane b), was then subjected to a slow cooling (a couple of hours at room temperature) leading to reversible RNA denaturation, prior to the 3′5′ RACE procedure: the RT-PCR products obtained under these conditions are similar to those obtained in the absence of any RNA denaturation step (lane a).
    Figure Legend Snippet: ( A ) Preparative sucrose gradient sedimentation of ribosomal particles from strain MC4100 labelled with [ 3 H]-uridine at 45°C. Fractions 18–21 were pooled, and 21S particles were concentrated by ultracentrifugation. ( B ) An aliquot of the 21S particles isolated from ( A ) was mixed with unlabelled 50S and 30S subunits from a wt strain and rerun on a new sucrose gradient under the same conditions. Sedimentation is from right to left. A 260 /ml, open circles. [ 3 H] c.p.m. × 10 3 , filled circles. ( C ) 16S rRNA phenol-extracted from the 21S particles isolated from ( A ) were subjected to 3′5′ RACE analysis, with (lane b) or without (lane a) a thermodenaturation step prior to the 3′5′ RNA ligation. The RT-PCR products of 305 bp and 188 bp were purified by preparative agarose gel electrophoresis (from lanes a and b, respectively) and sequenced. In lane c, an aliquot of the 16S rRNA was thermodenatured, but instead of rapid freezing leading to irreversible RNA denaturation (as shown in lane b), was then subjected to a slow cooling (a couple of hours at room temperature) leading to reversible RNA denaturation, prior to the 3′5′ RACE procedure: the RT-PCR products obtained under these conditions are similar to those obtained in the absence of any RNA denaturation step (lane a).

    Techniques Used: Sedimentation, Isolation, Ligation, Reverse Transcription Polymerase Chain Reaction, Purification, Agarose Gel Electrophoresis

    15) Product Images from "The RNA Helicase Rm62 Cooperates with SU(VAR)3-9 to Re-Silence Active Transcription in Drosophila melanogaster"

    Article Title: The RNA Helicase Rm62 Cooperates with SU(VAR)3-9 to Re-Silence Active Transcription in Drosophila melanogaster

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0020761

    Prolonged RNA production from the hsp70 locus after reduction of SU(VAR)3-9 or Rm62. ( A ) RNAi against SU(VAR)3-9 and/or Rm62 specifically eliminates the respective proteins as well as it's mRNA from SL2 whole cell extract (WCE). SL2 cells were transfected with specific double stranded RNAs against SU(VAR)3-9, Rm62 or GST (control). WCEs and total RNA were prepared 6 days after transfection. Left : Proteins were analyzed by SDS-PAGE followed by western blotting with the indicated antibodies (GST: control RNAi, SU(VAR)3-9: RNAi against Su(var)3-9, Rm62: RNAi against Rm62, SU(VAR)3-9/Rm62: double knockdown of Su(var)3-9 and Rm62). Right : 1 µg oft total RNA, was reverse transcribed (Superscript™, Reverse Transcriptase, Invitrogen) using gene specific primers for Su(var)3-9 or Rm62, respectively. 10% of the obtained cDNA were analyzed by standard PCR using specific primers for Su(var)3-9 or Rm62, and separated by agarose gel electrophoresis. To discriminate between genomic and cDNA, we used intron spanning primer pairs in the PCR reaction. ( B ) Quantitative RT-PCR of the hsp70 m RNA from SL2 cells before (no HS), after heat shock (30 min HS) and a 180 min of recovery phase (+180 min recovery). SL2 cells, cultured under standard conditions, were subjected to RNAi against GST (control), Su(var)3-9 or Rm62. After 6 days of culturing, cells were either not treated (no HS) or treated with a heat shock (30 min HS) followed by a recovery for 180 min at 26°C (+180 min recovery), respectively. RNA from these cells was isolated, reverse transcribed and analyzed by quantitative real time PCR. Bars represent relative hsp70 RNA expression levels (in percent) normalized to an internal control (U6 snRNA), which does not respond to heat shock. Percent expression was calculated to the maximal amount of RNA measured after heat shock. The inlet graph shows an enlargement of the calculated values after 180 min recovery upon heat shock. The observed difference between the SU(VAR)3-9/GST and Rm62/GST is significant as calculated with an unpaired two sided Student's t-test (p = 0.041 and p = 0.014, respectively). Error bars indicate the standard deviation of two replicates. ( C ) Relative expression of hsp70 RNA from wild type (WT) Rm62 ( CBO2119/LipF ) and Su(var)3-9 heteroalleic flies ( Su(var)3-9 1 /Su(var)3-9 2 ) before (no HS) or after heat shock (30 min HS) followed by 120 min of recovery (+120 min recovery). RNAs were extracted from the flies either not treated or treated with a heat impulse and “recovered” for 120 min at 25°C and further subjected to quantitative real time PCR. Bars represent relative hsp70 RNA expression levels (in percent) normalized to an internal control (18S rRNA), which has a minimal effect on heat shock. The observed difference between Su(var)3-9 mutant and Rm62 mutants compared to wildtype flies is significant as calculated with an unpaired two sided Student's t-test (p = 0.047 and p = 0.003, respectively). Error bars indicate the standard deviation of three replicates.
    Figure Legend Snippet: Prolonged RNA production from the hsp70 locus after reduction of SU(VAR)3-9 or Rm62. ( A ) RNAi against SU(VAR)3-9 and/or Rm62 specifically eliminates the respective proteins as well as it's mRNA from SL2 whole cell extract (WCE). SL2 cells were transfected with specific double stranded RNAs against SU(VAR)3-9, Rm62 or GST (control). WCEs and total RNA were prepared 6 days after transfection. Left : Proteins were analyzed by SDS-PAGE followed by western blotting with the indicated antibodies (GST: control RNAi, SU(VAR)3-9: RNAi against Su(var)3-9, Rm62: RNAi against Rm62, SU(VAR)3-9/Rm62: double knockdown of Su(var)3-9 and Rm62). Right : 1 µg oft total RNA, was reverse transcribed (Superscript™, Reverse Transcriptase, Invitrogen) using gene specific primers for Su(var)3-9 or Rm62, respectively. 10% of the obtained cDNA were analyzed by standard PCR using specific primers for Su(var)3-9 or Rm62, and separated by agarose gel electrophoresis. To discriminate between genomic and cDNA, we used intron spanning primer pairs in the PCR reaction. ( B ) Quantitative RT-PCR of the hsp70 m RNA from SL2 cells before (no HS), after heat shock (30 min HS) and a 180 min of recovery phase (+180 min recovery). SL2 cells, cultured under standard conditions, were subjected to RNAi against GST (control), Su(var)3-9 or Rm62. After 6 days of culturing, cells were either not treated (no HS) or treated with a heat shock (30 min HS) followed by a recovery for 180 min at 26°C (+180 min recovery), respectively. RNA from these cells was isolated, reverse transcribed and analyzed by quantitative real time PCR. Bars represent relative hsp70 RNA expression levels (in percent) normalized to an internal control (U6 snRNA), which does not respond to heat shock. Percent expression was calculated to the maximal amount of RNA measured after heat shock. The inlet graph shows an enlargement of the calculated values after 180 min recovery upon heat shock. The observed difference between the SU(VAR)3-9/GST and Rm62/GST is significant as calculated with an unpaired two sided Student's t-test (p = 0.041 and p = 0.014, respectively). Error bars indicate the standard deviation of two replicates. ( C ) Relative expression of hsp70 RNA from wild type (WT) Rm62 ( CBO2119/LipF ) and Su(var)3-9 heteroalleic flies ( Su(var)3-9 1 /Su(var)3-9 2 ) before (no HS) or after heat shock (30 min HS) followed by 120 min of recovery (+120 min recovery). RNAs were extracted from the flies either not treated or treated with a heat impulse and “recovered” for 120 min at 25°C and further subjected to quantitative real time PCR. Bars represent relative hsp70 RNA expression levels (in percent) normalized to an internal control (18S rRNA), which has a minimal effect on heat shock. The observed difference between Su(var)3-9 mutant and Rm62 mutants compared to wildtype flies is significant as calculated with an unpaired two sided Student's t-test (p = 0.047 and p = 0.003, respectively). Error bars indicate the standard deviation of three replicates.

    Techniques Used: Transfection, SDS Page, Western Blot, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Quantitative RT-PCR, Cell Culture, Isolation, Real-time Polymerase Chain Reaction, RNA Expression, Expressing, Standard Deviation, Mutagenesis

    16) Product Images from "Intra-species recombination among strains of the ampelovirus Grapevine leafroll-associated virus 4"

    Article Title: Intra-species recombination among strains of the ampelovirus Grapevine leafroll-associated virus 4

    Journal: Virology Journal

    doi: 10.1186/s12985-019-1243-4

    Analysis of recombination events in the genome of GLRaV-4 strains. ( a ) Graphical representation (not drawn to scale) of the generalized genome map of GLRaV-4. Individual open reading frames (ORFs) are shown as boxes with associated protein designations used for closteroviruses [ 2 ] and numbered 1 to 6 above the diagram. Abbreviations of ORFs: L-Pro , papain-like leader protease; MET , methyltransferase domain; HEL , RNA helicase domain; AlkB , the AlkB domain; RdRp , RNA-dependent RNA polymerase; p5, 5 kDa protein; Hsp70h , heat shock protein 70 homolog; CP , coat protein; p23, 23 kDa protein. Lines at the genome extremities represent non-translated regions. b Putative recombinant events in isolates LR106 and Estellat. (B-1) and (B-2) represent, respectively, recombination event-1 (nt 4105–5240) and event-2 (nt 627–1551) in ORF1a of the LR106 isolate and (B-3) represents recombinant event (nt 1–6312) in the genome of the Estellat isolate identified by the RDP. The X-axis indicates the nucleotide position in the alignment and the Y-axis shows informative nucleotide pairwise identity between parental and recombinant isolates. The color key of the parental isolates is shown next to the plots
    Figure Legend Snippet: Analysis of recombination events in the genome of GLRaV-4 strains. ( a ) Graphical representation (not drawn to scale) of the generalized genome map of GLRaV-4. Individual open reading frames (ORFs) are shown as boxes with associated protein designations used for closteroviruses [ 2 ] and numbered 1 to 6 above the diagram. Abbreviations of ORFs: L-Pro , papain-like leader protease; MET , methyltransferase domain; HEL , RNA helicase domain; AlkB , the AlkB domain; RdRp , RNA-dependent RNA polymerase; p5, 5 kDa protein; Hsp70h , heat shock protein 70 homolog; CP , coat protein; p23, 23 kDa protein. Lines at the genome extremities represent non-translated regions. b Putative recombinant events in isolates LR106 and Estellat. (B-1) and (B-2) represent, respectively, recombination event-1 (nt 4105–5240) and event-2 (nt 627–1551) in ORF1a of the LR106 isolate and (B-3) represents recombinant event (nt 1–6312) in the genome of the Estellat isolate identified by the RDP. The X-axis indicates the nucleotide position in the alignment and the Y-axis shows informative nucleotide pairwise identity between parental and recombinant isolates. The color key of the parental isolates is shown next to the plots

    Techniques Used: Recombinant

    17) Product Images from "qDRIP: Quantitative differential RNA:DNA hybrid immunoprecipitation sequencing"

    Article Title: qDRIP: Quantitative differential RNA:DNA hybrid immunoprecipitation sequencing

    Journal: bioRxiv

    doi: 10.1101/811208

    Preparing and evaluating synthetic RNA:DNA hybrids as spike-ins for DRIP. (A) Experimental scheme showing how hybrids were synthesized. Briefly, target regions were amplified from E. coli genomic DNA with a flanking T7 promoter. RNA was prepared from these templates by in vitro transcription, then hybridized to a synthetic ssDNA oligo. Hybrids were purified by agarose gel electrophoresis. (B) Length and GC content for the positive and negative control spike-ins used in this study. (C) Gel showing RNase H (RH) reversible size-shifts after hybridization of RNA and DNA. (D) qPCR of genomic (left) and spike-in (right) hybrids following transcription inhibition with DRB. RNase H treatment demonstrates specificity. Error bars represent 95% confidence interval (CI) of the mean. Results are significantly different as determined by non-overlapping 95% CIs.
    Figure Legend Snippet: Preparing and evaluating synthetic RNA:DNA hybrids as spike-ins for DRIP. (A) Experimental scheme showing how hybrids were synthesized. Briefly, target regions were amplified from E. coli genomic DNA with a flanking T7 promoter. RNA was prepared from these templates by in vitro transcription, then hybridized to a synthetic ssDNA oligo. Hybrids were purified by agarose gel electrophoresis. (B) Length and GC content for the positive and negative control spike-ins used in this study. (C) Gel showing RNase H (RH) reversible size-shifts after hybridization of RNA and DNA. (D) qPCR of genomic (left) and spike-in (right) hybrids following transcription inhibition with DRB. RNase H treatment demonstrates specificity. Error bars represent 95% confidence interval (CI) of the mean. Results are significantly different as determined by non-overlapping 95% CIs.

    Techniques Used: Synthesized, Amplification, In Vitro, Purification, Agarose Gel Electrophoresis, Negative Control, Hybridization, Real-time Polymerase Chain Reaction, Inhibition

    qDRIP provides strand-specific, high resolution RNA:DNA hybrid mapping. (A) Schematic of qDRIP experimental process. (B) Representative genome browser view of qDRIP-seq signal. From top to bottom: two qDRIP-seq biological replicates, input pooled from replicates, and RNase H digested pool prior to IP. All tracks normalized by reads per million mapped. Negative strand singal in red, positive in blue. (C) Read counts from template strand (TS) and non-template strand (NTS) of hybrids, as well as combined from ssDNA and dsDNA negative controls. (D) GC (green) and AT (red) skew across coding strand of qDRIP peaks, including 600 bp flanking 5’- and 3’-ends. (E) Fractions of qDRIP peaks overlapping noted genomic features. ( F) qDRIP-seq signal distribution around transcription start sites (TSS).
    Figure Legend Snippet: qDRIP provides strand-specific, high resolution RNA:DNA hybrid mapping. (A) Schematic of qDRIP experimental process. (B) Representative genome browser view of qDRIP-seq signal. From top to bottom: two qDRIP-seq biological replicates, input pooled from replicates, and RNase H digested pool prior to IP. All tracks normalized by reads per million mapped. Negative strand singal in red, positive in blue. (C) Read counts from template strand (TS) and non-template strand (NTS) of hybrids, as well as combined from ssDNA and dsDNA negative controls. (D) GC (green) and AT (red) skew across coding strand of qDRIP peaks, including 600 bp flanking 5’- and 3’-ends. (E) Fractions of qDRIP peaks overlapping noted genomic features. ( F) qDRIP-seq signal distribution around transcription start sites (TSS).

    Techniques Used:

    18) Product Images from "The Cap Snatching of Segmented Negative Sense RNA Viruses as a Tool to Map the Transcription Start Sites of Heterologous Co-infecting Viruses"

    Article Title: The Cap Snatching of Segmented Negative Sense RNA Viruses as a Tool to Map the Transcription Start Sites of Heterologous Co-infecting Viruses

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.02519

    A diagram showing the procedure of identifying the 5′ termini of begomoviral mRNAs using the cap snatching of rice stripe tenuivirus (RSV). In a co-infected host cell, RSV snatches capped RNA leaders from host mRNAs (black) as well as those of the co-infecting begomovirus (red). RSV mRNAs (blue) with heterogeneous 5′ terminal sequences were extracted from the co-infected host plant (step 1); the RNA was treated with alkaline phosphatase to remove the 5′-phosphate groups of some RNA species (step 2) and RppH (NEB) to de-cap mRNAs and leave a monophosphate at their 5′ ends (step 3); a RNA oligo was added to the 5′ monophosphate-bearing mRNAs (step 4) and the oligo-tagged mRNAs were reverse transcribed and PCR amplified (Step 5); The PCR products were used for library construction and high-throughput sequencing. The primer sequences obtained were mapped to the genome of a begomovirus (Step 6).
    Figure Legend Snippet: A diagram showing the procedure of identifying the 5′ termini of begomoviral mRNAs using the cap snatching of rice stripe tenuivirus (RSV). In a co-infected host cell, RSV snatches capped RNA leaders from host mRNAs (black) as well as those of the co-infecting begomovirus (red). RSV mRNAs (blue) with heterogeneous 5′ terminal sequences were extracted from the co-infected host plant (step 1); the RNA was treated with alkaline phosphatase to remove the 5′-phosphate groups of some RNA species (step 2) and RppH (NEB) to de-cap mRNAs and leave a monophosphate at their 5′ ends (step 3); a RNA oligo was added to the 5′ monophosphate-bearing mRNAs (step 4) and the oligo-tagged mRNAs were reverse transcribed and PCR amplified (Step 5); The PCR products were used for library construction and high-throughput sequencing. The primer sequences obtained were mapped to the genome of a begomovirus (Step 6).

    Techniques Used: Infection, Polymerase Chain Reaction, Amplification, Next-Generation Sequencing

    19) Product Images from "Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples"

    Article Title: Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples

    Journal: Microbiome

    doi: 10.1186/s40168-017-0317-z

    Optimized workflow for metagenomic virus sequencing. A workflow for metagenomic virus sequencing for diagnostic use was developed. Sample pre-processing included low-speed centrifugation, 0.45-μm filtration, storage at −80 °C, and DNase and RNase digestion. Random reverse transcription with an 8N primer including an anchor sequence, second strand synthesis, and anchor PCR amplification was performed separately for an RNA and DNA workflow. The two workflows were pooled in equal concentration for library preparation with NexteraXT
    Figure Legend Snippet: Optimized workflow for metagenomic virus sequencing. A workflow for metagenomic virus sequencing for diagnostic use was developed. Sample pre-processing included low-speed centrifugation, 0.45-μm filtration, storage at −80 °C, and DNase and RNase digestion. Random reverse transcription with an 8N primer including an anchor sequence, second strand synthesis, and anchor PCR amplification was performed separately for an RNA and DNA workflow. The two workflows were pooled in equal concentration for library preparation with NexteraXT

    Techniques Used: Sequencing, Diagnostic Assay, Centrifugation, Filtration, Polymerase Chain Reaction, Amplification, Concentration Assay

    Separate workflows for RNA and DNA yielded higher sequencing reads for DNA viruses. Plasma samples were spiked with four different viruses (adenovirus, HHV-4, influenzavirus, poliovirus) and processed and sequenced with the combined and the new separate workflow. In the separate workflow, random amplification products were pooled before NexteraXT library preparation in equal concentrations. The experiment was performed in triplicates. a Distribution of sequencing reads into the different taxonomic categories viral, human, bacterial, and unknown origin. b Number of reads ( upper panels ) and fraction of all quality passing reads ( lower panels ) obtained for each individual virus
    Figure Legend Snippet: Separate workflows for RNA and DNA yielded higher sequencing reads for DNA viruses. Plasma samples were spiked with four different viruses (adenovirus, HHV-4, influenzavirus, poliovirus) and processed and sequenced with the combined and the new separate workflow. In the separate workflow, random amplification products were pooled before NexteraXT library preparation in equal concentrations. The experiment was performed in triplicates. a Distribution of sequencing reads into the different taxonomic categories viral, human, bacterial, and unknown origin. b Number of reads ( upper panels ) and fraction of all quality passing reads ( lower panels ) obtained for each individual virus

    Techniques Used: Sequencing, Amplification

    20) Product Images from "Robust transcriptome-wide discovery of RNA binding protein binding sites with enhanced CLIP (eCLIP)"

    Article Title: Robust transcriptome-wide discovery of RNA binding protein binding sites with enhanced CLIP (eCLIP)

    Journal: Nature methods

    doi: 10.1038/nmeth.3810

    Improved identification of RNA binding protein (RBP) targets by enhanced C ross L inking and I mmuno P recipitation followed by high-throughput sequencing (eCLIP-seq) (a) RBP-RNA interactions are stabilized with UV crosslinking, followed by limited RNase I digestion, immunoprecipitation of RBP-RNA complexes with a specific antibody of interest, and stringent washes. After dephosphorylation of RNA fragments, an “inline barcoded” RNA adapter is ligated to the 3′ end. After protein gel electrophoresis and nitrocellulose membrane transfer, a region 75 kDa (~220 nt of RNA) above the protein size is excised and proteinase K treated to isolate RNA. RNA is further prepared into paired-end high-throughput sequencing libraries, where read 1 begins with the inline barcode and read 2 begins with a random-mer sequence (added during the 3′ DNA adapter ligation) followed by sequence corresponding to the 5′ end of the original RNA fragment (which often marks reverse transcriptase termination at the crosslink site (red X)). (b) Bars indicate the number of reads remaining after processing steps. PCR duplicate reads that map to the same genomic position and have the same random-mer as previously considered reads are discarded, leaving only “Usable reads”. (c) Varying numbers of uniquely mapped reads were randomly sampled from RBFOX2 iCLIP and eCLIP experiments and PCR duplicate removal was performed. Points indicate the mean of 100 downsampling experiments (for all, s.e.m. is less than 0.1% of mean value). (d) RBFOX2 read density in reads per million usable (RPM). Shown are iCLIP, two biological replicates for eCLIP with paired size-matched input (SMInput) and IgG-only controls. CLIPper-identified clusters indicated as boxes below, with dark colored boxes indicating binding sites enriched above SMInput.
    Figure Legend Snippet: Improved identification of RNA binding protein (RBP) targets by enhanced C ross L inking and I mmuno P recipitation followed by high-throughput sequencing (eCLIP-seq) (a) RBP-RNA interactions are stabilized with UV crosslinking, followed by limited RNase I digestion, immunoprecipitation of RBP-RNA complexes with a specific antibody of interest, and stringent washes. After dephosphorylation of RNA fragments, an “inline barcoded” RNA adapter is ligated to the 3′ end. After protein gel electrophoresis and nitrocellulose membrane transfer, a region 75 kDa (~220 nt of RNA) above the protein size is excised and proteinase K treated to isolate RNA. RNA is further prepared into paired-end high-throughput sequencing libraries, where read 1 begins with the inline barcode and read 2 begins with a random-mer sequence (added during the 3′ DNA adapter ligation) followed by sequence corresponding to the 5′ end of the original RNA fragment (which often marks reverse transcriptase termination at the crosslink site (red X)). (b) Bars indicate the number of reads remaining after processing steps. PCR duplicate reads that map to the same genomic position and have the same random-mer as previously considered reads are discarded, leaving only “Usable reads”. (c) Varying numbers of uniquely mapped reads were randomly sampled from RBFOX2 iCLIP and eCLIP experiments and PCR duplicate removal was performed. Points indicate the mean of 100 downsampling experiments (for all, s.e.m. is less than 0.1% of mean value). (d) RBFOX2 read density in reads per million usable (RPM). Shown are iCLIP, two biological replicates for eCLIP with paired size-matched input (SMInput) and IgG-only controls. CLIPper-identified clusters indicated as boxes below, with dark colored boxes indicating binding sites enriched above SMInput.

    Techniques Used: RNA Binding Assay, Next-Generation Sequencing, Immunoprecipitation, De-Phosphorylation Assay, Nucleic Acid Electrophoresis, Sequencing, Ligation, Polymerase Chain Reaction, Binding Assay

    21) Product Images from "Inhibition of mitochondrial respiration under hypoxia and increased antioxidant activity after reoxygenation of Tribolium castaneum"

    Article Title: Inhibition of mitochondrial respiration under hypoxia and increased antioxidant activity after reoxygenation of Tribolium castaneum

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0199056

    qRT-PCR analysis of selected transcripts to confirm expression profiles identified by RNA-seq. Tc ELOVL, elongation of very long chain fatty acids protein 7; Tc MRP, ATP-binding cassette subfamily C (CFTR/MRP) member 4; Tc HSP70, heat shock 70kDa protein; Tc MAPK, MAP kinase; Tc DUSP, Dual specificity MAP kinase phosphatase; Tc SD, superoxide dismutase, Cu-Zn family; Tc AG, alpha-glucosidase; Tc DACHS, DACHS, Hippo signling pathway; Tc FJBP Four-jointed box protein 1 (FJBP). Value represents mean ± SE of three independent PCR amplifications and quantifications.
    Figure Legend Snippet: qRT-PCR analysis of selected transcripts to confirm expression profiles identified by RNA-seq. Tc ELOVL, elongation of very long chain fatty acids protein 7; Tc MRP, ATP-binding cassette subfamily C (CFTR/MRP) member 4; Tc HSP70, heat shock 70kDa protein; Tc MAPK, MAP kinase; Tc DUSP, Dual specificity MAP kinase phosphatase; Tc SD, superoxide dismutase, Cu-Zn family; Tc AG, alpha-glucosidase; Tc DACHS, DACHS, Hippo signling pathway; Tc FJBP Four-jointed box protein 1 (FJBP). Value represents mean ± SE of three independent PCR amplifications and quantifications.

    Techniques Used: Quantitative RT-PCR, Expressing, RNA Sequencing Assay, Binding Assay, Polymerase Chain Reaction

    Gene expression pattern of glycolytic (A) and Krebs (B) cycle enzymes of Tribolium castaneum larvae in response to hypoxia. Total RNA was isolated from the larvae after 12 hours’ hypoxia treatment. qRT-PCR was used to illustrate gene expression. Tc HK, hexokinase; Tc PGI, phosphoglucose isomerase; Tc PFKFB, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; Tc FBP, fructose 1,6-bisphosphate aldolase; Tc TPI, triosephosphate isomerase; Tc GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Tc PGK, phosphoglycerate kinase; Tc PGM, phosphoglycerate mutase; Tc ENO, Enolase; Tc PK, Pyruvate kinase; Tc LDH, L-lactate dehydrogenase; Tc PDC, pyruvate dehydrogenase; Tc ACO, aconitate hydratase, mitochondria; Tc IDH, isocitrate dehydrogenase; Tc SCSb, succinyl-CoA synthetase beta chain; Tc SDH, succinate dehydrogenase; Tc FH, fumarate hydratase; Tc MDH, malate dehydrogenase. Red color represents upregulate, green color represents downregulate and black color represents no change.
    Figure Legend Snippet: Gene expression pattern of glycolytic (A) and Krebs (B) cycle enzymes of Tribolium castaneum larvae in response to hypoxia. Total RNA was isolated from the larvae after 12 hours’ hypoxia treatment. qRT-PCR was used to illustrate gene expression. Tc HK, hexokinase; Tc PGI, phosphoglucose isomerase; Tc PFKFB, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; Tc FBP, fructose 1,6-bisphosphate aldolase; Tc TPI, triosephosphate isomerase; Tc GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Tc PGK, phosphoglycerate kinase; Tc PGM, phosphoglycerate mutase; Tc ENO, Enolase; Tc PK, Pyruvate kinase; Tc LDH, L-lactate dehydrogenase; Tc PDC, pyruvate dehydrogenase; Tc ACO, aconitate hydratase, mitochondria; Tc IDH, isocitrate dehydrogenase; Tc SCSb, succinyl-CoA synthetase beta chain; Tc SDH, succinate dehydrogenase; Tc FH, fumarate hydratase; Tc MDH, malate dehydrogenase. Red color represents upregulate, green color represents downregulate and black color represents no change.

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR

    22) Product Images from "Recurrent rearrangements of FOS and FOSB define osteoblastoma"

    Article Title: Recurrent rearrangements of FOS and FOSB define osteoblastoma

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04530-z

    FOS fusions in osteoblastoma. a Clustered break points in FOS . b Central Circos plot showing the clustering of break points in FOS -mutant samples. All structural variants involving chromosome 14 are shown, demonstrating the paucity of genomic rearrangements. Surrounding panels demonstrate normalised RNA-Seq read counts for each fusion partner. Horizontal line segments reflect mean sequencing counts. The arrow above shows the direction of transcription of the fusion. c Retroviral v-fos . d Schematic of FOS break points in benign bone and vascular tumours generating similarity with the murine retroviral transforming v-fos . Subscript numbers from left to right report the length of the transcript to the stop codon and the predicted cleavage and poly-adenylation site, respectively. e FOS immunohistochemistry demonstrating strong nuclear immunoreactivity in FOS -mutant osteoblastoma, PD13482 (centre with zoom inset), Haematoxylin and eosin (H E) (left), and a clear breakapart of FISH probes surrounding the FOS locus (right)
    Figure Legend Snippet: FOS fusions in osteoblastoma. a Clustered break points in FOS . b Central Circos plot showing the clustering of break points in FOS -mutant samples. All structural variants involving chromosome 14 are shown, demonstrating the paucity of genomic rearrangements. Surrounding panels demonstrate normalised RNA-Seq read counts for each fusion partner. Horizontal line segments reflect mean sequencing counts. The arrow above shows the direction of transcription of the fusion. c Retroviral v-fos . d Schematic of FOS break points in benign bone and vascular tumours generating similarity with the murine retroviral transforming v-fos . Subscript numbers from left to right report the length of the transcript to the stop codon and the predicted cleavage and poly-adenylation site, respectively. e FOS immunohistochemistry demonstrating strong nuclear immunoreactivity in FOS -mutant osteoblastoma, PD13482 (centre with zoom inset), Haematoxylin and eosin (H E) (left), and a clear breakapart of FISH probes surrounding the FOS locus (right)

    Techniques Used: Mutagenesis, RNA Sequencing Assay, Sequencing, Immunohistochemistry, Fluorescence In Situ Hybridization

    23) Product Images from "Murine Gammaherpesvirus 68 Encodes a Functional Regulator of Complement Activation"

    Article Title: Murine Gammaherpesvirus 68 Encodes a Functional Regulator of Complement Activation

    Journal: Journal of Virology

    doi:

    ). The 5′ and 3′ RACE products are shown as arrows. (B, C, and D) Northern blots were probed with gene M4 (B)-, gene 4 (C)-, and gene 6 (D)-specific probes. Total RNA was harvested at 7 and 12 h postinfection from NIH 3T12 cells either mock (M) or γHV68 (V) infected at an MOI of 5 in the presence or absence of either a viral DNA synthesis inhibitor (PAA) or the protein synthesis inhibitors cycloheximide and anisomycin (C/A). Molecular size markers (in kilobase pairs) are shown to the left and right of Northern blot panels. ∗, transcripts referred to in the text. A probe to rat cyclophilin was used as a loading control. The data are representative of two independent experiments.
    Figure Legend Snippet: ). The 5′ and 3′ RACE products are shown as arrows. (B, C, and D) Northern blots were probed with gene M4 (B)-, gene 4 (C)-, and gene 6 (D)-specific probes. Total RNA was harvested at 7 and 12 h postinfection from NIH 3T12 cells either mock (M) or γHV68 (V) infected at an MOI of 5 in the presence or absence of either a viral DNA synthesis inhibitor (PAA) or the protein synthesis inhibitors cycloheximide and anisomycin (C/A). Molecular size markers (in kilobase pairs) are shown to the left and right of Northern blot panels. ∗, transcripts referred to in the text. A probe to rat cyclophilin was used as a loading control. The data are representative of two independent experiments.

    Techniques Used: Northern Blot, Infection, DNA Synthesis

    Transcripts in the region surrounding gene 4. Calculated mRNA sizes (in kilobase pairs) were based on the actual sequence between potential TATA box sequences and poly(A) recognition sequences (AATAAA) in the γHV68 genome as analyzed with the Vector NTI version 4.0 Deluxe program (Informax Inc.). RNA molecular size markers, in logarithmic scale, were plotted as a function of distance (in centimeters) from the Northern blots, and measured mRNA sizes were extrapolated from those graphs. For gene 4 and gene 6 transcript sizes, the sizes from the independently probed blots were averaged. The data are from a single experiment.
    Figure Legend Snippet: Transcripts in the region surrounding gene 4. Calculated mRNA sizes (in kilobase pairs) were based on the actual sequence between potential TATA box sequences and poly(A) recognition sequences (AATAAA) in the γHV68 genome as analyzed with the Vector NTI version 4.0 Deluxe program (Informax Inc.). RNA molecular size markers, in logarithmic scale, were plotted as a function of distance (in centimeters) from the Northern blots, and measured mRNA sizes were extrapolated from those graphs. For gene 4 and gene 6 transcript sizes, the sizes from the independently probed blots were averaged. The data are from a single experiment.

    Techniques Used: Sequencing, Plasmid Preparation, Northern Blot

    Identification of the γHV68 gene 4 transcriptional start site by S1 nuclease analysis. (A) Schematic representation of the genomic region surrounding the first ATG in the gene 4 ORF (shown as the black box with white letters). Arrows below the bases denote transcriptional start sites as deduced through S1 nuclease protection assay and 5′ RACE analysis. (B) The S1 oligonucleotide probe was incubated with total RNA isolated from mock- and γHV68-infected NIH 3T12 cells and various concentrations of S1 nuclease enzyme (100 to 500 U/ml). The G+A ladder for the probe is shown at the right. Arrowheads denote the major protected fragments (also indicated in panel A). The data are representative of three independent experiments.
    Figure Legend Snippet: Identification of the γHV68 gene 4 transcriptional start site by S1 nuclease analysis. (A) Schematic representation of the genomic region surrounding the first ATG in the gene 4 ORF (shown as the black box with white letters). Arrows below the bases denote transcriptional start sites as deduced through S1 nuclease protection assay and 5′ RACE analysis. (B) The S1 oligonucleotide probe was incubated with total RNA isolated from mock- and γHV68-infected NIH 3T12 cells and various concentrations of S1 nuclease enzyme (100 to 500 U/ml). The G+A ladder for the probe is shown at the right. Arrowheads denote the major protected fragments (also indicated in panel A). The data are representative of three independent experiments.

    Techniques Used: Incubation, Isolation, Infection

    24) Product Images from "Imprinting of the human L3MBTL gene, a polycomb family member located in a region of chromosome 20 deleted in human myeloid malignancies"

    Article Title: Imprinting of the human L3MBTL gene, a polycomb family member located in a region of chromosome 20 deleted in human myeloid malignancies

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0308195101

    Methylation of CpG islands 3 and 4 is associated with transcriptional silencing. ( A ) Diagram of 5′ region of L3MBTL locus showing the Msc I site, PCR primers (P7, P8) and PCR products derived from either RNA or genomic DNA. * , A/G SNP in exon 2. ( B ) PCR amplification of genomic DNA or RNA from individual 1 was followed by Msc I digestion. Similar results were obtained in two independent experiments. Lane 1, undigested PCR product of granulocyte genomic DNA; lanes 2 and 3, Msc I digested PCR products of genomic DNA from granulocytes and T cells, respectively; lane 4, undigested RT-PCR product from T cells; lane 5, Msc I digested RT-PCR product from T cells.
    Figure Legend Snippet: Methylation of CpG islands 3 and 4 is associated with transcriptional silencing. ( A ) Diagram of 5′ region of L3MBTL locus showing the Msc I site, PCR primers (P7, P8) and PCR products derived from either RNA or genomic DNA. * , A/G SNP in exon 2. ( B ) PCR amplification of genomic DNA or RNA from individual 1 was followed by Msc I digestion. Similar results were obtained in two independent experiments. Lane 1, undigested PCR product of granulocyte genomic DNA; lanes 2 and 3, Msc I digested PCR products of genomic DNA from granulocytes and T cells, respectively; lane 4, undigested RT-PCR product from T cells; lane 5, Msc I digested RT-PCR product from T cells.

    Techniques Used: Methylation, Polymerase Chain Reaction, Derivative Assay, Amplification, Reverse Transcription Polymerase Chain Reaction

    25) Product Images from "Primary and Secondary Sequence Structure Requirements for Recognition and Discrimination of Target RNAs by Pseudomonas aeruginosa RsmA and RsmF"

    Article Title: Primary and Secondary Sequence Structure Requirements for Recognition and Discrimination of Target RNAs by Pseudomonas aeruginosa RsmA and RsmF

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00343-16

    (A) Diagram of the SELEX strategy showing the three primers used to generate the 81-bp dsDNA library by PCR. The first strand primer incorporated a promoter for T7 RNA polymerase. In vitro transcription yielded a 62-nt RNA library consisting of constant
    Figure Legend Snippet: (A) Diagram of the SELEX strategy showing the three primers used to generate the 81-bp dsDNA library by PCR. The first strand primer incorporated a promoter for T7 RNA polymerase. In vitro transcription yielded a 62-nt RNA library consisting of constant

    Techniques Used: Polymerase Chain Reaction, In Vitro

    26) Product Images from "DDB1 Stimulates Viral Transcription of Hepatitis B Virus via HBx-Independent Mechanisms"

    Article Title: DDB1 Stimulates Viral Transcription of Hepatitis B Virus via HBx-Independent Mechanisms

    Journal: Journal of Virology

    doi: 10.1128/JVI.00977-16

    DDB1 does not affect HBV RNA stability. (A) Northern blot analysis following actinomycin D (ActD) treatment. HepG2 cells were transduced with a lentivirus expressing the indicated shRNAs. Cells were then transfected with the 1.3-mer WT replicon construct.
    Figure Legend Snippet: DDB1 does not affect HBV RNA stability. (A) Northern blot analysis following actinomycin D (ActD) treatment. HepG2 cells were transduced with a lentivirus expressing the indicated shRNAs. Cells were then transfected with the 1.3-mer WT replicon construct.

    Techniques Used: Northern Blot, Transduction, Expressing, Transfection, Construct

    27) Product Images from "Glycogen synthase kinase-3 (GSK-3) activity regulates mRNA methylation in mouse embryonic stem cells"

    Article Title: Glycogen synthase kinase-3 (GSK-3) activity regulates mRNA methylation in mouse embryonic stem cells

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA117.001298

    GSK-3 activity regulates FTO protein levels. A , Western blot analysis of FTO protein in WT and Gsk-3 DKO ESCs grown under standard conditions in the presence of LIF. Blots were then stripped and reprobed with α-tubulin antibody to ensure equal loading of protein lysates. B , qPCR data showing the expression of pluripotency-related genes when grown in the absence of LIF. WT and Gsk-3 DKO ESCs were grown in the absence of LIF for 14 days before collecting RNA. mRNA levels of pluripotency markers Nanog and Esrrb are shown as relative with respect to Gapdh mRNA levels. Each experiment shown was performed in triplicate. Error bars , S.D. *, statistical significance between the indicated groups ( p
    Figure Legend Snippet: GSK-3 activity regulates FTO protein levels. A , Western blot analysis of FTO protein in WT and Gsk-3 DKO ESCs grown under standard conditions in the presence of LIF. Blots were then stripped and reprobed with α-tubulin antibody to ensure equal loading of protein lysates. B , qPCR data showing the expression of pluripotency-related genes when grown in the absence of LIF. WT and Gsk-3 DKO ESCs were grown in the absence of LIF for 14 days before collecting RNA. mRNA levels of pluripotency markers Nanog and Esrrb are shown as relative with respect to Gapdh mRNA levels. Each experiment shown was performed in triplicate. Error bars , S.D. *, statistical significance between the indicated groups ( p

    Techniques Used: Activity Assay, Western Blot, Real-time Polymerase Chain Reaction, Expressing

    28) Product Images from "The m6A Reader ECT2 Controls Trichome Morphology by Affecting mRNA Stability in Arabidopsis [OPEN]"

    Article Title: The m6A Reader ECT2 Controls Trichome Morphology by Affecting mRNA Stability in Arabidopsis [OPEN]

    Journal: The Plant Cell

    doi: 10.1105/tpc.17.00934

    ECT2 Recognizes URUAY, a Plant-Specific 3′ UTR m 6 A Motif That Can Be Methylated by Arabidopsis Endogenous m 6 A Writer Proteins. (A) TLC results from an in vitro methylation assay using nuclear extracts with site specific labeled substrates. pA indicates [γ- 32 P]-labeled adenosine. Oligo 1, 5′-CUCGAUCCUUUUUGUpAGUUUCCGAC-3′; Oligo 2, 5′-UAUGCGUCUACUGUpACGGUUGAAUUU-3′. (B) TLC results from an in vitro methylation assay using nuclear extracts with site-specific labeled oligo RNA substrates. The RNA probes were as follows, and pA indicates [γ- 32 P]-labeled adenosine. UGUpA, 5′-CUCGAUCCUUUUUGUpAGUUUCCGAC-3′; GGpACU, 5′-CUCGAUCCUUUUGGpACUGUUUCCGAC-3′; CUpAUG, 5′-CUCGAUCCUUUUCUpAUGGUUUCCGAC-3′. (C) Quantification of m 6 A/A ratios in (B) , as calculated by densitometry using Image J. (D) EMSA measuring the dissociation constant ( K d , nM) of GST-ECT2 with methylated and unmethylated RNA probes. The 4 nmol RNA probe was labeled with [γ- 32 P], and GST-ECT2 concentration ranged from 10 to 2000 nM. Oligo RNA, 5′-AUGGGCCGUUCAUCUGCUAAAA(GGXCU/UGUXA/CUXUG) GCUUUUGGGGCUU*G*U-3′, X = A/m 6 A. The asterisk indicates that thiol-protected bases were used for the experiment.
    Figure Legend Snippet: ECT2 Recognizes URUAY, a Plant-Specific 3′ UTR m 6 A Motif That Can Be Methylated by Arabidopsis Endogenous m 6 A Writer Proteins. (A) TLC results from an in vitro methylation assay using nuclear extracts with site specific labeled substrates. pA indicates [γ- 32 P]-labeled adenosine. Oligo 1, 5′-CUCGAUCCUUUUUGUpAGUUUCCGAC-3′; Oligo 2, 5′-UAUGCGUCUACUGUpACGGUUGAAUUU-3′. (B) TLC results from an in vitro methylation assay using nuclear extracts with site-specific labeled oligo RNA substrates. The RNA probes were as follows, and pA indicates [γ- 32 P]-labeled adenosine. UGUpA, 5′-CUCGAUCCUUUUUGUpAGUUUCCGAC-3′; GGpACU, 5′-CUCGAUCCUUUUGGpACUGUUUCCGAC-3′; CUpAUG, 5′-CUCGAUCCUUUUCUpAUGGUUUCCGAC-3′. (C) Quantification of m 6 A/A ratios in (B) , as calculated by densitometry using Image J. (D) EMSA measuring the dissociation constant ( K d , nM) of GST-ECT2 with methylated and unmethylated RNA probes. The 4 nmol RNA probe was labeled with [γ- 32 P], and GST-ECT2 concentration ranged from 10 to 2000 nM. Oligo RNA, 5′-AUGGGCCGUUCAUCUGCUAAAA(GGXCU/UGUXA/CUXUG) GCUUUUGGGGCUU*G*U-3′, X = A/m 6 A. The asterisk indicates that thiol-protected bases were used for the experiment.

    Techniques Used: Methylation, Thin Layer Chromatography, In Vitro, Labeling, Concentration Assay

    29) Product Images from "CRISPR RNAs trigger innate immune responses in human cells"

    Article Title: CRISPR RNAs trigger innate immune responses in human cells

    Journal: Genome Research

    doi: 10.1101/gr.231936.117

    RNA-sensing immune responses activated by the AsCpf1-associated crRNA. ( A ) Schematics of DNMT1 -targeting crRNAs, which vary according to their preparation methods. crRNA sequences complementary to the DNMT1 -target site are shown in bold. ( B – D ) Relative IFNB1 ( B ), DDX58 ( C ), and OAS2 ( D ) mRNA levels at 24 h after transfection. Error bars, SEM; n = 3. ( E – G ) Relative IFNB1 ( E ), DDX58 ( F ), and OAS2 ( G ) mRNA levels in WT and DDX58 KO HeLa cell lines at 24 h post-transfection. Error bars, SEM; n = 3. ( H ) Indel frequencies induced by the DNMT1 -targeting AsCpf1 RNP were measured using next-generation sequencing (NGS). Statistical significances were calculated by t -test. (n.s.) Not significant, (Syn) chemically synthesized crRNA, (IVT) in vitro–transcribed crRNA, (± CIP) in vitro–transcribed crRNA with or without CIP treatment.
    Figure Legend Snippet: RNA-sensing immune responses activated by the AsCpf1-associated crRNA. ( A ) Schematics of DNMT1 -targeting crRNAs, which vary according to their preparation methods. crRNA sequences complementary to the DNMT1 -target site are shown in bold. ( B – D ) Relative IFNB1 ( B ), DDX58 ( C ), and OAS2 ( D ) mRNA levels at 24 h after transfection. Error bars, SEM; n = 3. ( E – G ) Relative IFNB1 ( E ), DDX58 ( F ), and OAS2 ( G ) mRNA levels in WT and DDX58 KO HeLa cell lines at 24 h post-transfection. Error bars, SEM; n = 3. ( H ) Indel frequencies induced by the DNMT1 -targeting AsCpf1 RNP were measured using next-generation sequencing (NGS). Statistical significances were calculated by t -test. (n.s.) Not significant, (Syn) chemically synthesized crRNA, (IVT) in vitro–transcribed crRNA, (± CIP) in vitro–transcribed crRNA with or without CIP treatment.

    Techniques Used: Transfection, Next-Generation Sequencing, Synthesized, In Vitro

    30) Product Images from "RIG-I like receptor sensing of host RNAs facilitates the cell-intrinsic immune response to KSHV infection"

    Article Title: RIG-I like receptor sensing of host RNAs facilitates the cell-intrinsic immune response to KSHV infection

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07314-7

    Accumulation of immunostimulatory 5′-ppp-vtRNAs during lytic reactivation. a Predicted secondary structure of vtRNAs generated by RNAfold. b SYBR-Gold staining of in vitro transcribed vtRNAs with or without CIP treatment. c HCT116 ISG54-luciferase reporter cells were transfected with 100 ng in vitro transcribed vtRNAs with or without CIP treatment. Cells were harvested 24 h posttransfection and subjected to luciferase assay. Mock indicated cells without RNA transfection and was set as 1. d BC-3 cells were reactivated for 3 days and expression of DUSP11 was quantified by RT-qPCR. L latency, D1–D3 lytic reactivation for 1 day to 3 days. The DUSP11 expression was normalized to the level of 18S rRNA and L was set as 1. e Cell lysates were prepared from BC-3 cells described in ( d ) and DUSP11 protein levels were monitored by Western blot. GAPDH was run as a loading control. f Latent and lytic BC-3 cells were subjected to RNAP II ChIP-qPCR analysis. Signals were normalized to input. g ). h HCT116 ISG54-luciferase reporter cells were transfected with vtRNA or U1 RNA isolated by antisense oligonucleotide affinity selection from either latent or lytic BC-3 cells. Cells were harvested 12 h posttransfection and subjected to luciferase assay. Mock indicated cells without RNA transfection and was set as 1. Error bars in all panels represent mean ± SD from three independent experiments. p Values were determined by the Student’s t test, * p
    Figure Legend Snippet: Accumulation of immunostimulatory 5′-ppp-vtRNAs during lytic reactivation. a Predicted secondary structure of vtRNAs generated by RNAfold. b SYBR-Gold staining of in vitro transcribed vtRNAs with or without CIP treatment. c HCT116 ISG54-luciferase reporter cells were transfected with 100 ng in vitro transcribed vtRNAs with or without CIP treatment. Cells were harvested 24 h posttransfection and subjected to luciferase assay. Mock indicated cells without RNA transfection and was set as 1. d BC-3 cells were reactivated for 3 days and expression of DUSP11 was quantified by RT-qPCR. L latency, D1–D3 lytic reactivation for 1 day to 3 days. The DUSP11 expression was normalized to the level of 18S rRNA and L was set as 1. e Cell lysates were prepared from BC-3 cells described in ( d ) and DUSP11 protein levels were monitored by Western blot. GAPDH was run as a loading control. f Latent and lytic BC-3 cells were subjected to RNAP II ChIP-qPCR analysis. Signals were normalized to input. g ). h HCT116 ISG54-luciferase reporter cells were transfected with vtRNA or U1 RNA isolated by antisense oligonucleotide affinity selection from either latent or lytic BC-3 cells. Cells were harvested 12 h posttransfection and subjected to luciferase assay. Mock indicated cells without RNA transfection and was set as 1. Error bars in all panels represent mean ± SD from three independent experiments. p Values were determined by the Student’s t test, * p

    Techniques Used: Generated, Staining, In Vitro, Luciferase, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Isolation, Selection

    5′-triphosphate containing vtRNAs block KSHV lytic reactivation. a iSLK.219 cells were mock transfected, or transfected with 100 ng in vitro transcribed vtRNAs with or without CIP treatment, or a RIG-I ligand RNA (3pRNA) and reactivated by adding Dox 4 h posttransfection. GFP and RFP images were captured 48 h postreactivation. Bar indicates 300 μm. b Quantification of RFP positive cells in ( a ). c Expression of the indicated viral genes was determined in latent and 48 h post-Dox treatment cells by RT-qPCR. d Expression of the indicated genes was quantified in latent and 24 h post-Dox treatment cells by RT-qPCR. c , d The gene expression was normalized to the level of 18S rRNA and Mock in latent cells was set as 1. Error bars in all panels represent mean ± SD from three independent experiments. p Values were determined by the Student’s t test, * p
    Figure Legend Snippet: 5′-triphosphate containing vtRNAs block KSHV lytic reactivation. a iSLK.219 cells were mock transfected, or transfected with 100 ng in vitro transcribed vtRNAs with or without CIP treatment, or a RIG-I ligand RNA (3pRNA) and reactivated by adding Dox 4 h posttransfection. GFP and RFP images were captured 48 h postreactivation. Bar indicates 300 μm. b Quantification of RFP positive cells in ( a ). c Expression of the indicated viral genes was determined in latent and 48 h post-Dox treatment cells by RT-qPCR. d Expression of the indicated genes was quantified in latent and 24 h post-Dox treatment cells by RT-qPCR. c , d The gene expression was normalized to the level of 18S rRNA and Mock in latent cells was set as 1. Error bars in all panels represent mean ± SD from three independent experiments. p Values were determined by the Student’s t test, * p

    Techniques Used: Blocking Assay, Transfection, In Vitro, Expressing, Quantitative RT-PCR

    RIG-I and MDA5 bind host RNAs during lytic reactivation in PEL. a HCT116 dual cells were transfected with RNA purified from latent and lytic BC-3 cells either by IgG or J2 antibody. Cells were harvested 24 h posttransfection and subjected to luciferase assay. Mock indicates no antibody. b Pie chart representation of gene biotypes identified by F-RIG-I and F-MDA5 fRIP-seq. c PCA analysis. d , e The gene ontology overrepresentation analysis of enriched RNAs from F-MDA5 ( d ) and F-RIG-I ( e ) fRIP-seq. f Distribution of MDA5 fRIP-seq reads on the NOP14 locus. g fRIP-qPCR analysis of NOP14 and GINS. h Distribution RIG-I fRIP-seq reads at the vtRNA loci. i fRIP-qPCR analysis of vtRNAs. Error bars in all panels represent mean ± SD from three independent experiments. p Values were determined by the Student’s t test, * p
    Figure Legend Snippet: RIG-I and MDA5 bind host RNAs during lytic reactivation in PEL. a HCT116 dual cells were transfected with RNA purified from latent and lytic BC-3 cells either by IgG or J2 antibody. Cells were harvested 24 h posttransfection and subjected to luciferase assay. Mock indicates no antibody. b Pie chart representation of gene biotypes identified by F-RIG-I and F-MDA5 fRIP-seq. c PCA analysis. d , e The gene ontology overrepresentation analysis of enriched RNAs from F-MDA5 ( d ) and F-RIG-I ( e ) fRIP-seq. f Distribution of MDA5 fRIP-seq reads on the NOP14 locus. g fRIP-qPCR analysis of NOP14 and GINS. h Distribution RIG-I fRIP-seq reads at the vtRNA loci. i fRIP-qPCR analysis of vtRNAs. Error bars in all panels represent mean ± SD from three independent experiments. p Values were determined by the Student’s t test, * p

    Techniques Used: Transfection, Purification, Luciferase, Real-time Polymerase Chain Reaction

    31) Product Images from "Encapsidated Host Factors in Alphavirus Particles Influence Midgut Infection of Aedes aegypti"

    Article Title: Encapsidated Host Factors in Alphavirus Particles Influence Midgut Infection of Aedes aegypti

    Journal: Viruses

    doi: 10.3390/v10050263

    Midgut immune response. Infected midguts were dissected out at 1 DPF and homogenized. RNA was extracted with TRIzol and cDNA was synthesized to quantify RNA levels of ( A ) nsP1 and indicator genes for Toll ( B ), IMD ( C ), JAK-STAT ( D ), apoptosis ( E ), and RNAi ( F ) pathways. P values were calculated by Mann–Whitney test. All error bars represent standard error of mean (SEM). The dashed line indicates gene expression levels in mosquitoes following a noninfectious blood meal.
    Figure Legend Snippet: Midgut immune response. Infected midguts were dissected out at 1 DPF and homogenized. RNA was extracted with TRIzol and cDNA was synthesized to quantify RNA levels of ( A ) nsP1 and indicator genes for Toll ( B ), IMD ( C ), JAK-STAT ( D ), apoptosis ( E ), and RNAi ( F ) pathways. P values were calculated by Mann–Whitney test. All error bars represent standard error of mean (SEM). The dashed line indicates gene expression levels in mosquitoes following a noninfectious blood meal.

    Techniques Used: Infection, Synthesized, Radial Immuno Diffusion, MANN-WHITNEY, Expressing

    32) Product Images from "Identification of an Important Orphan Histidine Kinase for the Initiation of Sporulation and Enterotoxin Production by Clostridium perfringens Type F Strain SM101"

    Article Title: Identification of an Important Orphan Histidine Kinase for the Initiation of Sporulation and Enterotoxin Production by Clostridium perfringens Type F Strain SM101

    Journal: mBio

    doi: 10.1128/mBio.02674-18

    Characterization of the SM101-CPR1055KO null mutant and analysis of sporulation and CPE production. (A) PCR confirming insertional mutagenesis of th e cpr1055 gene in SM101-CPR1055. Shown is the cpr1055 PCR product amplified using DNA from wild-type SM101 (left lane) or the SM101-CPR1055KO mutant (right lane). Note that DNA from the null mutant strain supported amplification of a larger product due to the insertion of an intron into its cpr1055 gene. (B) Southern blot hybridization with an intron-specific probe with DNA from SM101 or SM101-CPR1055KO. The blot shows results of intron-specific Southern blot hybridization with DNA from wild-type SM101 (left lane) or the cpr1055 null mutant (middle lane). DNA from each strain was digested overnight with EcoRI at 37°C and then electrophoresed on a 1% agarose gel. The size of the hybridizing band in the right lane is shown to the left. Using DNA from wild-type SM101, no intron-specific band was detected. However, a single intron-specific band was detected for the SM101-CPR1055KO mutant. (C) RT-PCR analysis for cpr1055 (top panel) or polC (middle panel) transcription in wild-type SM101 or the SM101-CPR1055KO mutant. SM101 DNA was used as a positive control (gDNA). PCRs lacking template DNA acted as a negative control. To show that the RNA preparations from both strains were free from DNA contamination, the samples were also subjected to PCR without reverse transcription (bottom panel). (D) Growth curves for wild-type SM101 versus the SM101-CPR1055KO mutant cultured at 37°C in MDS medium for up to 8 h. Aliquots of each culture were measured every 2 h for their OD 600 . (E) Comparison of results of sporulation by WT SM101 versus SM101-CPR1055KO. Both strains were grown overnight at 37°C in MDS and then subjected to heat shock treatment and plated on BHI agar. After overnight incubation in an anaerobic jar, the resultant colonies were counted and the counts were converted to numbers of spores per milliliter. (F) Comparison of levels of CPE production by SM101 versus the SM101-CPR1055KO mutant. Supernatants of WT SM101 or SM101-CPR1055KO were grown overnight at 37°C in MDS and then assessed by Western blotting for CPE. The results showed that CPE production remained strong after inactivation of the cpr1055 gene. All experiments were repeated three times, and mean representative values are shown. The markers used in panels A and C were Thermo Fisher 1-kb DNA ladders.
    Figure Legend Snippet: Characterization of the SM101-CPR1055KO null mutant and analysis of sporulation and CPE production. (A) PCR confirming insertional mutagenesis of th e cpr1055 gene in SM101-CPR1055. Shown is the cpr1055 PCR product amplified using DNA from wild-type SM101 (left lane) or the SM101-CPR1055KO mutant (right lane). Note that DNA from the null mutant strain supported amplification of a larger product due to the insertion of an intron into its cpr1055 gene. (B) Southern blot hybridization with an intron-specific probe with DNA from SM101 or SM101-CPR1055KO. The blot shows results of intron-specific Southern blot hybridization with DNA from wild-type SM101 (left lane) or the cpr1055 null mutant (middle lane). DNA from each strain was digested overnight with EcoRI at 37°C and then electrophoresed on a 1% agarose gel. The size of the hybridizing band in the right lane is shown to the left. Using DNA from wild-type SM101, no intron-specific band was detected. However, a single intron-specific band was detected for the SM101-CPR1055KO mutant. (C) RT-PCR analysis for cpr1055 (top panel) or polC (middle panel) transcription in wild-type SM101 or the SM101-CPR1055KO mutant. SM101 DNA was used as a positive control (gDNA). PCRs lacking template DNA acted as a negative control. To show that the RNA preparations from both strains were free from DNA contamination, the samples were also subjected to PCR without reverse transcription (bottom panel). (D) Growth curves for wild-type SM101 versus the SM101-CPR1055KO mutant cultured at 37°C in MDS medium for up to 8 h. Aliquots of each culture were measured every 2 h for their OD 600 . (E) Comparison of results of sporulation by WT SM101 versus SM101-CPR1055KO. Both strains were grown overnight at 37°C in MDS and then subjected to heat shock treatment and plated on BHI agar. After overnight incubation in an anaerobic jar, the resultant colonies were counted and the counts were converted to numbers of spores per milliliter. (F) Comparison of levels of CPE production by SM101 versus the SM101-CPR1055KO mutant. Supernatants of WT SM101 or SM101-CPR1055KO were grown overnight at 37°C in MDS and then assessed by Western blotting for CPE. The results showed that CPE production remained strong after inactivation of the cpr1055 gene. All experiments were repeated three times, and mean representative values are shown. The markers used in panels A and C were Thermo Fisher 1-kb DNA ladders.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Southern Blot, Hybridization, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Cell Culture, Incubation, Western Blot

    Characterization of the SM101-CPR0195KO null mutant and SM101-CPR0195comp complementing strain. (A) PCR confirming insertional mutagenesis of the cpr0195 gene in SM101-0195KO. Shown is the cpr0195 PCR product amplified using DNA from wild-type SM101 (lane 2), the SM101-CPR0195KO mutant (lane 3), or the SM101-CPR0195comp complementing strain (lane 4). Note that, compared to the ∼300-bp product amplified using DNA containing a wild-type cpr0195 gene, DNA from the null mutant strain supported amplification of a larger (∼1,200-bp) product due to the insertion of an intron into its cpr0195 gene. (B) Southern blot hybridization of an intron-specific probe with DNA from SM101 (left), SM101-CPR0195KO (middle), or SM101-CPR0195comp (right). DNA from each strain was digested overnight with EcoRI at 37°C and then electrophoresed on a 1% agarose gel. The size of the hybridizing band in the middle and right lanes is shown to the left. Using DNA from wild-type SM101, no intron-specific band was detected, while a single intron-specific band was detected for the SM101-CPR0195KO mutant and complementing strain. (C) RT-PCR analysis for cpr019 5 (top panel) or polC (middle panel) transcription in wild-type SM101, the SM101-CPR0195KO mutant, or the complementing strain. SM101 DNA was used as a positive control (gDNA [genomic DNA]). PCRs lacking template DNA acted as a negative control. To show that the RNA preparations from the three strains were free from DNA contamination, these samples were also subjected to PCR without reverse transcription (bottom panel). (D) Growth curves for wild-type SM101, the SM101-CPR0195KO mutant, and the SM101-CPR0195comp strain cultured at 37°C in MDS medium for up to 8 h. Aliquots of each culture were measured every 2 h for their OD 600 . All experiments were repeated three times, and mean representative values are shown. The markers used in panels A and C were Thermo Fisher 1-kb DNA ladders.
    Figure Legend Snippet: Characterization of the SM101-CPR0195KO null mutant and SM101-CPR0195comp complementing strain. (A) PCR confirming insertional mutagenesis of the cpr0195 gene in SM101-0195KO. Shown is the cpr0195 PCR product amplified using DNA from wild-type SM101 (lane 2), the SM101-CPR0195KO mutant (lane 3), or the SM101-CPR0195comp complementing strain (lane 4). Note that, compared to the ∼300-bp product amplified using DNA containing a wild-type cpr0195 gene, DNA from the null mutant strain supported amplification of a larger (∼1,200-bp) product due to the insertion of an intron into its cpr0195 gene. (B) Southern blot hybridization of an intron-specific probe with DNA from SM101 (left), SM101-CPR0195KO (middle), or SM101-CPR0195comp (right). DNA from each strain was digested overnight with EcoRI at 37°C and then electrophoresed on a 1% agarose gel. The size of the hybridizing band in the middle and right lanes is shown to the left. Using DNA from wild-type SM101, no intron-specific band was detected, while a single intron-specific band was detected for the SM101-CPR0195KO mutant and complementing strain. (C) RT-PCR analysis for cpr019 5 (top panel) or polC (middle panel) transcription in wild-type SM101, the SM101-CPR0195KO mutant, or the complementing strain. SM101 DNA was used as a positive control (gDNA [genomic DNA]). PCRs lacking template DNA acted as a negative control. To show that the RNA preparations from the three strains were free from DNA contamination, these samples were also subjected to PCR without reverse transcription (bottom panel). (D) Growth curves for wild-type SM101, the SM101-CPR0195KO mutant, and the SM101-CPR0195comp strain cultured at 37°C in MDS medium for up to 8 h. Aliquots of each culture were measured every 2 h for their OD 600 . All experiments were repeated three times, and mean representative values are shown. The markers used in panels A and C were Thermo Fisher 1-kb DNA ladders.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Southern Blot, Hybridization, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Cell Culture

    33) Product Images from "High-resolution structure of Cas13b and biochemical characterization of RNA targeting and cleavage"

    Article Title: High-resolution structure of Cas13b and biochemical characterization of RNA targeting and cleavage

    Journal: Cell reports

    doi: 10.1016/j.celrep.2019.02.094

    Biochemical characterization of RNA targeting by Cas13b. A. Thermal melting curves of PbuCas13b complexed with crRNA and target RNA. To the right of the figure legends are cartoons of the substrate used with red lines indicating mismatches (tandem mismatches are shown on the left panel; larger mismatches are shown on the right panel). Curves shifted to the left indicate a destabilized complex. B. The effect of single nucleotide mismatches between target and crRNA spacer on collateral nuclease activity activation in FLUORESCENT COLLATERAL RNA–CLEAVAGE assays. Two different guide sequences (upper, guide 1; lower, guide 2) are shown. C. . D. Diagram of the active site of PbuCas13b in the crystal structure. Citrate (shown in yellow) is bound in the active site and loop 938–951 is shown in the lower left labeled as ‘Loop’.
    Figure Legend Snippet: Biochemical characterization of RNA targeting by Cas13b. A. Thermal melting curves of PbuCas13b complexed with crRNA and target RNA. To the right of the figure legends are cartoons of the substrate used with red lines indicating mismatches (tandem mismatches are shown on the left panel; larger mismatches are shown on the right panel). Curves shifted to the left indicate a destabilized complex. B. The effect of single nucleotide mismatches between target and crRNA spacer on collateral nuclease activity activation in FLUORESCENT COLLATERAL RNA–CLEAVAGE assays. Two different guide sequences (upper, guide 1; lower, guide 2) are shown. C. . D. Diagram of the active site of PbuCas13b in the crystal structure. Citrate (shown in yellow) is bound in the active site and loop 938–951 is shown in the lower left labeled as ‘Loop’.

    Techniques Used: Activity Assay, Activation Assay, Labeling

    PbuCas13b crRNA recognition and processing. A. Diagram of crRNA substrate co-crystallized with PbuCas13b. Direct repeat nucleotides are colored red and spacer nucleotides in light blue (full spacer is not shown). Watson-Crick base pairing denoted by black lines; non-Watson-Crick base pairing denoted by gray lines. B. The structure of crRNA within the crystallized PbuCas13b complex. The coloring is consistent with panel (A) and individual bases are numbered (−1 to −36 in the crRNA, 1 for spacer). C. Model of the 3′ end of the crRNA showing the catalytic residue K393 of the crRNA processing site and additional PbuCas13b residues that coordinate the crRNA. D. Analysis of Lid domain residues predicted to coordinate and process crRNA within PbuCas13b. Right, schematic shows Cas13b-mediated RNA degradation. The upper panel shows collateral RNase activity in FLUORESCENT COLLATERAL RNA–CLEAVAGE assays with Lid domain mutants (asterisk indicates nearly undetectable levels of fluorescence); lower panel shows processing of crRNA by these mutants. Cartoons of the expected cleavage products are shown to the left of the gel; cleavage bands and expected sizes indicated by red triangles to the right of the gel.
    Figure Legend Snippet: PbuCas13b crRNA recognition and processing. A. Diagram of crRNA substrate co-crystallized with PbuCas13b. Direct repeat nucleotides are colored red and spacer nucleotides in light blue (full spacer is not shown). Watson-Crick base pairing denoted by black lines; non-Watson-Crick base pairing denoted by gray lines. B. The structure of crRNA within the crystallized PbuCas13b complex. The coloring is consistent with panel (A) and individual bases are numbered (−1 to −36 in the crRNA, 1 for spacer). C. Model of the 3′ end of the crRNA showing the catalytic residue K393 of the crRNA processing site and additional PbuCas13b residues that coordinate the crRNA. D. Analysis of Lid domain residues predicted to coordinate and process crRNA within PbuCas13b. Right, schematic shows Cas13b-mediated RNA degradation. The upper panel shows collateral RNase activity in FLUORESCENT COLLATERAL RNA–CLEAVAGE assays with Lid domain mutants (asterisk indicates nearly undetectable levels of fluorescence); lower panel shows processing of crRNA by these mutants. Cartoons of the expected cleavage products are shown to the left of the gel; cleavage bands and expected sizes indicated by red triangles to the right of the gel.

    Techniques Used: Activity Assay, Fluorescence

    34) Product Images from "Capping-RACE: a simple, accurate, and sensitive 5′ RACE method for use in prokaryotes"

    Article Title: Capping-RACE: a simple, accurate, and sensitive 5′ RACE method for use in prokaryotes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky739

    Experimental verification of Capping-RACE with in vitro transcribed RNA. ( A ) Different treatments of in vitro transcribed RNA. ( B ) The cDNA products from the differently treated RNA described in (A). Lane 1, the negative control, performed without adding reverse transcriptase (RT). Lane 2, in vitro transcribed RNA that was not subjected to any treatment. Lane 3, in vitro transcribed RNA subjected to RppH treatment. Lane 4, in vitro transcribed RNA subjected to dual treatment, i.e. RppH treatment prior to VCE treatment. Lane 5, in vitro transcribed RNA subjected to vaccinia capping enzyme (VCE) treatment. The reaction products were analysed on a 12% non-denaturing polyacrylamide gel and detected by a fluorescence image analyser (FUJIFILM, FLA-5100).
    Figure Legend Snippet: Experimental verification of Capping-RACE with in vitro transcribed RNA. ( A ) Different treatments of in vitro transcribed RNA. ( B ) The cDNA products from the differently treated RNA described in (A). Lane 1, the negative control, performed without adding reverse transcriptase (RT). Lane 2, in vitro transcribed RNA that was not subjected to any treatment. Lane 3, in vitro transcribed RNA subjected to RppH treatment. Lane 4, in vitro transcribed RNA subjected to dual treatment, i.e. RppH treatment prior to VCE treatment. Lane 5, in vitro transcribed RNA subjected to vaccinia capping enzyme (VCE) treatment. The reaction products were analysed on a 12% non-denaturing polyacrylamide gel and detected by a fluorescence image analyser (FUJIFILM, FLA-5100).

    Techniques Used: In Vitro, Negative Control, Fluorescence

    35) Product Images from "Capping-RACE: a simple, accurate, and sensitive 5′ RACE method for use in prokaryotes"

    Article Title: Capping-RACE: a simple, accurate, and sensitive 5′ RACE method for use in prokaryotes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky739

    Experimental verification of Capping-RACE with in vitro transcribed RNA. ( A ) Different treatments of in vitro transcribed RNA. ( B ) The cDNA products from the differently treated RNA described in (A). Lane 1, the negative control, performed without adding reverse transcriptase (RT). Lane 2, in vitro transcribed RNA that was not subjected to any treatment. Lane 3, in vitro transcribed RNA subjected to RppH treatment. Lane 4, in vitro transcribed RNA subjected to dual treatment, i.e. RppH treatment prior to VCE treatment. Lane 5, in vitro transcribed RNA subjected to vaccinia capping enzyme (VCE) treatment. The reaction products were analysed on a 12% non-denaturing polyacrylamide gel and detected by a fluorescence image analyser (FUJIFILM, FLA-5100).
    Figure Legend Snippet: Experimental verification of Capping-RACE with in vitro transcribed RNA. ( A ) Different treatments of in vitro transcribed RNA. ( B ) The cDNA products from the differently treated RNA described in (A). Lane 1, the negative control, performed without adding reverse transcriptase (RT). Lane 2, in vitro transcribed RNA that was not subjected to any treatment. Lane 3, in vitro transcribed RNA subjected to RppH treatment. Lane 4, in vitro transcribed RNA subjected to dual treatment, i.e. RppH treatment prior to VCE treatment. Lane 5, in vitro transcribed RNA subjected to vaccinia capping enzyme (VCE) treatment. The reaction products were analysed on a 12% non-denaturing polyacrylamide gel and detected by a fluorescence image analyser (FUJIFILM, FLA-5100).

    Techniques Used: In Vitro, Negative Control, Fluorescence

    36) Product Images from "A Secreted RNA Binding Protein Forms RNA-Stabilizing Granules in the Honeybee Royal Jelly"

    Article Title: A Secreted RNA Binding Protein Forms RNA-Stabilizing Granules in the Honeybee Royal Jelly

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2019.03.010

    Multivalent RNA Binding Stimulates Super-order Assembly of Dynamic MRJP-3 RNPs and Isolation of RJ RNA Partners of MRJP-3 (A) The multivalent interaction of MRJP-3 with RNA is reversible. MRJP-3-bound ssRNA ∗ was introduced to increasing quantities of unlabeled ssRNA. ssRNA ∗ (0.04 μM) and MRJP-3 (31.3 μM) were used in all ssRNA ∗ - and protein-containing treatments. Unlabeled DNA ladder served as labeling control. (B) MRJP-3 RNPs are affected by the protein/ssRNA mole ratio. Images of RNPs formed at various mole ratios of MRJP-3 and Alexa Fluor-488 labeled ssRNA (ssRNA ∗ ). Scale bar represents 10 μm. (C) RNA mediates super-order assembly of MRJP-3 oligomers, resulting in RNP formation in soluble RJ fraction. RJ buffer or 4.28 μM Alexa Fluor-633 labeled MRJP-3 (MRJP-3 ∗ ) was introduced to 50% soluble RJ fraction. ssRNA (0.15 μM) or ssRNA ∗ was used in RNA-containing treatments. Scale bar represents 2 μm. (D) MRJP-3 binds both endogenous ( Apis mellifera ) and exogenous (viral) RNA. Only viruses with a mapped fraction of at least 1% are shown. Points are individual biological replicates. Bars represent the mean across replicates. Horizontal lines indicate tests for significant enrichment of viral RNA over bee RNA in the MRJP-3 bound fraction, but not in RJ ( ∗ p
    Figure Legend Snippet: Multivalent RNA Binding Stimulates Super-order Assembly of Dynamic MRJP-3 RNPs and Isolation of RJ RNA Partners of MRJP-3 (A) The multivalent interaction of MRJP-3 with RNA is reversible. MRJP-3-bound ssRNA ∗ was introduced to increasing quantities of unlabeled ssRNA. ssRNA ∗ (0.04 μM) and MRJP-3 (31.3 μM) were used in all ssRNA ∗ - and protein-containing treatments. Unlabeled DNA ladder served as labeling control. (B) MRJP-3 RNPs are affected by the protein/ssRNA mole ratio. Images of RNPs formed at various mole ratios of MRJP-3 and Alexa Fluor-488 labeled ssRNA (ssRNA ∗ ). Scale bar represents 10 μm. (C) RNA mediates super-order assembly of MRJP-3 oligomers, resulting in RNP formation in soluble RJ fraction. RJ buffer or 4.28 μM Alexa Fluor-633 labeled MRJP-3 (MRJP-3 ∗ ) was introduced to 50% soluble RJ fraction. ssRNA (0.15 μM) or ssRNA ∗ was used in RNA-containing treatments. Scale bar represents 2 μm. (D) MRJP-3 binds both endogenous ( Apis mellifera ) and exogenous (viral) RNA. Only viruses with a mapped fraction of at least 1% are shown. Points are individual biological replicates. Bars represent the mean across replicates. Horizontal lines indicate tests for significant enrichment of viral RNA over bee RNA in the MRJP-3 bound fraction, but not in RJ ( ∗ p

    Techniques Used: RNA Binding Assay, Isolation, Labeling

    MRJP-3 RNP Granules Protect RNA From Degradation and Enhance RNA Bioavailability (A) MRJP-3-bound RNA is protected from RNase-A digestion. Treatments included ssRNA mixed with MRJP-3, ssRNA mixed with MRJP-3 followed by incubation with PK, ssRNA mixed with MRJP-3 and RNase-A, ssRNA mixed with MRJP-3 and RNase-A followed by incubation with PK, ssRNA mixed with MRJP-1, and ssRNA mixed with MRJP-1 and RNase-A. ssRNA (0.3 μM) and MRJP-3 or MRJP-1 (42.8 μM) were used in all ssRNA- and protein-containing treatments. RNase challenge was performed by introducing 5 μg RNase-A followed by 1 h incubation at room temperature. (B) RNase-A presence does not affect MRJP-3 RNPs. Images of RNPs formed with ssRNA ∗ with or without RNase-A. ssRNA ∗ (0.3 μM) and MRJP-3 or MRJP-1 (42.8 μM) were used in all ssRNA ∗ - and protein-containing treatments. RNase challenge was performed by introducing 5 μg RNase-A followed by 1–3 h incubation at room temperature. Scale bar represents 2 μm. (C) dsRNA-MRJP-3 RNPs enhance unc-22 RNAi phenotype in C. elegans . Each treatment contained three biological repeats (n = 150 animals per treatment). (D) MRJP-3 RNPs enhances ingested dsRNA uptake in C. elegans . Animals were soaked in the presence of MRJP-3 RNPs formed with Alexa Fluor-647-labeled dsRNA-Fluc (dsRNA ∗∗ ). Control groups included soaking animals with dsRNA ∗∗ mixed with MRJP-1, dsRNA ∗∗ mixed in RJ buffer, and MRJP-3 alone. dsRNA ∗∗ (2.15 nM) and MRJP-3 or MRJP-1 (42.8 μM) were used in all dsRNA- and protein-containing treatments. (E) A working model describing the role of MRJP-3 in the transmissible RNA pathway in honeybees. Nurse bees secrete jelly-containing RNPs that comprise endogenous and exogenous (e.g., viral, fungi, bacteria, plant) RNAs. Bee larvae ingest environmental MRJP-3 RNPs through jelly consumption. The ingested RNA is taken up to the hemolymph, is systemically spread, and affects gene expression including an antiviral response. .
    Figure Legend Snippet: MRJP-3 RNP Granules Protect RNA From Degradation and Enhance RNA Bioavailability (A) MRJP-3-bound RNA is protected from RNase-A digestion. Treatments included ssRNA mixed with MRJP-3, ssRNA mixed with MRJP-3 followed by incubation with PK, ssRNA mixed with MRJP-3 and RNase-A, ssRNA mixed with MRJP-3 and RNase-A followed by incubation with PK, ssRNA mixed with MRJP-1, and ssRNA mixed with MRJP-1 and RNase-A. ssRNA (0.3 μM) and MRJP-3 or MRJP-1 (42.8 μM) were used in all ssRNA- and protein-containing treatments. RNase challenge was performed by introducing 5 μg RNase-A followed by 1 h incubation at room temperature. (B) RNase-A presence does not affect MRJP-3 RNPs. Images of RNPs formed with ssRNA ∗ with or without RNase-A. ssRNA ∗ (0.3 μM) and MRJP-3 or MRJP-1 (42.8 μM) were used in all ssRNA ∗ - and protein-containing treatments. RNase challenge was performed by introducing 5 μg RNase-A followed by 1–3 h incubation at room temperature. Scale bar represents 2 μm. (C) dsRNA-MRJP-3 RNPs enhance unc-22 RNAi phenotype in C. elegans . Each treatment contained three biological repeats (n = 150 animals per treatment). (D) MRJP-3 RNPs enhances ingested dsRNA uptake in C. elegans . Animals were soaked in the presence of MRJP-3 RNPs formed with Alexa Fluor-647-labeled dsRNA-Fluc (dsRNA ∗∗ ). Control groups included soaking animals with dsRNA ∗∗ mixed with MRJP-1, dsRNA ∗∗ mixed in RJ buffer, and MRJP-3 alone. dsRNA ∗∗ (2.15 nM) and MRJP-3 or MRJP-1 (42.8 μM) were used in all dsRNA- and protein-containing treatments. (E) A working model describing the role of MRJP-3 in the transmissible RNA pathway in honeybees. Nurse bees secrete jelly-containing RNPs that comprise endogenous and exogenous (e.g., viral, fungi, bacteria, plant) RNAs. Bee larvae ingest environmental MRJP-3 RNPs through jelly consumption. The ingested RNA is taken up to the hemolymph, is systemically spread, and affects gene expression including an antiviral response. .

    Techniques Used: Incubation, Labeling, Expressing

    The Honeybee Jelly Harbors RNA-Binding Activity (A) Experimental design for RNA detection in RJ. Hives were fed with a 10% sucrose solution with or without the addition of Alexa Fluor-488-labeled dsRNA (dsRNA ∗ ). (B) Immunohistochemistry-based detection of dsRNA ∗ in RJ samples, which were reacted with Alexa Fluor-488 antibody. Scale bar represents 25 μm. (C) RJ proteins bind dsRNA. dsRNA-binding activity was tested using EMSA. Treatments included dsRNA mixed in RJ buffer, 10% RJ mixed with dsRNA, 10% RJ digested by Proteinase K (PK) and then mixed with dsRNA, 10% RJ mixed with dsRNA and then digested by PK, 10% RJ mixed with dsRNA and PK buffer, 27.3 μM purified BSA mixed with dsRNA, 10% RJ only, and 10% RJ only digested by PK. dsRNA (0.05 μM) was applied in all dsRNA-containing treatments. (D) Precipitation dynamics of dsRNA-protein complexes in RJ. Two percent RJ was mixed with increasing dsRNA concentrations. (E) MRJP-3 and its prion-like TRR. Amino acid sequence in bold: secretion signal peptide. Amino acid sequence highlighted in color: tandem repeats. Alignment of the tandem repeats, QN (in gray) and positively charged amino acids (in red). .
    Figure Legend Snippet: The Honeybee Jelly Harbors RNA-Binding Activity (A) Experimental design for RNA detection in RJ. Hives were fed with a 10% sucrose solution with or without the addition of Alexa Fluor-488-labeled dsRNA (dsRNA ∗ ). (B) Immunohistochemistry-based detection of dsRNA ∗ in RJ samples, which were reacted with Alexa Fluor-488 antibody. Scale bar represents 25 μm. (C) RJ proteins bind dsRNA. dsRNA-binding activity was tested using EMSA. Treatments included dsRNA mixed in RJ buffer, 10% RJ mixed with dsRNA, 10% RJ digested by Proteinase K (PK) and then mixed with dsRNA, 10% RJ mixed with dsRNA and then digested by PK, 10% RJ mixed with dsRNA and PK buffer, 27.3 μM purified BSA mixed with dsRNA, 10% RJ only, and 10% RJ only digested by PK. dsRNA (0.05 μM) was applied in all dsRNA-containing treatments. (D) Precipitation dynamics of dsRNA-protein complexes in RJ. Two percent RJ was mixed with increasing dsRNA concentrations. (E) MRJP-3 and its prion-like TRR. Amino acid sequence in bold: secretion signal peptide. Amino acid sequence highlighted in color: tandem repeats. Alignment of the tandem repeats, QN (in gray) and positively charged amino acids (in red). .

    Techniques Used: RNA Binding Assay, Activity Assay, RNA Detection, Labeling, Immunohistochemistry, Binding Assay, Purification, Sequencing

    MRJP-3 Is a Multivalent RNA-Binding Oligomer (A) Taxonomy tree analysis suggests that the MRJP-3 tandem-repeats region evolved in the genus Apis and is associated with jelly secretion. (B) Purified MRJP-3 binds dsRNA and ssRNA as demonstrated by EMSA. MRJP-3 was incubated with increasing concentrations of dsRNA or ssRNA. Additional controls: dsRNA and ssRNA only, MRJP-1 mixed with 43.1 nM dsRNA or 0.3 μM ssRNA, and MRJP-3 only. Protein (42.8 μM) was applied in all MRJP-3- or MRJP-1-containing treatments. (C) MRJP-3 efficiently binds ssRNA that is 18 nt and longer. Binding activity was tested using ssRNA substrates with different lengths and analyzed by EMSA. ssRNA (19.4 pmol) and proteins (42.8 μM) were applied in all ssRNA- and/or protein-containing treatments. (D) Binding curves of Alexa Fluor-488-labeled 22 nt ssRNA and dsRNA to MRJP-3 in RJ buffer conditions (left and right curves, respectively). Calculated and estimated equilibrium K d values are shown as dashed lines. (E) The TRR of MRJP-3 is predicted to be intrinsically disordered by the PONDR VSL2 and IUPred algorithms. (F) The TRR of MRJP-3 is required for RNP formation. Proteins (13.6 μM) and ssRNA ∗ (0.2 μM) were applied in all RNA- and/or protein-containing treatments. Scale bar represents 1 μm. .
    Figure Legend Snippet: MRJP-3 Is a Multivalent RNA-Binding Oligomer (A) Taxonomy tree analysis suggests that the MRJP-3 tandem-repeats region evolved in the genus Apis and is associated with jelly secretion. (B) Purified MRJP-3 binds dsRNA and ssRNA as demonstrated by EMSA. MRJP-3 was incubated with increasing concentrations of dsRNA or ssRNA. Additional controls: dsRNA and ssRNA only, MRJP-1 mixed with 43.1 nM dsRNA or 0.3 μM ssRNA, and MRJP-3 only. Protein (42.8 μM) was applied in all MRJP-3- or MRJP-1-containing treatments. (C) MRJP-3 efficiently binds ssRNA that is 18 nt and longer. Binding activity was tested using ssRNA substrates with different lengths and analyzed by EMSA. ssRNA (19.4 pmol) and proteins (42.8 μM) were applied in all ssRNA- and/or protein-containing treatments. (D) Binding curves of Alexa Fluor-488-labeled 22 nt ssRNA and dsRNA to MRJP-3 in RJ buffer conditions (left and right curves, respectively). Calculated and estimated equilibrium K d values are shown as dashed lines. (E) The TRR of MRJP-3 is predicted to be intrinsically disordered by the PONDR VSL2 and IUPred algorithms. (F) The TRR of MRJP-3 is required for RNP formation. Proteins (13.6 μM) and ssRNA ∗ (0.2 μM) were applied in all RNA- and/or protein-containing treatments. Scale bar represents 1 μm. .

    Techniques Used: RNA Binding Assay, Purification, Incubation, Binding Assay, Activity Assay, Labeling

    37) Product Images from "Reovirus Nonstructural Protein σNS Acts as an RNA Stability Factor Promoting Viral Genome Replication"

    Article Title: Reovirus Nonstructural Protein σNS Acts as an RNA Stability Factor Promoting Viral Genome Replication

    Journal: Journal of Virology

    doi: 10.1128/JVI.00563-18

    The σNS protein forms filamentous structures in the presence of RNA. Uncapped and nonpolyadenylated s4 RNA was incubated with 50× molar excess of purified recombinant WT or Δ38 σNS on ice for 1 h, followed by plunge freezing. Frozen specimens were imaged at ×40,000 magnification with defocus levels ranging from −2.0 μm to −3.5 μm. Representative images for WT σNS, WT σNS complexed with s4 RNA, an enlargement of WT σNS and s4 RNA filaments, Δ38 σNS, Δ38 σNS incubated with s4 RNA, and s4 RNA alone are shown.
    Figure Legend Snippet: The σNS protein forms filamentous structures in the presence of RNA. Uncapped and nonpolyadenylated s4 RNA was incubated with 50× molar excess of purified recombinant WT or Δ38 σNS on ice for 1 h, followed by plunge freezing. Frozen specimens were imaged at ×40,000 magnification with defocus levels ranging from −2.0 μm to −3.5 μm. Representative images for WT σNS, WT σNS complexed with s4 RNA, an enlargement of WT σNS and s4 RNA filaments, Δ38 σNS, Δ38 σNS incubated with s4 RNA, and s4 RNA alone are shown.

    Techniques Used: Incubation, Purification, Recombinant

    The σNS protein diminishes translation of viral and nonviral RNAs. (A) HEK293T cells were transfected with increasing concentrations of plasmids encoding either WT or Δ38 σNS and a fixed concentration of renilla luciferase-encoding plasmid and incubated for 24 h. The cells were lysed, and luciferase levels were quantified. The results are presented as mean luminescence percentages normalized to luciferase levels in the absence of σNS plasmid for at least three independent experiments. The error bars indicate SD. (B) Firefly luciferase uncapped and polyadenylated RNA was incubated with increasing concentrations of purified recombinant WT or Δ38 σNS or BSA at room temperature for 10 min. Protein-RNA complexes were added to wheat germ extracts and incubated at 25°C for 1 h. Luciferase synthesis was quantified by luciferase assay. The results are presented as mean luminescence percentages normalized to luciferase levels in the absence of protein for at least three independent experiments. The error bars indicate SD. (C) Capped, 2′-O-methylated, and nonpolyadenylated s4 RNA was incubated with increasing concentrations of purified recombinant WT or Δ38 σNS at room temperature for 10 min. Protein-RNA complexes were added to wheat germ extracts and incubated at 25°C for 1 h in the presence of [ 35 S]methionine. Samples were resolved by SDS-PAGE and visualized by phosphorimaging. (D) Pixel intensity analysis of the σ3 protein band for at least three independent experiments. The error bars indicate SD. Values that differ significantly from the no-protein condition values by one-sample t test for each time point are indicated (*, P
    Figure Legend Snippet: The σNS protein diminishes translation of viral and nonviral RNAs. (A) HEK293T cells were transfected with increasing concentrations of plasmids encoding either WT or Δ38 σNS and a fixed concentration of renilla luciferase-encoding plasmid and incubated for 24 h. The cells were lysed, and luciferase levels were quantified. The results are presented as mean luminescence percentages normalized to luciferase levels in the absence of σNS plasmid for at least three independent experiments. The error bars indicate SD. (B) Firefly luciferase uncapped and polyadenylated RNA was incubated with increasing concentrations of purified recombinant WT or Δ38 σNS or BSA at room temperature for 10 min. Protein-RNA complexes were added to wheat germ extracts and incubated at 25°C for 1 h. Luciferase synthesis was quantified by luciferase assay. The results are presented as mean luminescence percentages normalized to luciferase levels in the absence of protein for at least three independent experiments. The error bars indicate SD. (C) Capped, 2′-O-methylated, and nonpolyadenylated s4 RNA was incubated with increasing concentrations of purified recombinant WT or Δ38 σNS at room temperature for 10 min. Protein-RNA complexes were added to wheat germ extracts and incubated at 25°C for 1 h in the presence of [ 35 S]methionine. Samples were resolved by SDS-PAGE and visualized by phosphorimaging. (D) Pixel intensity analysis of the σ3 protein band for at least three independent experiments. The error bars indicate SD. Values that differ significantly from the no-protein condition values by one-sample t test for each time point are indicated (*, P

    Techniques Used: Transfection, Concentration Assay, Luciferase, Plasmid Preparation, Incubation, Purification, Recombinant, Methylation, SDS Page

    The σNS protein protects viral RNA from degradation. (A) HEK293T cells were transfected with plasmids encoding either WT or Δ38 σNS and incubated for 20 h, followed by a second transfection with a σ3-encoding plasmid and incubation for 4 h. The cells were treated with 10 μg/ml of actinomycin D and lysed at the intervals shown, and σ3-encoding s4 RNA was quantified by RT-qPCR. The results are presented as mean RNA levels normalized to the RNA levels at 0 h for at least three independent experiments. The error bars indicate SD. (B and C) Radiolabeled uncapped and nonpolyadenylated s4 RNA was incubated without protein (No prot) or with 1 or 10 μM purified recombinant WT or Δ38 σNS at room temperature for 10 min, followed by addition of HeLa S100 lysates. (B) RNA was purified at the intervals shown, resolved by electrophoresis, and visualized by phosphorimaging. (C) Pixel intensity analysis of the s4 RNA bands. The results are presented as mean RNA levels normalized to the RNA levels at 0 min for three independent experiments. The error bars indicate SD. Values that differ significantly from the values at the start of the time course by one-sample t test for each time point are indicated (*, P
    Figure Legend Snippet: The σNS protein protects viral RNA from degradation. (A) HEK293T cells were transfected with plasmids encoding either WT or Δ38 σNS and incubated for 20 h, followed by a second transfection with a σ3-encoding plasmid and incubation for 4 h. The cells were treated with 10 μg/ml of actinomycin D and lysed at the intervals shown, and σ3-encoding s4 RNA was quantified by RT-qPCR. The results are presented as mean RNA levels normalized to the RNA levels at 0 h for at least three independent experiments. The error bars indicate SD. (B and C) Radiolabeled uncapped and nonpolyadenylated s4 RNA was incubated without protein (No prot) or with 1 or 10 μM purified recombinant WT or Δ38 σNS at room temperature for 10 min, followed by addition of HeLa S100 lysates. (B) RNA was purified at the intervals shown, resolved by electrophoresis, and visualized by phosphorimaging. (C) Pixel intensity analysis of the s4 RNA bands. The results are presented as mean RNA levels normalized to the RNA levels at 0 min for three independent experiments. The error bars indicate SD. Values that differ significantly from the values at the start of the time course by one-sample t test for each time point are indicated (*, P

    Techniques Used: Transfection, Incubation, Plasmid Preparation, Quantitative RT-PCR, Purification, Recombinant, Electrophoresis

    Reovirus T3D does not synthesize dsRNA in σNS-siRNA cells. T3D or T3D-R virus was allowed to adsorb onto σNS-siRNA cells at an MOI of 1 PFU/cell. Cells were lysed at the intervals shown, and negative (−)-sense s4 RNA was quantified by single-strand RT-qPCR. The results are presented as mean RNA levels at each time point normalized to the RNA levels at 0 h for at least three independent experiments. The error bars indicate SD. Values that differ significantly between T3D- and T3D-R-infected σNS-siRNA cells at each time point by two-way ANOVA followed by Sidak's multiple-comparison test are indicated (***, P
    Figure Legend Snippet: Reovirus T3D does not synthesize dsRNA in σNS-siRNA cells. T3D or T3D-R virus was allowed to adsorb onto σNS-siRNA cells at an MOI of 1 PFU/cell. Cells were lysed at the intervals shown, and negative (−)-sense s4 RNA was quantified by single-strand RT-qPCR. The results are presented as mean RNA levels at each time point normalized to the RNA levels at 0 h for at least three independent experiments. The error bars indicate SD. Values that differ significantly between T3D- and T3D-R-infected σNS-siRNA cells at each time point by two-way ANOVA followed by Sidak's multiple-comparison test are indicated (***, P

    Techniques Used: Quantitative RT-PCR, Infection

    The σNS protein binds viral and nonviral ssRNAs. (A) Reovirus T3D was allowed to adsorb onto HEK293T cells at an MOI of 100 PFU/cell and was incubated for 24 h. The cells were lysed and treated with 0.25 μg/ml of RNase A at 30°C for 1 h. Proteins were resolved by native PAGE or SDS-PAGE, followed by immunoblotting using antibodies specific for σNS and α-tubulin (SDS-PAGE only). Representative immunoblots from triplicate experiments are shown. Pixel intensity is depicted in a rainbow scale (native PAGE only). (B) T1L and T3D WT and Δ38 σNS proteins were translated in vitro in the presence of [ 35 S]methionine and incubated with 0.25 μg/ml of RNase A at 30°C for 1 h after translation, heated at 95°C for 10 min in SDS-PAGE sample buffer, or left untreated. Samples were resolved by native PAGE or SDS-PAGE and visualized by phosphorimaging. (C) Increasing concentrations of purified recombinant WT or Δ38 σNS were incubated with radiolabeled uncapped and nonpolyadenylated 7SK stem I RNA at room temperature for 10 min, followed by native electrophoresis and visualization by phosphorimaging. The arrowhead indicates RNA that failed to enter the gel. (D) Increasing concentrations of purified recombinant WT σNS protein were incubated with radiolabeled uncapped and nonpolyadenylated s4 RNA at room temperature for 10 min. σNS-RNA complexes were spotted onto nitrocellulose membranes, and unbound free RNA was collected on nylon membranes. The membranes were visualized by phosphorimaging. (E) K D , SD, and R 2 values for WT σNS and 7SK stem I (panel C, top) or s4 RNA (panel D) were determined using KaleidaGraph. (F) Langmuir isotherm curve fitting for WT σNS and 7SK stem I or s4 RNA. The results are presented as the mean percent shift at each concentration of σNS for at least three independent experiments (7SK stem I RNA, n = 5; s4 RNA, n = 3). The error bars indicate SD.
    Figure Legend Snippet: The σNS protein binds viral and nonviral ssRNAs. (A) Reovirus T3D was allowed to adsorb onto HEK293T cells at an MOI of 100 PFU/cell and was incubated for 24 h. The cells were lysed and treated with 0.25 μg/ml of RNase A at 30°C for 1 h. Proteins were resolved by native PAGE or SDS-PAGE, followed by immunoblotting using antibodies specific for σNS and α-tubulin (SDS-PAGE only). Representative immunoblots from triplicate experiments are shown. Pixel intensity is depicted in a rainbow scale (native PAGE only). (B) T1L and T3D WT and Δ38 σNS proteins were translated in vitro in the presence of [ 35 S]methionine and incubated with 0.25 μg/ml of RNase A at 30°C for 1 h after translation, heated at 95°C for 10 min in SDS-PAGE sample buffer, or left untreated. Samples were resolved by native PAGE or SDS-PAGE and visualized by phosphorimaging. (C) Increasing concentrations of purified recombinant WT or Δ38 σNS were incubated with radiolabeled uncapped and nonpolyadenylated 7SK stem I RNA at room temperature for 10 min, followed by native electrophoresis and visualization by phosphorimaging. The arrowhead indicates RNA that failed to enter the gel. (D) Increasing concentrations of purified recombinant WT σNS protein were incubated with radiolabeled uncapped and nonpolyadenylated s4 RNA at room temperature for 10 min. σNS-RNA complexes were spotted onto nitrocellulose membranes, and unbound free RNA was collected on nylon membranes. The membranes were visualized by phosphorimaging. (E) K D , SD, and R 2 values for WT σNS and 7SK stem I (panel C, top) or s4 RNA (panel D) were determined using KaleidaGraph. (F) Langmuir isotherm curve fitting for WT σNS and 7SK stem I or s4 RNA. The results are presented as the mean percent shift at each concentration of σNS for at least three independent experiments (7SK stem I RNA, n = 5; s4 RNA, n = 3). The error bars indicate SD.

    Techniques Used: Incubation, Clear Native PAGE, SDS Page, Western Blot, In Vitro, Purification, Recombinant, Electrophoresis, Concentration Assay

    38) Product Images from "RNA Polymerase II Transcription Attenuation at the Yeast DNA Repair Gene, DEF1, Involves Sen1-Dependent and Polyadenylation Site-Dependent Termination"

    Article Title: RNA Polymerase II Transcription Attenuation at the Yeast DNA Repair Gene, DEF1, Involves Sen1-Dependent and Polyadenylation Site-Dependent Termination

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.118.200072

    DEF1 promoter-proximal pA site (pA 1 ) is sufficient to confer Pol II transcription attenuation in a CUP1 /lacZ reporter assay. (A) Schematic of CUP1/lacZ reporter gene used to measure transcription termination (not to scale). The pGAC24 plasmid contains the actin exon(E1)-intron-exon(E2) fused to a CUP1 or lacZ reporter gene. The promoter-proximal DEF1 pA site, CYC1 pA site, or SNR13 transcription termination site were inserted within the intron. The DEF1 attenuator includes the 5′-UTR and upstream ORF but not the consensus TATA box promoter element. In the absence of a pA/terminator insert (No Term.), full-length mRNA production confers copper-resistance and high β-galactosidase activity. In the presence of a pA/terminator insert, attenuated non-coding RNA (ncRNA) production confers copper-sensitivity and low β-galactosidase activity. Trans -acting mutants that prevent pA/terminator recognition promote copper-resistance and higher β-galactosidase activity. (B) DEF1 pA 1 site confers copper-sensitivity in a DEF1-CUP1 reporter, and sen1 , ssu72 , and hrp1 mutants confer copper-resistance. (C) DEF1 pA 1 site reduces expression of CUP1 mRNA due to accumulation of attenuated ncRNA. Note that based on the RT-PCR primer locations (F1, R1, and R2 in panel A), the RT-PCR product from spliced mRNA (231 bp) is shorter than the PCR product from attenuated, unspliced transcript (387 bp). The % attenuated vs. full-length was determined by adding up signal intensities for both bands and determining the relative ratio. No Term. = No Terminator. (D) DEF1 pA 1 site reduces expression of a lacZ reporter similarly to known transcription terminators from CYC1 and SNR13 . β-galactosidase activity was measured following cell lysis and incubation with ONPG substrate, using absorption at OD 600 for cell density and OD 420 for product production. Experiments were performed in biological triplicate, and errors bars show standard deviation.
    Figure Legend Snippet: DEF1 promoter-proximal pA site (pA 1 ) is sufficient to confer Pol II transcription attenuation in a CUP1 /lacZ reporter assay. (A) Schematic of CUP1/lacZ reporter gene used to measure transcription termination (not to scale). The pGAC24 plasmid contains the actin exon(E1)-intron-exon(E2) fused to a CUP1 or lacZ reporter gene. The promoter-proximal DEF1 pA site, CYC1 pA site, or SNR13 transcription termination site were inserted within the intron. The DEF1 attenuator includes the 5′-UTR and upstream ORF but not the consensus TATA box promoter element. In the absence of a pA/terminator insert (No Term.), full-length mRNA production confers copper-resistance and high β-galactosidase activity. In the presence of a pA/terminator insert, attenuated non-coding RNA (ncRNA) production confers copper-sensitivity and low β-galactosidase activity. Trans -acting mutants that prevent pA/terminator recognition promote copper-resistance and higher β-galactosidase activity. (B) DEF1 pA 1 site confers copper-sensitivity in a DEF1-CUP1 reporter, and sen1 , ssu72 , and hrp1 mutants confer copper-resistance. (C) DEF1 pA 1 site reduces expression of CUP1 mRNA due to accumulation of attenuated ncRNA. Note that based on the RT-PCR primer locations (F1, R1, and R2 in panel A), the RT-PCR product from spliced mRNA (231 bp) is shorter than the PCR product from attenuated, unspliced transcript (387 bp). The % attenuated vs. full-length was determined by adding up signal intensities for both bands and determining the relative ratio. No Term. = No Terminator. (D) DEF1 pA 1 site reduces expression of a lacZ reporter similarly to known transcription terminators from CYC1 and SNR13 . β-galactosidase activity was measured following cell lysis and incubation with ONPG substrate, using absorption at OD 600 for cell density and OD 420 for product production. Experiments were performed in biological triplicate, and errors bars show standard deviation.

    Techniques Used: Reporter Assay, Plasmid Preparation, Activity Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Lysis, Incubation, Standard Deviation

    Hrp1 overexpression partially restores recognition of cis -acting DEF1 attenuator mutants. (A) Reporter strains containing WT or mutant DEF1-CUP1 reporters were transformed with an empty vector (pRS314) or a plasmid containing HRP1 ( pRS314- HRP1) and grown on –Leu/Trp copper plates to assess CUP1 expression. (B) Total RNA was collected from indicated strains grown at 30°C, and attenuated and read-through mRNAs were detected via RT-PCR. (C) Summary of genetic interactions between hyperactive HRP1 allele and cis -acting DEF1 attenuator mutants. The copper-sensitivity of mutants indicated in red was partially suppressed by HRP1 overexpression, consistent with improved attenuator recognition and Hrp1-binding at RNA regions I and II.
    Figure Legend Snippet: Hrp1 overexpression partially restores recognition of cis -acting DEF1 attenuator mutants. (A) Reporter strains containing WT or mutant DEF1-CUP1 reporters were transformed with an empty vector (pRS314) or a plasmid containing HRP1 ( pRS314- HRP1) and grown on –Leu/Trp copper plates to assess CUP1 expression. (B) Total RNA was collected from indicated strains grown at 30°C, and attenuated and read-through mRNAs were detected via RT-PCR. (C) Summary of genetic interactions between hyperactive HRP1 allele and cis -acting DEF1 attenuator mutants. The copper-sensitivity of mutants indicated in red was partially suppressed by HRP1 overexpression, consistent with improved attenuator recognition and Hrp1-binding at RNA regions I and II.

    Techniques Used: Over Expression, Mutagenesis, Transformation Assay, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction, Binding Assay

    The def1 A-1G attenuator mutant increases Def1 mRNA and protein and reduces cell viability when overexpressing pr-Def1. (A) Schematic of the DEF1 gene (not to scale). The relevant pA sites, efficiency element (EE), mutations (A-1G, C1590A), and RT-PCR primers are indicated. (B) Yeast strains containing chromosomal (CHR) DEF1 or def1 Δ were transformed with empty vector (pRS426), WT DEF1 (pRS426- DEF1 ), or mutant (pRS426- DEF1 , A-1G ) plasmids. Total RNA was isolated, and Def1 read-through mRNA was detected via RT-PCR (using blue primers indicated in (A). The signal intensity of the DEF1 mRNA bands was normalized to the 18S loading control and then the def1 Δ pRS426- DEF1 sample. RT: Reverse Transcriptase. (C) Yeast strains containing WT chromosomal DEF1 or def1 Δ were transformed with empty vector (pRS426), WT DEF1 (pRS426- DEF1 ), attenuator mutant (pRS426- DEF1 , A-1G ), pr-Def1 mutant (pRS426- DEF1-C1590A ), or double mutant plasmids. Strains were spotted on -Ura plates and growth was assessed after 1 week at the indicated temperatures. (D) Total RNA was collected from strains in (C) containing def1 mutants C1590A and C1590A/A-1G in a def1 Δ strain grown at 30°C. DEF1 read-through mRNA was detected via RT-PCR and quantified as in (B). (E) Western blot of extracts from strains in (C) following growth at 30°C and a temperature shift to 39°C for 0, 1, or 2 hr. Def1 protein levels were normalized to the actin loading control, and signal from def1 C1590A/A-1G was normalized to def1 C1590A at consistent time points.
    Figure Legend Snippet: The def1 A-1G attenuator mutant increases Def1 mRNA and protein and reduces cell viability when overexpressing pr-Def1. (A) Schematic of the DEF1 gene (not to scale). The relevant pA sites, efficiency element (EE), mutations (A-1G, C1590A), and RT-PCR primers are indicated. (B) Yeast strains containing chromosomal (CHR) DEF1 or def1 Δ were transformed with empty vector (pRS426), WT DEF1 (pRS426- DEF1 ), or mutant (pRS426- DEF1 , A-1G ) plasmids. Total RNA was isolated, and Def1 read-through mRNA was detected via RT-PCR (using blue primers indicated in (A). The signal intensity of the DEF1 mRNA bands was normalized to the 18S loading control and then the def1 Δ pRS426- DEF1 sample. RT: Reverse Transcriptase. (C) Yeast strains containing WT chromosomal DEF1 or def1 Δ were transformed with empty vector (pRS426), WT DEF1 (pRS426- DEF1 ), attenuator mutant (pRS426- DEF1 , A-1G ), pr-Def1 mutant (pRS426- DEF1-C1590A ), or double mutant plasmids. Strains were spotted on -Ura plates and growth was assessed after 1 week at the indicated temperatures. (D) Total RNA was collected from strains in (C) containing def1 mutants C1590A and C1590A/A-1G in a def1 Δ strain grown at 30°C. DEF1 read-through mRNA was detected via RT-PCR and quantified as in (B). (E) Western blot of extracts from strains in (C) following growth at 30°C and a temperature shift to 39°C for 0, 1, or 2 hr. Def1 protein levels were normalized to the actin loading control, and signal from def1 C1590A/A-1G was normalized to def1 C1590A at consistent time points.

    Techniques Used: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Plasmid Preparation, Isolation, Western Blot

    39) Product Images from "Clcn7F318L/+ as a new mouse model of Albers-Schönberg disease"

    Article Title: Clcn7F318L/+ as a new mouse model of Albers-Schönberg disease

    Journal: Bone

    doi: 10.1016/j.bone.2017.09.007

    Generation of mice with a Clcn7 F318L knockin allele that is expressed and translated into protein (A) Schematic depiction of the wildtype (wt) Clcn7 locus and the gene targeting construct (not drawn to scale) indicating the locations of PCR primers used to amplify DNA for the homologous recombination arms within the targeting vector (P1–P4) and to confirm correct targeting of the locus (P5–P8). The targeting construct includes a neomycin resistance cassette (Neo R ) flanked by loxP sites for positive selection, the p.F318L mutation in exon 11 (*), and a thymidine kinase (TK) cassette for negative selection. After achieving germline transmission, the Neo R cassette was removed by Cre-recombination. The bottom schematic depicts the mutant Clcn7 locus used in all animal experiments, with the location of primers (P9 and P10) used for genotyping indicated. (B) Representative image of a genotyping gel showing a 407 bp wildtype amplimer and a 530 bp mutant amplimer. (C) Sequencing electropherogram of PCR amplimer generated from a Clcn7 F318L/+ mouse confirming heterozygosity for the T→C transition. (D) RT-PCR using RNA from Clcn7 +/+ , Clcn7 F318L/+ and Clcn7 F318L/F318L tibial bone showing the expected 677 bp amplimer in each sample, with no evidence of altered splicing due to the Clcn7 F318L allele. (E) Bar graph depicting droplet digital PCR results using cDNA from Clcn7 +/+ , Clcn7 F318L/+ and Clcn7 F318L/F318L tibial bone showing the percentage of droplets than contain mutant or wildtype amplimers. Samples from 3 mice with each genotype were studied, and ddPCR reactions were performed in duplicate. An average of 163 amplimer containing droplets were detected per reaction. (F) Fluorescence photomicrographs of cultured bone marrow cells isolated from Clcn7 +/+ and Clcn7 F318L/F318L mice that had been incubated with an anti-CLC-7 primary antibody and a FITC-labeled (green fluorescence) secondary antibody. Nuclei were stained with Hoechst 33342 dye and fluoresce blue. Note Clcn7 +/+ and Clcn7 F318L/F318L cells have similar patterns of CLC-7 immunofluorescence. Importantly, in the absence of primary antibody, these immunofluorescence signals were not observed in these cells (data not shown). Scale bar equals 10 μm.
    Figure Legend Snippet: Generation of mice with a Clcn7 F318L knockin allele that is expressed and translated into protein (A) Schematic depiction of the wildtype (wt) Clcn7 locus and the gene targeting construct (not drawn to scale) indicating the locations of PCR primers used to amplify DNA for the homologous recombination arms within the targeting vector (P1–P4) and to confirm correct targeting of the locus (P5–P8). The targeting construct includes a neomycin resistance cassette (Neo R ) flanked by loxP sites for positive selection, the p.F318L mutation in exon 11 (*), and a thymidine kinase (TK) cassette for negative selection. After achieving germline transmission, the Neo R cassette was removed by Cre-recombination. The bottom schematic depicts the mutant Clcn7 locus used in all animal experiments, with the location of primers (P9 and P10) used for genotyping indicated. (B) Representative image of a genotyping gel showing a 407 bp wildtype amplimer and a 530 bp mutant amplimer. (C) Sequencing electropherogram of PCR amplimer generated from a Clcn7 F318L/+ mouse confirming heterozygosity for the T→C transition. (D) RT-PCR using RNA from Clcn7 +/+ , Clcn7 F318L/+ and Clcn7 F318L/F318L tibial bone showing the expected 677 bp amplimer in each sample, with no evidence of altered splicing due to the Clcn7 F318L allele. (E) Bar graph depicting droplet digital PCR results using cDNA from Clcn7 +/+ , Clcn7 F318L/+ and Clcn7 F318L/F318L tibial bone showing the percentage of droplets than contain mutant or wildtype amplimers. Samples from 3 mice with each genotype were studied, and ddPCR reactions were performed in duplicate. An average of 163 amplimer containing droplets were detected per reaction. (F) Fluorescence photomicrographs of cultured bone marrow cells isolated from Clcn7 +/+ and Clcn7 F318L/F318L mice that had been incubated with an anti-CLC-7 primary antibody and a FITC-labeled (green fluorescence) secondary antibody. Nuclei were stained with Hoechst 33342 dye and fluoresce blue. Note Clcn7 +/+ and Clcn7 F318L/F318L cells have similar patterns of CLC-7 immunofluorescence. Importantly, in the absence of primary antibody, these immunofluorescence signals were not observed in these cells (data not shown). Scale bar equals 10 μm.

    Techniques Used: Mouse Assay, Knock-In, Construct, Polymerase Chain Reaction, Homologous Recombination, Plasmid Preparation, Selection, Mutagenesis, Transmission Assay, Sequencing, Generated, Reverse Transcription Polymerase Chain Reaction, Digital PCR, Fluorescence, Cell Culture, Isolation, Incubation, Labeling, Staining, Immunofluorescence

    40) Product Images from "A fluorescent analog of a HBV core protein-directed drug is used to interrogate antiviral mechanism"

    Article Title: A fluorescent analog of a HBV core protein-directed drug is used to interrogate antiviral mechanism

    Journal: Journal of the American Chemical Society

    doi: 10.1021/jacs.8b07988

    Detection of polymerase defective and empty HBV intracellular cores by HAP-ALEX. (A) HuH7-H1 cells were transfected with genomic clone of HBV that makes no envelope protein and encodes a Y63F mutant polymerase. These transfections will yield cores that contain pgRNA but are unable to synthesize rcDNA. 3 days post-transfection cells were treated with HAP-ALEX for 16 hours after which the cells were fixed and stained with anti Cp antibodies (Dako) for IF. (B) A parallel control experiment with expression of clone with defective Y63F polymerase and the V124W Cp mutant where Cp does not bind HAP-ALEX. (C) Expression of empty cores was accomplished by transfection of pTruf-HBc which encodes Cp but no other component of HBV. HAP-ALEX binds Cp and induces formation of large puncta under expression conditions that normally yield RNA-filled capsids (A) and empty capsids (C).
    Figure Legend Snippet: Detection of polymerase defective and empty HBV intracellular cores by HAP-ALEX. (A) HuH7-H1 cells were transfected with genomic clone of HBV that makes no envelope protein and encodes a Y63F mutant polymerase. These transfections will yield cores that contain pgRNA but are unable to synthesize rcDNA. 3 days post-transfection cells were treated with HAP-ALEX for 16 hours after which the cells were fixed and stained with anti Cp antibodies (Dako) for IF. (B) A parallel control experiment with expression of clone with defective Y63F polymerase and the V124W Cp mutant where Cp does not bind HAP-ALEX. (C) Expression of empty cores was accomplished by transfection of pTruf-HBc which encodes Cp but no other component of HBV. HAP-ALEX binds Cp and induces formation of large puncta under expression conditions that normally yield RNA-filled capsids (A) and empty capsids (C).

    Techniques Used: Transfection, Mutagenesis, Staining, Expressing

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    Real-time Polymerase Chain Reaction:

    Article Title: Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response
    Article Snippet: .. 1 μg of RNA was reverse-transcribed into cDNA using random hexamers and M-MuLV reverse transcriptase (New England Biolabs). qPCRs were assembled with Absolute QPCR ROX Mix (Thermo Scientific AB-1139), gene-specific oligonucleotides, double-dye probes (see ) and analysed using the StepOnePlus™ Real-Time PCR System (Applied Biosystem). ..

    Incubation:

    Article Title: Utility of the Trypanosoma cruzi Sequence Database for Identification of Potential Vaccine Candidates by In Silico and In Vitro Screening
    Article Snippet: .. First-strand cDNA was synthesized by incubation of total RNA (5 μg) with 2.5 U of Moloney murine leukemia virus reverse transcriptase (New England Biolabs) and oligo(dT)16 at 42°C for 1 h in a 20-μl reaction volume and stored at −20°C until further use. .. Genes TcG1-TcG8 were amplified from epimastigote cDNA in a PCR with universal splice leader, present upstream to the start codon (ATG) in mRNA-cDNA of trypanosomes , as forward primer and gene-specific reverse primer (listed in Table ).

    Article Title: Capping-RACE: a simple, accurate, and sensitive 5′ RACE method for use in prokaryotes
    Article Snippet: .. Different treatments of the 111 nt in vitro transcribed RNA The 111 nt in vitro transcribed RNA was pre-treated to modify the 5′ ends as follows: (i) 1 μg of RNA was incubated with 1 μl of RNA 5′ pyrophosphohydrolase (RppH) (NEB, Ipswich, MA, USA) for 1 h at 37°C; (ii) 1 μg of RNA was incubated with 1 μl of VCE (NEB) for 30 min at 37°C; or (iii) 1 μg of RNA was treated with RppH for 1 h at 37°C and purified by ethanol precipitation, followed by incubation with VCE for 30 min at 37°C. .. All samples were purified by ethanol precipitation and evaluated using a Nano Drop (Thermo Fisher Scientific).

    Expressing:

    Article Title: Histidine-Rich Glycoprotein Can Prevent Development of Mouse Experimental Glioblastoma
    Article Snippet: .. RNA Extraction and Detection of RCAS Mediated Gene Expression Total RNA was extracted from frozen brain tumor tissue, eight slides of 20 µm tissue sections per sample using TRIzol® reagent (Invitrogen). cDNA was made through reverse transcription of 1 µg of total RNA using random primers (S12545, New England Biolabs, Ipswich, MA) and M-MuLV reverse transcriptase (New England Biolabs) and subsequently 100 ng of cDNA was used for PCR detection of PDGF-B and HRG of human origin used in the RCAS vectors utilizing the PuReTaq Ready-To-Go™ PCR Beads (GE Healthcare, Uppsala, Sweden) system. .. RNA from U-343MG-a was used as positive control for PDGF-B, DF-1 RCAS-HRG as positive control for HRG and frozen brain tissue from an FVB/N mouse as negative control.

    Ethanol Precipitation:

    Article Title: Capping-RACE: a simple, accurate, and sensitive 5′ RACE method for use in prokaryotes
    Article Snippet: .. Different treatments of the 111 nt in vitro transcribed RNA The 111 nt in vitro transcribed RNA was pre-treated to modify the 5′ ends as follows: (i) 1 μg of RNA was incubated with 1 μl of RNA 5′ pyrophosphohydrolase (RppH) (NEB, Ipswich, MA, USA) for 1 h at 37°C; (ii) 1 μg of RNA was incubated with 1 μl of VCE (NEB) for 30 min at 37°C; or (iii) 1 μg of RNA was treated with RppH for 1 h at 37°C and purified by ethanol precipitation, followed by incubation with VCE for 30 min at 37°C. .. All samples were purified by ethanol precipitation and evaluated using a Nano Drop (Thermo Fisher Scientific).

    Polymerase Chain Reaction:

    Article Title: Histidine-Rich Glycoprotein Can Prevent Development of Mouse Experimental Glioblastoma
    Article Snippet: .. RNA Extraction and Detection of RCAS Mediated Gene Expression Total RNA was extracted from frozen brain tumor tissue, eight slides of 20 µm tissue sections per sample using TRIzol® reagent (Invitrogen). cDNA was made through reverse transcription of 1 µg of total RNA using random primers (S12545, New England Biolabs, Ipswich, MA) and M-MuLV reverse transcriptase (New England Biolabs) and subsequently 100 ng of cDNA was used for PCR detection of PDGF-B and HRG of human origin used in the RCAS vectors utilizing the PuReTaq Ready-To-Go™ PCR Beads (GE Healthcare, Uppsala, Sweden) system. .. RNA from U-343MG-a was used as positive control for PDGF-B, DF-1 RCAS-HRG as positive control for HRG and frozen brain tissue from an FVB/N mouse as negative control.

    De-Phosphorylation Assay:

    Article Title: PRC2 binds to active promoters and contacts nascent RNAs in embryonic stem cells
    Article Snippet: .. For PAR-CLIP-seq experiments, 3′-blocked DNA adapter (100 pmol/μl) was ligated to the RNA after dephosphorylation and before 5′ 32 P end-labeling by incubating the beads with T4 RNA ligase 1 (New England Biolabs) for 1 hour at 25°C. .. Labeled material was resolved on 8% bis-tris gels, transferred to nitrocellulose membranes and exposed to autoradiography films for ∼4 hours.

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    The Monarch RNA Cleanup Kit 500 µg reliably purifies up to 500 μg of concentrated high quality RNA 25 nt from enzymatic reactions and in vitro transcription IVT reactions The
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    New England Biolabs ptb rna complexes
    Isolation of <t>PTB</t> – <t>RNA</t> complexes without gel purifications A. Comparison of Halo fusion proteins (input vs . unbound fractions) for the indicated samples. The Halo fusion proteins are labeled with HaloTag Alexa Fluor® 660 ligand and resolved on SDS–PAGE. Bottom panel, Western blot analysis of tubulin as a loading control. B. Western blot analysis showing that similar amounts of PTB–RNA complexes are released by TEV protease following the indicated washing conditions after Halo bead isolation from the HEK 293T Halo-PTB stable cells. Non-transfected HEK 293T cells are used as control. C. Autoradiogram (upper panel) of 32 P-labeled RNA crosslinked to PTB purified by HaloTag and released by TEV protease. RNA–protein complexes of 60–70 kD are seen only with the UV crosslinking condition. Western blot analysis (bottom panel) of TEV-released PTB proteins from equal amounts of samples prepared from the lysate of the Halo-PTB stable cell line.
    Ptb Rna Complexes, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs rna
    Acetylation of H2A.Z is required for upregulation of Hes1 Notch target gene. ( A ) Schematic representation of the Flag-RBP-J/Tip60 fusion proteins used in Figure 4B and C and in Supplementary Figures S9 and S10 . Amino acid numbering is accordingly to accession NP_033061.3 for RBP-J and NP_874368.1 for Tip60. RBP-J domain: LAG1-DNAbind, LAG1 DNA binding (CDD:255260); Tip60 domain: MOZ/SAS, MOZ/SAS family (CDD:250916). ( B ) RBP-J/Tip60 wildtype (wt) but not its catalytic dead (cd) mutant upregulates Hes1 expression in MT cells. MT cells were infected with retroviral particles delivering plasmids encoding Flag-tagged RBP-J/Tip60-wt, cd mutant or empty vector (Control). Total <t>RNA</t> was reverse transcribed into <t>cDNA</t> and analysed by qPCR using primers specific for Tbp or Hes1 . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD ([**] P
    Rna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs hela rna
    ERCC reads to determine library preparation quality and back-calculate <t>RNA</t> input mass. (A) After normalizing to RNA input mass, reads aligning to the 92 ERCC spike-in transcripts correlate linearly with ERCC spike-in concentration across six orders of magnitude in all libraries prepared with the miniaturized protocol (R 2 E R C C m a s s ( p g ) T o t a l m a s s ( p g ) = E R C C r e a d s T o t a l r e a d s Back-calculated masses of <t>HeLa</t> libraries correlated strongly with QuBit quantification (R 2 = 0.9954).
    Hela Rna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Isolation of PTB – RNA complexes without gel purifications A. Comparison of Halo fusion proteins (input vs . unbound fractions) for the indicated samples. The Halo fusion proteins are labeled with HaloTag Alexa Fluor® 660 ligand and resolved on SDS–PAGE. Bottom panel, Western blot analysis of tubulin as a loading control. B. Western blot analysis showing that similar amounts of PTB–RNA complexes are released by TEV protease following the indicated washing conditions after Halo bead isolation from the HEK 293T Halo-PTB stable cells. Non-transfected HEK 293T cells are used as control. C. Autoradiogram (upper panel) of 32 P-labeled RNA crosslinked to PTB purified by HaloTag and released by TEV protease. RNA–protein complexes of 60–70 kD are seen only with the UV crosslinking condition. Western blot analysis (bottom panel) of TEV-released PTB proteins from equal amounts of samples prepared from the lysate of the Halo-PTB stable cell line.

    Journal: Genomics, Proteomics & Bioinformatics

    Article Title: GoldCLIP: Gel-omitted Ligation-dependent CLIP

    doi: 10.1016/j.gpb.2018.04.003

    Figure Lengend Snippet: Isolation of PTB – RNA complexes without gel purifications A. Comparison of Halo fusion proteins (input vs . unbound fractions) for the indicated samples. The Halo fusion proteins are labeled with HaloTag Alexa Fluor® 660 ligand and resolved on SDS–PAGE. Bottom panel, Western blot analysis of tubulin as a loading control. B. Western blot analysis showing that similar amounts of PTB–RNA complexes are released by TEV protease following the indicated washing conditions after Halo bead isolation from the HEK 293T Halo-PTB stable cells. Non-transfected HEK 293T cells are used as control. C. Autoradiogram (upper panel) of 32 P-labeled RNA crosslinked to PTB purified by HaloTag and released by TEV protease. RNA–protein complexes of 60–70 kD are seen only with the UV crosslinking condition. Western blot analysis (bottom panel) of TEV-released PTB proteins from equal amounts of samples prepared from the lysate of the Halo-PTB stable cell line.

    Article Snippet: Finally, PTB–RNA complexes were cleaved off the beads by TEV protease and digested with protease K (catalog No. P8102S; New England Biolabs) at 37 °C for 30 min.

    Techniques: Isolation, Labeling, SDS Page, Western Blot, Transfection, Purification, Stable Transfection

    HaloTag based GoldCLIP technology A. Schematic flow chart of GoldCLIP technology. Cells stably expressing Halo-tagged fusion RBPs are crosslinked by UV irradiation. After cell lysis, Halo-RBP complexes are then captured by magnetic beads coated with Halo ligand under native conditions and a specific 3′ linker is ligated to RNAs bound by RBPs. Following denaturing washes, purified RNAs are cloned via an iCLIP protocol for high-throughput sequencing. B. Western blot analysis showing the expression level of Halo-PTB in the HEK 293T Halo-PTB stable cells compared to endogenous PTB using a monoclonal anti-PTB antibody (BB7). Non-transfected HEK 293T cells are used as control. A diagram of Halo-PTB fusion protein is shown below. C. Localization of Halo-PTB fusion proteins in 293T cell line. HaloTag TMR ligand staining of Halo-PTB fusion protein is shown in the top panel, and immunofluorescent staining of endogenous PTB using a monoclonal PTB antibody (BB7) is shown in the bottom panel. RBP, RNA-binding protein; iCLIP, individual-nucleotide resolution CLIP; PTB, polypyrimidine tract-binding protein; TMR, tetramethylrhodamine; TEV, tobacco etch virus.

    Journal: Genomics, Proteomics & Bioinformatics

    Article Title: GoldCLIP: Gel-omitted Ligation-dependent CLIP

    doi: 10.1016/j.gpb.2018.04.003

    Figure Lengend Snippet: HaloTag based GoldCLIP technology A. Schematic flow chart of GoldCLIP technology. Cells stably expressing Halo-tagged fusion RBPs are crosslinked by UV irradiation. After cell lysis, Halo-RBP complexes are then captured by magnetic beads coated with Halo ligand under native conditions and a specific 3′ linker is ligated to RNAs bound by RBPs. Following denaturing washes, purified RNAs are cloned via an iCLIP protocol for high-throughput sequencing. B. Western blot analysis showing the expression level of Halo-PTB in the HEK 293T Halo-PTB stable cells compared to endogenous PTB using a monoclonal anti-PTB antibody (BB7). Non-transfected HEK 293T cells are used as control. A diagram of Halo-PTB fusion protein is shown below. C. Localization of Halo-PTB fusion proteins in 293T cell line. HaloTag TMR ligand staining of Halo-PTB fusion protein is shown in the top panel, and immunofluorescent staining of endogenous PTB using a monoclonal PTB antibody (BB7) is shown in the bottom panel. RBP, RNA-binding protein; iCLIP, individual-nucleotide resolution CLIP; PTB, polypyrimidine tract-binding protein; TMR, tetramethylrhodamine; TEV, tobacco etch virus.

    Article Snippet: Finally, PTB–RNA complexes were cleaved off the beads by TEV protease and digested with protease K (catalog No. P8102S; New England Biolabs) at 37 °C for 30 min.

    Techniques: Flow Cytometry, Stable Transfection, Expressing, Irradiation, Lysis, Magnetic Beads, Purification, Clone Assay, Next-Generation Sequencing, Western Blot, Transfection, Staining, RNA Binding Assay, Cross-linking Immunoprecipitation, Binding Assay

    GoldCLIP identified endogenous RNA targets of PTB A. Comparison of genomic distribution of the uniquely-mapped reads identified by Halo-PTB GoldCLIP or the published iCLIP datasets. Color code is indicated in the legend box on the right. TTS, transcription termination site. B. Total number of peaks identified by GoldCLIP. Two different crosslinking conditions (UVC in blue, 254 nm and UVA in orange, 365 nm) are shown with two negative controls: Halo-PTB without UV crosslinking (PTB_No UV) and Halo-YFP crosslinked with UVC (YFP_UVC). Peaks identified using the published datasets (PTB_iCLIP2) are shown in gray. C. Comparison of Halo-PTB clusters identified by GoldCLIP at the PTBP1 locus. Genomic tracks of reverse transcriptase stops are shown for the different samples, i.e. , Halo-PTB crosslinked with either UVC (PTB_UVC) or UVA (PTB_UVA), iCLIP from endogenous PTB (PTB_iCLIP2), Halo-PTB without UV crosslinking (PTB_No UV) and Halo-YFP crosslinked with UVC (YFP_UVC). D. and E. show the highly correlated Pearson’s coefficient between the number of reads obtained from two biological replicates in a 500 bp window across the whole genome for UVC crosslinking (D) and UVA (E), respectively. F. Top HOMER motifs calculated from the peak reads after UVC crosslinking are shown. G. Over-represented Halo-PTB binding motifs identified by GoldCLIP after UVC crosslinking. Histogram of Z-scores indicates the enrichment of hexamers in GoldCLIP clusters compared to randomly chosen regions of similar sizes in the same genes. Z-scores of the top three hexamers are indicated. H. Heatmap showing the coverage of Halo-PTB binding motifs at crosslink clusters that are defined with a 3-nt clustering window. The clusters are sorted from the shortest to the longest. The nucleotide preceding the start and the nucleotide following the median end of all clusters are marked by white lines in the plot. A color key for the coverage per nucleotide of the PTB-binding motifs is shown on the right. I. Similar to F except UVA crosslinking condition was used. J. Similar to G except UVA crosslinking condition was used. K. Similar to H except UVA crosslinking condition was used.

    Journal: Genomics, Proteomics & Bioinformatics

    Article Title: GoldCLIP: Gel-omitted Ligation-dependent CLIP

    doi: 10.1016/j.gpb.2018.04.003

    Figure Lengend Snippet: GoldCLIP identified endogenous RNA targets of PTB A. Comparison of genomic distribution of the uniquely-mapped reads identified by Halo-PTB GoldCLIP or the published iCLIP datasets. Color code is indicated in the legend box on the right. TTS, transcription termination site. B. Total number of peaks identified by GoldCLIP. Two different crosslinking conditions (UVC in blue, 254 nm and UVA in orange, 365 nm) are shown with two negative controls: Halo-PTB without UV crosslinking (PTB_No UV) and Halo-YFP crosslinked with UVC (YFP_UVC). Peaks identified using the published datasets (PTB_iCLIP2) are shown in gray. C. Comparison of Halo-PTB clusters identified by GoldCLIP at the PTBP1 locus. Genomic tracks of reverse transcriptase stops are shown for the different samples, i.e. , Halo-PTB crosslinked with either UVC (PTB_UVC) or UVA (PTB_UVA), iCLIP from endogenous PTB (PTB_iCLIP2), Halo-PTB without UV crosslinking (PTB_No UV) and Halo-YFP crosslinked with UVC (YFP_UVC). D. and E. show the highly correlated Pearson’s coefficient between the number of reads obtained from two biological replicates in a 500 bp window across the whole genome for UVC crosslinking (D) and UVA (E), respectively. F. Top HOMER motifs calculated from the peak reads after UVC crosslinking are shown. G. Over-represented Halo-PTB binding motifs identified by GoldCLIP after UVC crosslinking. Histogram of Z-scores indicates the enrichment of hexamers in GoldCLIP clusters compared to randomly chosen regions of similar sizes in the same genes. Z-scores of the top three hexamers are indicated. H. Heatmap showing the coverage of Halo-PTB binding motifs at crosslink clusters that are defined with a 3-nt clustering window. The clusters are sorted from the shortest to the longest. The nucleotide preceding the start and the nucleotide following the median end of all clusters are marked by white lines in the plot. A color key for the coverage per nucleotide of the PTB-binding motifs is shown on the right. I. Similar to F except UVA crosslinking condition was used. J. Similar to G except UVA crosslinking condition was used. K. Similar to H except UVA crosslinking condition was used.

    Article Snippet: Finally, PTB–RNA complexes were cleaved off the beads by TEV protease and digested with protease K (catalog No. P8102S; New England Biolabs) at 37 °C for 30 min.

    Techniques: Binding Assay

    Acetylation of H2A.Z is required for upregulation of Hes1 Notch target gene. ( A ) Schematic representation of the Flag-RBP-J/Tip60 fusion proteins used in Figure 4B and C and in Supplementary Figures S9 and S10 . Amino acid numbering is accordingly to accession NP_033061.3 for RBP-J and NP_874368.1 for Tip60. RBP-J domain: LAG1-DNAbind, LAG1 DNA binding (CDD:255260); Tip60 domain: MOZ/SAS, MOZ/SAS family (CDD:250916). ( B ) RBP-J/Tip60 wildtype (wt) but not its catalytic dead (cd) mutant upregulates Hes1 expression in MT cells. MT cells were infected with retroviral particles delivering plasmids encoding Flag-tagged RBP-J/Tip60-wt, cd mutant or empty vector (Control). Total RNA was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp or Hes1 . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD ([**] P

    Journal: Nucleic Acids Research

    Article Title: Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response

    doi: 10.1093/nar/gky551

    Figure Lengend Snippet: Acetylation of H2A.Z is required for upregulation of Hes1 Notch target gene. ( A ) Schematic representation of the Flag-RBP-J/Tip60 fusion proteins used in Figure 4B and C and in Supplementary Figures S9 and S10 . Amino acid numbering is accordingly to accession NP_033061.3 for RBP-J and NP_874368.1 for Tip60. RBP-J domain: LAG1-DNAbind, LAG1 DNA binding (CDD:255260); Tip60 domain: MOZ/SAS, MOZ/SAS family (CDD:250916). ( B ) RBP-J/Tip60 wildtype (wt) but not its catalytic dead (cd) mutant upregulates Hes1 expression in MT cells. MT cells were infected with retroviral particles delivering plasmids encoding Flag-tagged RBP-J/Tip60-wt, cd mutant or empty vector (Control). Total RNA was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp or Hes1 . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD ([**] P

    Article Snippet: 1 μg of RNA was reverse-transcribed into cDNA using random hexamers and M-MuLV reverse transcriptase (New England Biolabs). qPCRs were assembled with Absolute QPCR ROX Mix (Thermo Scientific AB-1139), gene-specific oligonucleotides, double-dye probes (see ) and analysed using the StepOnePlus™ Real-Time PCR System (Applied Biosystem).

    Techniques: Binding Assay, Mutagenesis, Expressing, Infection, Plasmid Preparation, Real-time Polymerase Chain Reaction

    H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. ( A ) Schematic representation of the NICD-inducible system established in MT cells. The NICD was fused to the estrogen receptor binding domain (NICD-ER) and retrovirally introduced into MT cells. The NICD-ER fusion protein is retained into the cytoplasm unless cells are treated with ( Z )-4-hydroxytamoxifen (4-OHT) that induces its nuclear translocation and activation of Notch target genes. ( B ) Hes1 and Il2ra Notch target genes are induced upon 4-OHT treatment of MT NICD-ER cells. Total RNA from MT NICD-ER cells, treated for 24 h with 4-OHT or EtOH as control, was reverse transcribed into cDNA and analyzed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of three independent experiments. ( C ) H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. MT NICD-ER cells were treated for 24 h with 4-OHT or EtOH as control and subjected to ChIP analysis using antibodies against H2A.Z, H2A.Zac, H3 or IgG as control. The qPCR analysis was focused at the Notch-dependent enhancers (red squares) represented on the left ( Hes1 +0.6 kb and Il2ra -26 kb ). Chrom X was used as negative control ( Control ). Data were normalized to the positive control ( GAPDH 0 kb ) and, in the case of H2A.Zac/H2A.Z, the H2A.Zac signals were further normalized to H2A.Z. Shown is the mean ± SD of two independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response

    doi: 10.1093/nar/gky551

    Figure Lengend Snippet: H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. ( A ) Schematic representation of the NICD-inducible system established in MT cells. The NICD was fused to the estrogen receptor binding domain (NICD-ER) and retrovirally introduced into MT cells. The NICD-ER fusion protein is retained into the cytoplasm unless cells are treated with ( Z )-4-hydroxytamoxifen (4-OHT) that induces its nuclear translocation and activation of Notch target genes. ( B ) Hes1 and Il2ra Notch target genes are induced upon 4-OHT treatment of MT NICD-ER cells. Total RNA from MT NICD-ER cells, treated for 24 h with 4-OHT or EtOH as control, was reverse transcribed into cDNA and analyzed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of three independent experiments. ( C ) H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. MT NICD-ER cells were treated for 24 h with 4-OHT or EtOH as control and subjected to ChIP analysis using antibodies against H2A.Z, H2A.Zac, H3 or IgG as control. The qPCR analysis was focused at the Notch-dependent enhancers (red squares) represented on the left ( Hes1 +0.6 kb and Il2ra -26 kb ). Chrom X was used as negative control ( Control ). Data were normalized to the positive control ( GAPDH 0 kb ) and, in the case of H2A.Zac/H2A.Z, the H2A.Zac signals were further normalized to H2A.Z. Shown is the mean ± SD of two independent experiments.

    Article Snippet: 1 μg of RNA was reverse-transcribed into cDNA using random hexamers and M-MuLV reverse transcriptase (New England Biolabs). qPCRs were assembled with Absolute QPCR ROX Mix (Thermo Scientific AB-1139), gene-specific oligonucleotides, double-dye probes (see ) and analysed using the StepOnePlus™ Real-Time PCR System (Applied Biosystem).

    Techniques: Activation Assay, Binding Assay, Translocation Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Negative Control, Positive Control

    Histone variant H2A.Z has a negative impact on the expression of Notch target genes. ( A ) Histone Variant H2A.Z is efficiently depleted by CRISPR/Cas9 in MT cells. Whole Cell Extract (WCE) was prepared from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells and analysed by Western blotting. GAPDH was used as loading control. ( B ) Hes1 and Il2ra Notch target genes are upregulated upon depletion of H2A.Z. Total RNA from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of five independent experiments ([*] P

    Journal: Nucleic Acids Research

    Article Title: Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response

    doi: 10.1093/nar/gky551

    Figure Lengend Snippet: Histone variant H2A.Z has a negative impact on the expression of Notch target genes. ( A ) Histone Variant H2A.Z is efficiently depleted by CRISPR/Cas9 in MT cells. Whole Cell Extract (WCE) was prepared from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells and analysed by Western blotting. GAPDH was used as loading control. ( B ) Hes1 and Il2ra Notch target genes are upregulated upon depletion of H2A.Z. Total RNA from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of five independent experiments ([*] P

    Article Snippet: 1 μg of RNA was reverse-transcribed into cDNA using random hexamers and M-MuLV reverse transcriptase (New England Biolabs). qPCRs were assembled with Absolute QPCR ROX Mix (Thermo Scientific AB-1139), gene-specific oligonucleotides, double-dye probes (see ) and analysed using the StepOnePlus™ Real-Time PCR System (Applied Biosystem).

    Techniques: Variant Assay, Expressing, CRISPR, Western Blot, Real-time Polymerase Chain Reaction

    Experimental verification of Capping-RACE with in vitro transcribed RNA. ( A ) Different treatments of in vitro transcribed RNA. ( B ) The cDNA products from the differently treated RNA described in (A). Lane 1, the negative control, performed without adding reverse transcriptase (RT). Lane 2, in vitro transcribed RNA that was not subjected to any treatment. Lane 3, in vitro transcribed RNA subjected to RppH treatment. Lane 4, in vitro transcribed RNA subjected to dual treatment, i.e. RppH treatment prior to VCE treatment. Lane 5, in vitro transcribed RNA subjected to vaccinia capping enzyme (VCE) treatment. The reaction products were analysed on a 12% non-denaturing polyacrylamide gel and detected by a fluorescence image analyser (FUJIFILM, FLA-5100).

    Journal: Nucleic Acids Research

    Article Title: Capping-RACE: a simple, accurate, and sensitive 5′ RACE method for use in prokaryotes

    doi: 10.1093/nar/gky739

    Figure Lengend Snippet: Experimental verification of Capping-RACE with in vitro transcribed RNA. ( A ) Different treatments of in vitro transcribed RNA. ( B ) The cDNA products from the differently treated RNA described in (A). Lane 1, the negative control, performed without adding reverse transcriptase (RT). Lane 2, in vitro transcribed RNA that was not subjected to any treatment. Lane 3, in vitro transcribed RNA subjected to RppH treatment. Lane 4, in vitro transcribed RNA subjected to dual treatment, i.e. RppH treatment prior to VCE treatment. Lane 5, in vitro transcribed RNA subjected to vaccinia capping enzyme (VCE) treatment. The reaction products were analysed on a 12% non-denaturing polyacrylamide gel and detected by a fluorescence image analyser (FUJIFILM, FLA-5100).

    Article Snippet: Different treatments of the 111 nt in vitro transcribed RNA The 111 nt in vitro transcribed RNA was pre-treated to modify the 5′ ends as follows: (i) 1 μg of RNA was incubated with 1 μl of RNA 5′ pyrophosphohydrolase (RppH) (NEB, Ipswich, MA, USA) for 1 h at 37°C; (ii) 1 μg of RNA was incubated with 1 μl of VCE (NEB) for 30 min at 37°C; or (iii) 1 μg of RNA was treated with RppH for 1 h at 37°C and purified by ethanol precipitation, followed by incubation with VCE for 30 min at 37°C.

    Techniques: In Vitro, Negative Control, Fluorescence

    ERCC reads to determine library preparation quality and back-calculate RNA input mass. (A) After normalizing to RNA input mass, reads aligning to the 92 ERCC spike-in transcripts correlate linearly with ERCC spike-in concentration across six orders of magnitude in all libraries prepared with the miniaturized protocol (R 2 E R C C m a s s ( p g ) T o t a l m a s s ( p g ) = E R C C r e a d s T o t a l r e a d s Back-calculated masses of HeLa libraries correlated strongly with QuBit quantification (R 2 = 0.9954).

    Journal: PLoS ONE

    Article Title: Miniaturization and optimization of 384-well compatible RNA sequencing library preparation

    doi: 10.1371/journal.pone.0206194

    Figure Lengend Snippet: ERCC reads to determine library preparation quality and back-calculate RNA input mass. (A) After normalizing to RNA input mass, reads aligning to the 92 ERCC spike-in transcripts correlate linearly with ERCC spike-in concentration across six orders of magnitude in all libraries prepared with the miniaturized protocol (R 2 E R C C m a s s ( p g ) T o t a l m a s s ( p g ) = E R C C r e a d s T o t a l r e a d s Back-calculated masses of HeLa libraries correlated strongly with QuBit quantification (R 2 = 0.9954).

    Article Snippet: To optimize miniaturization of our laboratory’s current library preparation protocol, we prepared libraries from varying concentrations of HeLa RNA using the New England Biolabs Ultra II RNA Library Prep Kit (E7770S/L).

    Techniques: Concentration Assay

    HeLa transcriptome coverage is comparable in full volume and miniaturized volume preparations. Rank-rank plots of the human transcriptome show strong correlation between the full-volume hand prepared protocol and the miniaturized, automated protocol for both (A) 1ng and (B) 5ng of HeLa RNA input (5ng RNA input: Spearman’s ρ = 0.79, p

    Journal: PLoS ONE

    Article Title: Miniaturization and optimization of 384-well compatible RNA sequencing library preparation

    doi: 10.1371/journal.pone.0206194

    Figure Lengend Snippet: HeLa transcriptome coverage is comparable in full volume and miniaturized volume preparations. Rank-rank plots of the human transcriptome show strong correlation between the full-volume hand prepared protocol and the miniaturized, automated protocol for both (A) 1ng and (B) 5ng of HeLa RNA input (5ng RNA input: Spearman’s ρ = 0.79, p

    Article Snippet: To optimize miniaturization of our laboratory’s current library preparation protocol, we prepared libraries from varying concentrations of HeLa RNA using the New England Biolabs Ultra II RNA Library Prep Kit (E7770S/L).

    Techniques:

    Dehydrated RNA demonstrates preserved integrity. Bioanalyzer traces and RNA Integrity Numbers (RINs) of biological replicates of HeLa RNA. (A) Before being dried in a vacuum evaporator. (B) After being dried for 30 minutes at 40°C. (C) After being dried for 25 minutes at 65°C. RINs indicate that RNA quality is not compromised during the dehydration process.

    Journal: PLoS ONE

    Article Title: Miniaturization and optimization of 384-well compatible RNA sequencing library preparation

    doi: 10.1371/journal.pone.0206194

    Figure Lengend Snippet: Dehydrated RNA demonstrates preserved integrity. Bioanalyzer traces and RNA Integrity Numbers (RINs) of biological replicates of HeLa RNA. (A) Before being dried in a vacuum evaporator. (B) After being dried for 30 minutes at 40°C. (C) After being dried for 25 minutes at 65°C. RINs indicate that RNA quality is not compromised during the dehydration process.

    Article Snippet: To optimize miniaturization of our laboratory’s current library preparation protocol, we prepared libraries from varying concentrations of HeLa RNA using the New England Biolabs Ultra II RNA Library Prep Kit (E7770S/L).

    Techniques:

    Final libraries produced by the full volume and miniaturized protocols have similar fragment distributions. (A) Bioanalyzer trace of a 5ng HeLa RNA final library prepared with the full volume protocol (average fragment size = 438bp, 95% between 200-1000bp). (B) Bioanalyzer trace of a 5ng HeLa RNA final library prepared with the miniaturized protocol (average fragment size = 457bp, 100% between 200-1000bp).

    Journal: PLoS ONE

    Article Title: Miniaturization and optimization of 384-well compatible RNA sequencing library preparation

    doi: 10.1371/journal.pone.0206194

    Figure Lengend Snippet: Final libraries produced by the full volume and miniaturized protocols have similar fragment distributions. (A) Bioanalyzer trace of a 5ng HeLa RNA final library prepared with the full volume protocol (average fragment size = 438bp, 95% between 200-1000bp). (B) Bioanalyzer trace of a 5ng HeLa RNA final library prepared with the miniaturized protocol (average fragment size = 457bp, 100% between 200-1000bp).

    Article Snippet: To optimize miniaturization of our laboratory’s current library preparation protocol, we prepared libraries from varying concentrations of HeLa RNA using the New England Biolabs Ultra II RNA Library Prep Kit (E7770S/L).

    Techniques: Produced