pnctr rna fragment  (New England Biolabs)


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    RNase Inhibitor Murine
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    RNase Inhibitor Murine 15 000 units
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    New England Biolabs pnctr rna fragment
    RNase Inhibitor Murine
    RNase Inhibitor Murine 15 000 units
    https://www.bioz.com/result/pnctr rna fragment/product/New England Biolabs
    Average 91 stars, based on 1265 article reviews
    Price from $9.99 to $1999.99
    pnctr rna fragment - by Bioz Stars, 2021-02
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    Images

    1) Product Images from "A Short Tandem Repeat-Enriched RNA Assembles a Nuclear Compartment to Control Alternative Splicing and Promote Cell Survival"

    Article Title: A Short Tandem Repeat-Enriched RNA Assembles a Nuclear Compartment to Control Alternative Splicing and Promote Cell Survival

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2018.08.041

    PNCTR Is a pol-I Transcript Interacting with Multiple Copies of PTBP1 Protein (A) Diagram of the predicted PNCTR locus also showing an adjacent 47S/45S rRNA gene and probes used in this study. Mapping to chr21 should be considered provisional since different IGS sequences share extensive regions of homology, and not all parts of human rDNA have been sequenced. (B) Top: northern blot analysis of PNCTR expression in HeLa cells using the probe introduced in (A). Bottom: methylene-blue-stained membrane showing that the lanes were loaded equally. (C) RIP carried out with a PTBP1-specific antibody or a non-immune IgG control. Immunoprecipitated RNAs were analyzed by qRT-PCR using primers specific to PNCTR, PTBP2 pre-mRNA (positive control), or U6 snRNA (negative control). Data are averaged from three experiments ± SD and compared by a two-tailed t test. (D) EMSA with purified PTBP1 protein and a PNCTR-specific RNA probe (sequence on the top). Bottom right: multivalent complexes assemble on incubating the probe with increasing amounts of PTBP1. Bottom left: no band shifts are detected when PTBP1 is substituted with BSA. (E) The PTBP1-PNCTR interaction in (D) is specific since it can be disrupted by increasing amounts of unlabeled PNCTR probe (bottom left), but not a control competitor (top, control RNA sequence; bottom right, the EMSA result). (F) IF-FISH staining of HeLa cells showing that PNCTR co-localizes with PTBP1 in the perinucleolar compartment (PNC). FBL, nucleolar marker fibrillarin. Scale bar, 10 μm. See also Figure S2 .
    Figure Legend Snippet: PNCTR Is a pol-I Transcript Interacting with Multiple Copies of PTBP1 Protein (A) Diagram of the predicted PNCTR locus also showing an adjacent 47S/45S rRNA gene and probes used in this study. Mapping to chr21 should be considered provisional since different IGS sequences share extensive regions of homology, and not all parts of human rDNA have been sequenced. (B) Top: northern blot analysis of PNCTR expression in HeLa cells using the probe introduced in (A). Bottom: methylene-blue-stained membrane showing that the lanes were loaded equally. (C) RIP carried out with a PTBP1-specific antibody or a non-immune IgG control. Immunoprecipitated RNAs were analyzed by qRT-PCR using primers specific to PNCTR, PTBP2 pre-mRNA (positive control), or U6 snRNA (negative control). Data are averaged from three experiments ± SD and compared by a two-tailed t test. (D) EMSA with purified PTBP1 protein and a PNCTR-specific RNA probe (sequence on the top). Bottom right: multivalent complexes assemble on incubating the probe with increasing amounts of PTBP1. Bottom left: no band shifts are detected when PTBP1 is substituted with BSA. (E) The PTBP1-PNCTR interaction in (D) is specific since it can be disrupted by increasing amounts of unlabeled PNCTR probe (bottom left), but not a control competitor (top, control RNA sequence; bottom right, the EMSA result). (F) IF-FISH staining of HeLa cells showing that PNCTR co-localizes with PTBP1 in the perinucleolar compartment (PNC). FBL, nucleolar marker fibrillarin. Scale bar, 10 μm. See also Figure S2 .

    Techniques Used: Northern Blot, Expressing, Staining, Immunoprecipitation, Quantitative RT-PCR, Positive Control, Negative Control, Two Tailed Test, Purification, Sequencing, Fluorescence In Situ Hybridization, Marker

    PNCTR Is Often Upregulated in Cancer Cells (A) qRT-PCR analyses showing that PNCTR expression is orders of magnitude higher in transformed cells (HeLa, HCT116, SW620, MCF7, and the SV40-transformed clone VA-13 of the normal lung fibroblast line WI-38) than in their non-transformed counterparts (ARPE-19 and WI-38). The data are averaged from 3 assays ± SD and the expression levels in WI-38 cells were set to 1. (B) qRT-PCR analyses carried out with and without reverse transcriptase (RT) show that the PNCTR signals in ARPE-19 and WI-38 correspond to bona fide expression of this strRNA at low but detectable levels. (C) qRT-PCR comparison of PNCTR expression in five invasive lung cancers and patient-matched normal lung samples ( Table S3 ). Data were obtained using RqP1 primers, normalized to β-actin, averaged from 3 amplification experiments ± SD and compared by a two-tailed t test. (D) PNCTR-positive nuclear dots are readily detectable by RNA-FISH in a lymph node metastasis sample collected for the case (a) in (C), but not in the matching normal lung control. (E) A close up of the boxed area in (D). Scale bars in (D) and (E), 10 μm. See also Figure S7 .
    Figure Legend Snippet: PNCTR Is Often Upregulated in Cancer Cells (A) qRT-PCR analyses showing that PNCTR expression is orders of magnitude higher in transformed cells (HeLa, HCT116, SW620, MCF7, and the SV40-transformed clone VA-13 of the normal lung fibroblast line WI-38) than in their non-transformed counterparts (ARPE-19 and WI-38). The data are averaged from 3 assays ± SD and the expression levels in WI-38 cells were set to 1. (B) qRT-PCR analyses carried out with and without reverse transcriptase (RT) show that the PNCTR signals in ARPE-19 and WI-38 correspond to bona fide expression of this strRNA at low but detectable levels. (C) qRT-PCR comparison of PNCTR expression in five invasive lung cancers and patient-matched normal lung samples ( Table S3 ). Data were obtained using RqP1 primers, normalized to β-actin, averaged from 3 amplification experiments ± SD and compared by a two-tailed t test. (D) PNCTR-positive nuclear dots are readily detectable by RNA-FISH in a lymph node metastasis sample collected for the case (a) in (C), but not in the matching normal lung control. (E) A close up of the boxed area in (D). Scale bars in (D) and (E), 10 μm. See also Figure S7 .

    Techniques Used: Quantitative RT-PCR, Expressing, Transformation Assay, Amplification, Two Tailed Test, Fluorescence In Situ Hybridization

    PNCTR Antagonizes Splicing Regulation Function of PTBP1 (A) RNA-seq analyses carried out to examine possible role of PNCTR in modulating PTBP1 activity as a regulator of alternative splicing. (B–E) Fisher’s exact tests showing that gmPNCTR-regulated alternative splicing events are significantly enriched among those regulated by (B) siPTBP1 or (C) siPTBP1/2, as compared to their occurrence in the entire list of alternative splicing events (Total) considered by ExpressionPlot. Note that alternative events controlled by both (D) gmPNCTR and siPTBP1 or (E) gmPNCTR and siPTBP1/2 are preferentially regulated in opposite directions (anti-regulated) rather than the same direction (co-regulated). (F) Regulation of CHEK2 exon 8 splicing by the PNCTR/PTBP1 circuitry. Left: the two alternative splicing possibilities. Right: RT-PCR analyses of HeLa cells showing that combined knockdown of PTBP1 and PTBP2 (siPTBP1/2) stimulates exon 8 inclusion, while knockdown of PNCTR (gmPNCTR, 400 nM) or overexpression of recombinant FLAG-tagged PTBP1 promotes its skipping. (G) Effects in (F) presented as differences in percent-spliced-in values (ΔΨ; Wang et al., 2008b ) between experimental treatments and the corresponding controls. Positive ΔΨ values indicate an increase and negative, a decrease in exon 8 inclusion. Similar quantifications were also done for cells transfected with 25 and 100 nM gapmers. All data are averaged from 3 experimentally independent comparisons ± SD and analyzed by a paired t test. (H) CLIP-seq and iCLIP analyses show that PTBP1 forms physical contacts with an extensive array of YUCUYY and YYUCUY motifs in front of CHEK2 exon 8. Functional significance of the PTBP1 interaction sequence highlighted in gray was validated in the minigene experiment in Figures S6 L and S6M. (I) CLIP/qRT-PCR experiment showing an increase in PTBP1 interaction efficiency with the CHEK2 exon 8 region (CLIP-CHEK2e8) in HeLa cells treated with gmPNCTR and a lack of this effect for an upstream (CLIP-CHEK2e2) and a downstream region (CLIP-CHEK2i8). PTBP2 pre-mRNA exon 10 region (CLIP-PTBP2e10) and ACTB mRNA are used as a positive and a negative control, respectively. Data are averaged from two triplicated CLIP/qRT-PCR experiments ± SD and compared by a two-tailed t test. (J) Left: immunoblot analysis showing a decrease in the CHEK2 protein levels in HeLa cells transfected for 24 hr with gmPNCTR compared to gmControl. CC3 is used as a sample identity marker and GAPDH as a lane-loading control. Right: immunoblot quantification showing GAPDH-normalized CHEK2 expression levels averaged from 3 experiments ± SD and compared by paired t test. (K) Left: HeLa cells treated with 50 nM of either siControl or siCHEK2 for 36 hr were post-transfected with 400 nM of gmPNCTR or gmControl for 12 hr and analyzed for CC3 expression. Note that the preemptive knockdown of CHEK2 facilitates induction of CC3 in the gmPNCTR samples. Right: GAPDH-normalized CC3 expression averaged from 6 experiments ± SD and compared by paired t test. (L) Expression of recombinant PTBP1 is sufficient to upregulate CC3 in HeLa cells. Left: immunoblot analysis of control and FLAG-PTBP1-transfected samples. Right: GAPDH-normalized CC3 expression averaged from 3 experiments ± SD and compared by paired t test. See also Figures S5 and S6 and Table S2 .
    Figure Legend Snippet: PNCTR Antagonizes Splicing Regulation Function of PTBP1 (A) RNA-seq analyses carried out to examine possible role of PNCTR in modulating PTBP1 activity as a regulator of alternative splicing. (B–E) Fisher’s exact tests showing that gmPNCTR-regulated alternative splicing events are significantly enriched among those regulated by (B) siPTBP1 or (C) siPTBP1/2, as compared to their occurrence in the entire list of alternative splicing events (Total) considered by ExpressionPlot. Note that alternative events controlled by both (D) gmPNCTR and siPTBP1 or (E) gmPNCTR and siPTBP1/2 are preferentially regulated in opposite directions (anti-regulated) rather than the same direction (co-regulated). (F) Regulation of CHEK2 exon 8 splicing by the PNCTR/PTBP1 circuitry. Left: the two alternative splicing possibilities. Right: RT-PCR analyses of HeLa cells showing that combined knockdown of PTBP1 and PTBP2 (siPTBP1/2) stimulates exon 8 inclusion, while knockdown of PNCTR (gmPNCTR, 400 nM) or overexpression of recombinant FLAG-tagged PTBP1 promotes its skipping. (G) Effects in (F) presented as differences in percent-spliced-in values (ΔΨ; Wang et al., 2008b ) between experimental treatments and the corresponding controls. Positive ΔΨ values indicate an increase and negative, a decrease in exon 8 inclusion. Similar quantifications were also done for cells transfected with 25 and 100 nM gapmers. All data are averaged from 3 experimentally independent comparisons ± SD and analyzed by a paired t test. (H) CLIP-seq and iCLIP analyses show that PTBP1 forms physical contacts with an extensive array of YUCUYY and YYUCUY motifs in front of CHEK2 exon 8. Functional significance of the PTBP1 interaction sequence highlighted in gray was validated in the minigene experiment in Figures S6 L and S6M. (I) CLIP/qRT-PCR experiment showing an increase in PTBP1 interaction efficiency with the CHEK2 exon 8 region (CLIP-CHEK2e8) in HeLa cells treated with gmPNCTR and a lack of this effect for an upstream (CLIP-CHEK2e2) and a downstream region (CLIP-CHEK2i8). PTBP2 pre-mRNA exon 10 region (CLIP-PTBP2e10) and ACTB mRNA are used as a positive and a negative control, respectively. Data are averaged from two triplicated CLIP/qRT-PCR experiments ± SD and compared by a two-tailed t test. (J) Left: immunoblot analysis showing a decrease in the CHEK2 protein levels in HeLa cells transfected for 24 hr with gmPNCTR compared to gmControl. CC3 is used as a sample identity marker and GAPDH as a lane-loading control. Right: immunoblot quantification showing GAPDH-normalized CHEK2 expression levels averaged from 3 experiments ± SD and compared by paired t test. (K) Left: HeLa cells treated with 50 nM of either siControl or siCHEK2 for 36 hr were post-transfected with 400 nM of gmPNCTR or gmControl for 12 hr and analyzed for CC3 expression. Note that the preemptive knockdown of CHEK2 facilitates induction of CC3 in the gmPNCTR samples. Right: GAPDH-normalized CC3 expression averaged from 6 experiments ± SD and compared by paired t test. (L) Expression of recombinant PTBP1 is sufficient to upregulate CC3 in HeLa cells. Left: immunoblot analysis of control and FLAG-PTBP1-transfected samples. Right: GAPDH-normalized CC3 expression averaged from 3 experiments ± SD and compared by paired t test. See also Figures S5 and S6 and Table S2 .

    Techniques Used: RNA Sequencing Assay, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Over Expression, Recombinant, Transfection, Cross-linking Immunoprecipitation, Functional Assay, Sequencing, Quantitative RT-PCR, Negative Control, Two Tailed Test, Marker, Expressing

    2) 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

    3) Product Images from "RNA targeting with CRISPR-Cas13a"

    Article Title: RNA targeting with CRISPR-Cas13a

    Journal: Nature

    doi: 10.1038/nature24049

    Biochemical characterization of LwaCas13a RNA cleavage activity a, LwaCas13a has more active RNAse activity than LshCas13a. b, Gel electrophoresis of ssRNA1 after incubation with LwaCas13a and with and without crRNA 1 for varying amounts of times. c, Gel electrophoresis of ssRNA1 after incubation with varying amounts of LwaCas13a-crRNA complex. d, Sequence and structure of ssRNA 4 and ssRNA 5. crRNA spacer sequence is highlighted in blue. e, Gel electrophoresis of ssRNA 4 and ssRNA 5 after incubation with LwaCas13a and crRNA 1. f, Sequence and structure of ssRNA 4 with sites of poly-x modifications highlighted in red. crRNA spacer sequence is highlighted in blue. g, Gel electrophoresis of ssRNA 4 with each of 4 possible poly-x modifications incubated with LwaCas13a and crRNA 1. h, LwaCas13a can process pre-crRNA from the L. wadei CRISPR-Cas locus. i, Cleavage efficiency of ssRNA 1 for crRNA spacer truncations after incubation with LwaCas13a.
    Figure Legend Snippet: Biochemical characterization of LwaCas13a RNA cleavage activity a, LwaCas13a has more active RNAse activity than LshCas13a. b, Gel electrophoresis of ssRNA1 after incubation with LwaCas13a and with and without crRNA 1 for varying amounts of times. c, Gel electrophoresis of ssRNA1 after incubation with varying amounts of LwaCas13a-crRNA complex. d, Sequence and structure of ssRNA 4 and ssRNA 5. crRNA spacer sequence is highlighted in blue. e, Gel electrophoresis of ssRNA 4 and ssRNA 5 after incubation with LwaCas13a and crRNA 1. f, Sequence and structure of ssRNA 4 with sites of poly-x modifications highlighted in red. crRNA spacer sequence is highlighted in blue. g, Gel electrophoresis of ssRNA 4 with each of 4 possible poly-x modifications incubated with LwaCas13a and crRNA 1. h, LwaCas13a can process pre-crRNA from the L. wadei CRISPR-Cas locus. i, Cleavage efficiency of ssRNA 1 for crRNA spacer truncations after incubation with LwaCas13a.

    Techniques Used: Activity Assay, Nucleic Acid Electrophoresis, Incubation, Sequencing, CRISPR

    4) Product Images from "A low-bias and sensitive small RNA library preparation method using randomized splint ligation"

    Article Title: A low-bias and sensitive small RNA library preparation method using randomized splint ligation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa480

    Schematic of randomized splint ligation library preparation. First the preadenylated 3′ adapter is ligated on using randomized splint ligation. Following adapter ligation, the excess adapter is depleted using 5′ deadenylase and lambda exonuclease, and the degenerate portion of the adapter is cleaved off by excising the deoxyuracil using USER. Next the 5′ adapter is ligated on using randomized splint ligation and cDNA is synthesized using the remaining portion of the 3′ adapter splint strand as a primer for the reverse transcription. Finally, library molecules containing both adapters are enriched and extended using PCR.
    Figure Legend Snippet: Schematic of randomized splint ligation library preparation. First the preadenylated 3′ adapter is ligated on using randomized splint ligation. Following adapter ligation, the excess adapter is depleted using 5′ deadenylase and lambda exonuclease, and the degenerate portion of the adapter is cleaved off by excising the deoxyuracil using USER. Next the 5′ adapter is ligated on using randomized splint ligation and cDNA is synthesized using the remaining portion of the 3′ adapter splint strand as a primer for the reverse transcription. Finally, library molecules containing both adapters are enriched and extended using PCR.

    Techniques Used: Ligation, Synthesized, Polymerase Chain Reaction

    5) Product Images from "Dicer cleaves 5′-extended microRNA precursors originating from RNA polymerase II transcription start sites"

    Article Title: Dicer cleaves 5′-extended microRNA precursors originating from RNA polymerase II transcription start sites

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky306

    Processing of 5′-extended pre-miRNAs is independent of Dicer 5′ pocket. ( A ) In vitro transcribed 5′-extended pre-miR-HSUR4s were incubated with four different human Dicers purified by Flag IP. [WT: wild type Dicer; TN: transdominant negative mutation Dicer; 3′ mut: 3′ pocket mutation Dicer (Y926A); 5′ mut: 5′ pocket mutation Dicer (R778A/R780A/H982A)]. Northern blots were performed using probes targeting miR-HSUR4-3p. ( B ) Quantitations of relative mature miRNA levels (mature miRNA/pre-miRNA) compared to WT Dicer processing in (A) (mean ± standard deviation) were derived from three independent experiments. FC: fold change. ( C ) In vitro transcribed 5′-monophosphate pre-miR-HSUR4s without 5′ extension (–2 nt) but with 1 nt or 2 nt 3′ overhang were incubated with four different human Dicers purified by Flag IP. Schematics of pre-miR-HSUR4s with 1 nt or 2 nt 3′ overhang are given. ( D ) Quantitations of relative mature miRNA levels compared to WT Dicer processing in (C) were derived from three independent experiments. ( E ) In vitro transcribed 5′-monophosphate pre-let-7a-1 and m 7 G-capped +15 nt extended pre-let-7a-1 were incubated with four different human Dicers purified by Flag IP. Northern blots were performed using probes targeting let-7a-1 (5p). Schematic of +15 nt extended pre-let-7a-1 is shown, with extended sequence underlined. ( F ) Quantitations of relative mature miRNA levels compared to WT Dicer processing in (E) were derived from three independent experiments.
    Figure Legend Snippet: Processing of 5′-extended pre-miRNAs is independent of Dicer 5′ pocket. ( A ) In vitro transcribed 5′-extended pre-miR-HSUR4s were incubated with four different human Dicers purified by Flag IP. [WT: wild type Dicer; TN: transdominant negative mutation Dicer; 3′ mut: 3′ pocket mutation Dicer (Y926A); 5′ mut: 5′ pocket mutation Dicer (R778A/R780A/H982A)]. Northern blots were performed using probes targeting miR-HSUR4-3p. ( B ) Quantitations of relative mature miRNA levels (mature miRNA/pre-miRNA) compared to WT Dicer processing in (A) (mean ± standard deviation) were derived from three independent experiments. FC: fold change. ( C ) In vitro transcribed 5′-monophosphate pre-miR-HSUR4s without 5′ extension (–2 nt) but with 1 nt or 2 nt 3′ overhang were incubated with four different human Dicers purified by Flag IP. Schematics of pre-miR-HSUR4s with 1 nt or 2 nt 3′ overhang are given. ( D ) Quantitations of relative mature miRNA levels compared to WT Dicer processing in (C) were derived from three independent experiments. ( E ) In vitro transcribed 5′-monophosphate pre-let-7a-1 and m 7 G-capped +15 nt extended pre-let-7a-1 were incubated with four different human Dicers purified by Flag IP. Northern blots were performed using probes targeting let-7a-1 (5p). Schematic of +15 nt extended pre-let-7a-1 is shown, with extended sequence underlined. ( F ) Quantitations of relative mature miRNA levels compared to WT Dicer processing in (E) were derived from three independent experiments.

    Techniques Used: In Vitro, Incubation, Purification, Mutagenesis, Northern Blot, Standard Deviation, Derivative Assay, Sequencing

    6) Product Images from "Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome"

    Article Title: Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190177

    Effect of the growth parameters on RiCF. (A) Schematics of a hypothetical scenario when RNA inhibits NAPs that could potentially cleave DNA. During lysis, quick RNA degradation removes the inhibition resulting in breakage of chromosomes. (B) Growth phase dependence of RiCF. AB1157 was grown at 37°C with periodic OD measurements, and samples for plugs were withdrawn at various times. The cells were made into plugs using lysis agarose and RNase (50 μg/plug) and the plugs were lysed and electrophoresed under standard conditions. Data points are means of at least three independent assays ± SEM. (C) Effect of translation and transcription inhibition on RiCF. Cells were grown till OD 0.5–0.6, split into three parts and chloramphenicol (40 μg/ml) or rifampicin (150 μg/ml) were added to two samples. All sample were shaken for another 2–3 hours at 37°C before making plugs as described in (B). Data points are means of four independent assays ± SEM. (D) Growth in minimal medium reduces RiCF. Cells were grown in LB or MOPS till the OD reached 0.6 and made into plugs using standard conditions. The values presented are means of six independent assays ± SEM. (E) Effect of growth temperature on RNase-induced chromosomal fragmentation. Cultures of AB1157 were grown at 20°C, 30°C, 37°C, 42°C or 45°C to same cell densities (A 600 = 0.6), and plugs were made in lysis agarose with RNAse A (50 μg/plug), as described in (A). Data are means of three to six independent measurements ± SEM.
    Figure Legend Snippet: Effect of the growth parameters on RiCF. (A) Schematics of a hypothetical scenario when RNA inhibits NAPs that could potentially cleave DNA. During lysis, quick RNA degradation removes the inhibition resulting in breakage of chromosomes. (B) Growth phase dependence of RiCF. AB1157 was grown at 37°C with periodic OD measurements, and samples for plugs were withdrawn at various times. The cells were made into plugs using lysis agarose and RNase (50 μg/plug) and the plugs were lysed and electrophoresed under standard conditions. Data points are means of at least three independent assays ± SEM. (C) Effect of translation and transcription inhibition on RiCF. Cells were grown till OD 0.5–0.6, split into three parts and chloramphenicol (40 μg/ml) or rifampicin (150 μg/ml) were added to two samples. All sample were shaken for another 2–3 hours at 37°C before making plugs as described in (B). Data points are means of four independent assays ± SEM. (D) Growth in minimal medium reduces RiCF. Cells were grown in LB or MOPS till the OD reached 0.6 and made into plugs using standard conditions. The values presented are means of six independent assays ± SEM. (E) Effect of growth temperature on RNase-induced chromosomal fragmentation. Cultures of AB1157 were grown at 20°C, 30°C, 37°C, 42°C or 45°C to same cell densities (A 600 = 0.6), and plugs were made in lysis agarose with RNAse A (50 μg/plug), as described in (A). Data are means of three to six independent measurements ± SEM.

    Techniques Used: Lysis, Inhibition

    RNA degradation causes chromosomal fragmentation. (A)  Schematics of a hypothetical scenario when RNA makes the central core of nucleoids, and its degradation results in collapse of the nucleoid structure, causing chromosomal fragmentation.  (B)  Radiogram of a pulsed field gel showing chromosomal fragmentation in AB1157 when cells were embedded in agarose plugs in the presence and absence of proteinase K (25 μg/plug) and/or RNase (50 μg/plug) and lysed overnight at 62°C.  (C)  Radiogram showing DNase I sensitivity of the signal entering the gel. Plugs were lysed at 62°C, washed extensively to remove traces of lysis buffer and then treated with DNase I at 37°C before PFGE.  (D)  A representative gel showing that RNA degradation by different enzymes causes chromosomal fragmentation. Plugs were made in the absence of proteinase K in 1x restriction enzyme buffer (NEBuffer 3 for RNase A, XRN-1 and RNAse I f  and NEBuffer 4 for Exo T). The concentrations of the enzymes used were, RNase, 50 μg/plug; XRN-1, 5 U/plug; RNAse I f , 100 U/plug and Exo T, 20 U/plug.  (E)  Quantification of the chromosomal fragmentation when plugs were made in the presence of various RNA degrading enzymes. The values presented are means of four independent assays ± SEM. CZ, compression zone.
    Figure Legend Snippet: RNA degradation causes chromosomal fragmentation. (A) Schematics of a hypothetical scenario when RNA makes the central core of nucleoids, and its degradation results in collapse of the nucleoid structure, causing chromosomal fragmentation. (B) Radiogram of a pulsed field gel showing chromosomal fragmentation in AB1157 when cells were embedded in agarose plugs in the presence and absence of proteinase K (25 μg/plug) and/or RNase (50 μg/plug) and lysed overnight at 62°C. (C) Radiogram showing DNase I sensitivity of the signal entering the gel. Plugs were lysed at 62°C, washed extensively to remove traces of lysis buffer and then treated with DNase I at 37°C before PFGE. (D) A representative gel showing that RNA degradation by different enzymes causes chromosomal fragmentation. Plugs were made in the absence of proteinase K in 1x restriction enzyme buffer (NEBuffer 3 for RNase A, XRN-1 and RNAse I f and NEBuffer 4 for Exo T). The concentrations of the enzymes used were, RNase, 50 μg/plug; XRN-1, 5 U/plug; RNAse I f , 100 U/plug and Exo T, 20 U/plug. (E) Quantification of the chromosomal fragmentation when plugs were made in the presence of various RNA degrading enzymes. The values presented are means of four independent assays ± SEM. CZ, compression zone.

    Techniques Used: Pulsed-Field Gel, Lysis

    7) Product Images from "eIF3d is an mRNA cap-binding protein required for specialized translation initiation"

    Article Title: eIF3d is an mRNA cap-binding protein required for specialized translation initiation

    Journal: Nature

    doi: 10.1038/nature18954

    eIF3d cap-binding activity is required for efficient 48S initiation complex formation on specific mRNAs a , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to eIF3 in the presence of competitor ligands. b , Electrostatic surface view of the eIF3d cap-binding domain colored by charge, with a zoomed view of single stranded RNA (ssRNA) and cap analog modeled according to their positions bound to DXO 15 . Positive charge is colored blue and negative charge is in red, and the RNA gate is removed for clarity. c , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to wild type or helix α5 or helix α11-mutant eIF3. eIF3d-helix α5 mutant (D249Q/V262I/Y263A), helix α11 mutant (T317E/N320E/H321A). WT, wild type. d , Incorporation of c-Jun and ACTB mRNA into initiation complexes by wild type, helix α5, or helix α11-mutant eIF3d as measured by quantitative RT-PCR. mRNA-ribosome association is expressed as the ratio between the quantity of mRNA transcripts to 18S rRNA and normalized to the wild type sample. The results are representative of three independent experiments and given as the mean ± s.d. from a representative quantitative RT-PCR experiment performed in duplicate.
    Figure Legend Snippet: eIF3d cap-binding activity is required for efficient 48S initiation complex formation on specific mRNAs a , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to eIF3 in the presence of competitor ligands. b , Electrostatic surface view of the eIF3d cap-binding domain colored by charge, with a zoomed view of single stranded RNA (ssRNA) and cap analog modeled according to their positions bound to DXO 15 . Positive charge is colored blue and negative charge is in red, and the RNA gate is removed for clarity. c , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to wild type or helix α5 or helix α11-mutant eIF3. eIF3d-helix α5 mutant (D249Q/V262I/Y263A), helix α11 mutant (T317E/N320E/H321A). WT, wild type. d , Incorporation of c-Jun and ACTB mRNA into initiation complexes by wild type, helix α5, or helix α11-mutant eIF3d as measured by quantitative RT-PCR. mRNA-ribosome association is expressed as the ratio between the quantity of mRNA transcripts to 18S rRNA and normalized to the wild type sample. The results are representative of three independent experiments and given as the mean ± s.d. from a representative quantitative RT-PCR experiment performed in duplicate.

    Techniques Used: Binding Assay, Activity Assay, SDS-Gel, Labeling, Mutagenesis, Quantitative RT-PCR

    eIF4E recognizes the 5′ end of the c-Jun mRNA less efficiently than ACTB mRNA a , Coomassie blue stained SDS gel of recombinant human eIF4E expressed in E. coli. b , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled ACTB or c-Jun 5′ UTR RNA crosslinked to eIF4E. The result is representative of three independent experiments. For gel source data, see Supplementary Figure 1 .
    Figure Legend Snippet: eIF4E recognizes the 5′ end of the c-Jun mRNA less efficiently than ACTB mRNA a , Coomassie blue stained SDS gel of recombinant human eIF4E expressed in E. coli. b , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled ACTB or c-Jun 5′ UTR RNA crosslinked to eIF4E. The result is representative of three independent experiments. For gel source data, see Supplementary Figure 1 .

    Techniques Used: Staining, SDS-Gel, Recombinant, Labeling

    5' end recognition of c-Jun mRNA is eIF4F-independent a , Distribution of c-Jun or ACTB mRNA-containing initiation complexes in programmed 293T cell in vitro translation extracts. The mRNA abundance (black line) is expressed as the fraction of total recovered transcripts. The results are given as the mean ± standard deviation (s.d.) of a representative quantitative RT-PCR experiment performed in duplicate. The polysome profile (gray line) is plotted as relative absorbance at 254 nm versus elution fractions. b , Western blot analysis of initiation factors in 48S translation complexes formed on c-Jun and ACTB mRNAs. 293T, total protein from 293T in vitro translation extracts. For gel source data, see Supplementary Figure 1 . c , Phosphorimage of SDS gel resolving RNase-protected 32 P-internal or 32 P-cap-labeled c-Jun 5' UTR RNA crosslinked to eIF3 subunits. Recombinant eIF3a migrates at ~100 kDa due to a C-terminal truncation 26 . The results of a - c are representative of three independent experiments.
    Figure Legend Snippet: 5' end recognition of c-Jun mRNA is eIF4F-independent a , Distribution of c-Jun or ACTB mRNA-containing initiation complexes in programmed 293T cell in vitro translation extracts. The mRNA abundance (black line) is expressed as the fraction of total recovered transcripts. The results are given as the mean ± standard deviation (s.d.) of a representative quantitative RT-PCR experiment performed in duplicate. The polysome profile (gray line) is plotted as relative absorbance at 254 nm versus elution fractions. b , Western blot analysis of initiation factors in 48S translation complexes formed on c-Jun and ACTB mRNAs. 293T, total protein from 293T in vitro translation extracts. For gel source data, see Supplementary Figure 1 . c , Phosphorimage of SDS gel resolving RNase-protected 32 P-internal or 32 P-cap-labeled c-Jun 5' UTR RNA crosslinked to eIF3 subunits. Recombinant eIF3a migrates at ~100 kDa due to a C-terminal truncation 26 . The results of a - c are representative of three independent experiments.

    Techniques Used: In Vitro, Standard Deviation, Quantitative RT-PCR, Western Blot, SDS-Gel, Labeling, Recombinant

    8) Product Images from "A Short Tandem Repeat-Enriched RNA Assembles a Nuclear Compartment to Control Alternative Splicing and Promote Cell Survival"

    Article Title: A Short Tandem Repeat-Enriched RNA Assembles a Nuclear Compartment to Control Alternative Splicing and Promote Cell Survival

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2018.08.041

    PNCTR Is a pol-I Transcript Interacting with Multiple Copies of PTBP1 Protein (A) Diagram of the predicted PNCTR locus also showing an adjacent 47S/45S rRNA gene and probes used in this study. Mapping to chr21 should be considered provisional since different IGS sequences share extensive regions of homology, and not all parts of human rDNA have been sequenced. (B) Top: northern blot analysis of PNCTR expression in HeLa cells using the probe introduced in (A). Bottom: methylene-blue-stained membrane showing that the lanes were loaded equally. (C) RIP carried out with a PTBP1-specific antibody or a non-immune IgG control. Immunoprecipitated RNAs were analyzed by qRT-PCR using primers specific to PNCTR, PTBP2 pre-mRNA (positive control), or U6 snRNA (negative control). Data are averaged from three experiments ± SD and compared by a two-tailed t test. (D) EMSA with purified PTBP1 protein and a PNCTR-specific RNA probe (sequence on the top). Bottom right: multivalent complexes assemble on incubating the probe with increasing amounts of PTBP1. Bottom left: no band shifts are detected when PTBP1 is substituted with BSA. (E) The PTBP1-PNCTR interaction in (D) is specific since it can be disrupted by increasing amounts of unlabeled PNCTR probe (bottom left), but not a control competitor (top, control RNA sequence; bottom right, the EMSA result). (F) IF-FISH staining of HeLa cells showing that PNCTR co-localizes with PTBP1 in the perinucleolar compartment (PNC). FBL, nucleolar marker fibrillarin. Scale bar, 10 μm. .
    Figure Legend Snippet: PNCTR Is a pol-I Transcript Interacting with Multiple Copies of PTBP1 Protein (A) Diagram of the predicted PNCTR locus also showing an adjacent 47S/45S rRNA gene and probes used in this study. Mapping to chr21 should be considered provisional since different IGS sequences share extensive regions of homology, and not all parts of human rDNA have been sequenced. (B) Top: northern blot analysis of PNCTR expression in HeLa cells using the probe introduced in (A). Bottom: methylene-blue-stained membrane showing that the lanes were loaded equally. (C) RIP carried out with a PTBP1-specific antibody or a non-immune IgG control. Immunoprecipitated RNAs were analyzed by qRT-PCR using primers specific to PNCTR, PTBP2 pre-mRNA (positive control), or U6 snRNA (negative control). Data are averaged from three experiments ± SD and compared by a two-tailed t test. (D) EMSA with purified PTBP1 protein and a PNCTR-specific RNA probe (sequence on the top). Bottom right: multivalent complexes assemble on incubating the probe with increasing amounts of PTBP1. Bottom left: no band shifts are detected when PTBP1 is substituted with BSA. (E) The PTBP1-PNCTR interaction in (D) is specific since it can be disrupted by increasing amounts of unlabeled PNCTR probe (bottom left), but not a control competitor (top, control RNA sequence; bottom right, the EMSA result). (F) IF-FISH staining of HeLa cells showing that PNCTR co-localizes with PTBP1 in the perinucleolar compartment (PNC). FBL, nucleolar marker fibrillarin. Scale bar, 10 μm. .

    Techniques Used: Northern Blot, Expressing, Staining, Immunoprecipitation, Quantitative RT-PCR, Positive Control, Negative Control, Two Tailed Test, Purification, Sequencing, Fluorescence In Situ Hybridization, Marker

    PNCTR Is Often Upregulated in Cancer Cells (A) qRT-PCR analyses showing that PNCTR expression is orders of magnitude higher in transformed cells (HeLa, HCT116, SW620, MCF7, and the SV40-transformed clone VA-13 of the normal lung fibroblast line WI-38) than in their non-transformed counterparts (ARPE-19 and WI-38). The data are averaged from 3 assays ± SD and the expression levels in WI-38 cells were set to 1. (B) qRT-PCR analyses carried out with and without reverse transcriptase (RT) show that the PNCTR signals in ARPE-19 and WI-38 correspond to bona fide expression of this strRNA at low but detectable levels. ). Data were obtained using RqP1 primers, normalized to β-actin, averaged from 3 amplification experiments ± SD and compared by a two-tailed t test. (D) PNCTR-positive nuclear dots are readily detectable by RNA-FISH in a lymph node metastasis sample collected for the case (a) in (C), but not in the matching normal lung control. (E) A close up of the boxed area in (D). Scale bars in (D) and (E), 10 μm. .
    Figure Legend Snippet: PNCTR Is Often Upregulated in Cancer Cells (A) qRT-PCR analyses showing that PNCTR expression is orders of magnitude higher in transformed cells (HeLa, HCT116, SW620, MCF7, and the SV40-transformed clone VA-13 of the normal lung fibroblast line WI-38) than in their non-transformed counterparts (ARPE-19 and WI-38). The data are averaged from 3 assays ± SD and the expression levels in WI-38 cells were set to 1. (B) qRT-PCR analyses carried out with and without reverse transcriptase (RT) show that the PNCTR signals in ARPE-19 and WI-38 correspond to bona fide expression of this strRNA at low but detectable levels. ). Data were obtained using RqP1 primers, normalized to β-actin, averaged from 3 amplification experiments ± SD and compared by a two-tailed t test. (D) PNCTR-positive nuclear dots are readily detectable by RNA-FISH in a lymph node metastasis sample collected for the case (a) in (C), but not in the matching normal lung control. (E) A close up of the boxed area in (D). Scale bars in (D) and (E), 10 μm. .

    Techniques Used: Quantitative RT-PCR, Expressing, Transformation Assay, Amplification, Two Tailed Test, Fluorescence In Situ Hybridization

    PNCTR Antagonizes Splicing Regulation Function of PTBP1 (A) RNA-seq analyses carried out to examine possible role of PNCTR in modulating PTBP1 activity as a regulator of alternative splicing. (B–E) Fisher’s exact tests showing that gmPNCTR-regulated alternative splicing events are significantly enriched among those regulated by (B) siPTBP1 or (C) siPTBP1/2, as compared to their occurrence in the entire list of alternative splicing events (Total) considered by ExpressionPlot. Note that alternative events controlled by both (D) gmPNCTR and siPTBP1 or (E) gmPNCTR and siPTBP1/2 are preferentially regulated in opposite directions (anti-regulated) rather than the same direction (co-regulated). (F) Regulation of CHEK2 exon 8 splicing by the PNCTR/PTBP1 circuitry. Left: the two alternative splicing possibilities. Right: RT-PCR analyses of HeLa cells showing that combined knockdown of PTBP1 and PTBP2 (siPTBP1/2) stimulates exon 8 inclusion, while knockdown of PNCTR (gmPNCTR, 400 nM) or overexpression of recombinant FLAG-tagged PTBP1 promotes its skipping. ) between experimental treatments and the corresponding controls. Positive ΔΨ values indicate an increase and negative, a decrease in exon 8 inclusion. Similar quantifications were also done for cells transfected with 25 and 100 nM gapmers. All data are averaged from 3 experimentally independent comparisons ± SD and analyzed by a paired t test. L and S6M. (I) CLIP/qRT-PCR experiment showing an increase in PTBP1 interaction efficiency with the CHEK2 exon 8 region (CLIP-CHEK2e8) in HeLa cells treated with gmPNCTR and a lack of this effect for an upstream (CLIP-CHEK2e2) and a downstream region (CLIP-CHEK2i8). PTBP2 pre-mRNA exon 10 region (CLIP-PTBP2e10) and ACTB mRNA are used as a positive and a negative control, respectively. Data are averaged from two triplicated CLIP/qRT-PCR experiments ± SD and compared by a two-tailed t test. (J) Left: immunoblot analysis showing a decrease in the CHEK2 protein levels in HeLa cells transfected for 24 hr with gmPNCTR compared to gmControl. CC3 is used as a sample identity marker and GAPDH as a lane-loading control. Right: immunoblot quantification showing GAPDH-normalized CHEK2 expression levels averaged from 3 experiments ± SD and compared by paired t test. (K) Left: HeLa cells treated with 50 nM of either siControl or siCHEK2 for 36 hr were post-transfected with 400 nM of gmPNCTR or gmControl for 12 hr and analyzed for CC3 expression. Note that the preemptive knockdown of CHEK2 facilitates induction of CC3 in the gmPNCTR samples. Right: GAPDH-normalized CC3 expression averaged from 6 experiments ± SD and compared by paired t test. (L) Expression of recombinant PTBP1 is sufficient to upregulate CC3 in HeLa cells. Left: immunoblot analysis of control and FLAG-PTBP1-transfected samples. Right: GAPDH-normalized CC3 expression averaged from 3 experiments ± SD and compared by paired t test. .
    Figure Legend Snippet: PNCTR Antagonizes Splicing Regulation Function of PTBP1 (A) RNA-seq analyses carried out to examine possible role of PNCTR in modulating PTBP1 activity as a regulator of alternative splicing. (B–E) Fisher’s exact tests showing that gmPNCTR-regulated alternative splicing events are significantly enriched among those regulated by (B) siPTBP1 or (C) siPTBP1/2, as compared to their occurrence in the entire list of alternative splicing events (Total) considered by ExpressionPlot. Note that alternative events controlled by both (D) gmPNCTR and siPTBP1 or (E) gmPNCTR and siPTBP1/2 are preferentially regulated in opposite directions (anti-regulated) rather than the same direction (co-regulated). (F) Regulation of CHEK2 exon 8 splicing by the PNCTR/PTBP1 circuitry. Left: the two alternative splicing possibilities. Right: RT-PCR analyses of HeLa cells showing that combined knockdown of PTBP1 and PTBP2 (siPTBP1/2) stimulates exon 8 inclusion, while knockdown of PNCTR (gmPNCTR, 400 nM) or overexpression of recombinant FLAG-tagged PTBP1 promotes its skipping. ) between experimental treatments and the corresponding controls. Positive ΔΨ values indicate an increase and negative, a decrease in exon 8 inclusion. Similar quantifications were also done for cells transfected with 25 and 100 nM gapmers. All data are averaged from 3 experimentally independent comparisons ± SD and analyzed by a paired t test. L and S6M. (I) CLIP/qRT-PCR experiment showing an increase in PTBP1 interaction efficiency with the CHEK2 exon 8 region (CLIP-CHEK2e8) in HeLa cells treated with gmPNCTR and a lack of this effect for an upstream (CLIP-CHEK2e2) and a downstream region (CLIP-CHEK2i8). PTBP2 pre-mRNA exon 10 region (CLIP-PTBP2e10) and ACTB mRNA are used as a positive and a negative control, respectively. Data are averaged from two triplicated CLIP/qRT-PCR experiments ± SD and compared by a two-tailed t test. (J) Left: immunoblot analysis showing a decrease in the CHEK2 protein levels in HeLa cells transfected for 24 hr with gmPNCTR compared to gmControl. CC3 is used as a sample identity marker and GAPDH as a lane-loading control. Right: immunoblot quantification showing GAPDH-normalized CHEK2 expression levels averaged from 3 experiments ± SD and compared by paired t test. (K) Left: HeLa cells treated with 50 nM of either siControl or siCHEK2 for 36 hr were post-transfected with 400 nM of gmPNCTR or gmControl for 12 hr and analyzed for CC3 expression. Note that the preemptive knockdown of CHEK2 facilitates induction of CC3 in the gmPNCTR samples. Right: GAPDH-normalized CC3 expression averaged from 6 experiments ± SD and compared by paired t test. (L) Expression of recombinant PTBP1 is sufficient to upregulate CC3 in HeLa cells. Left: immunoblot analysis of control and FLAG-PTBP1-transfected samples. Right: GAPDH-normalized CC3 expression averaged from 3 experiments ± SD and compared by paired t test. .

    Techniques Used: RNA Sequencing Assay, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Over Expression, Recombinant, Transfection, Cross-linking Immunoprecipitation, Quantitative RT-PCR, Negative Control, Two Tailed Test, Marker, Expressing

    9) Product Images from "SPRINT: a Cas13a-based platform for detection of small molecules"

    Article Title: SPRINT: a Cas13a-based platform for detection of small molecules

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa673

    Transcription and nuclease activities constitute SPRINT assays. ( A ) Certain allosteric transcription factors release their DNA binding site when they are bound by ligand, therefore enabling transcript elongation. ( B ) An ON-riboswitch allows transcript elongation by RNA polymerase when the riboswitch is bound to its cognate ligand. ( C ) Cas13a detects RNA transcripts that contain the target sequence (yellow). After binding its target, Cas13a collaterally cleaves RNA oligonucleotides and de-quenches fluorophores. ( D ) Workflow of a typical SPRINT experiment. A master mix containing components such as RNA polymerase, Cas13a and DNA template is incubated and then added to wells that contain compounds that regulate transcription. The transcriptional output is then measured via the fluorescence signal.
    Figure Legend Snippet: Transcription and nuclease activities constitute SPRINT assays. ( A ) Certain allosteric transcription factors release their DNA binding site when they are bound by ligand, therefore enabling transcript elongation. ( B ) An ON-riboswitch allows transcript elongation by RNA polymerase when the riboswitch is bound to its cognate ligand. ( C ) Cas13a detects RNA transcripts that contain the target sequence (yellow). After binding its target, Cas13a collaterally cleaves RNA oligonucleotides and de-quenches fluorophores. ( D ) Workflow of a typical SPRINT experiment. A master mix containing components such as RNA polymerase, Cas13a and DNA template is incubated and then added to wells that contain compounds that regulate transcription. The transcriptional output is then measured via the fluorescence signal.

    Techniques Used: Binding Assay, Sequencing, Incubation, Fluorescence

    10) Product Images from "Synthesis of low immunogenicity RNA with high-temperature in vitro transcription"

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    Journal: RNA

    doi: 10.1261/rna.073858.119

    High-temperature IVT does not affect antisense dsRNA by-product formation. ( A ) Native gel electrophoresis analysis of IVT reactions on 512B DNA template using wild-type T7 (37°C) with/without RNase III treatment. ( B ) dsRNA immunoblot with J2 antibody on IVT reactions (crude and purified) with 512B template. ( C ) Native gel electrophoresis analyses and dsRNA immunoblot analysis of 512B IVT reactions conducted with TsT7-1 at 37°C versus 50°C.
    Figure Legend Snippet: High-temperature IVT does not affect antisense dsRNA by-product formation. ( A ) Native gel electrophoresis analysis of IVT reactions on 512B DNA template using wild-type T7 (37°C) with/without RNase III treatment. ( B ) dsRNA immunoblot with J2 antibody on IVT reactions (crude and purified) with 512B template. ( C ) Native gel electrophoresis analyses and dsRNA immunoblot analysis of 512B IVT reactions conducted with TsT7-1 at 37°C versus 50°C.

    Techniques Used: Nucleic Acid Electrophoresis, Purification

    11) Product Images from "Polyvinylsulfonic acid: A Low-cost RNase inhibitor for enhanced RNA preservation and cell-free protein translation"

    Article Title: Polyvinylsulfonic acid: A Low-cost RNase inhibitor for enhanced RNA preservation and cell-free protein translation

    Journal: Bioengineered

    doi: 10.1080/21655979.2017.1313648

    Inhibition of RNase Activity with PVSA. The relative RNase Activity of both RNase A and E. coli lysate was measured at varying concentrations of PVSA using RNaseAlert® (Ambion). The amount of PVSA required for 50% inhibition (IC 50 , inset) was determined from normalized data fit to a reciprocal semi-log response curve (n = 3, error bars represent 1 standard deviation).
    Figure Legend Snippet: Inhibition of RNase Activity with PVSA. The relative RNase Activity of both RNase A and E. coli lysate was measured at varying concentrations of PVSA using RNaseAlert® (Ambion). The amount of PVSA required for 50% inhibition (IC 50 , inset) was determined from normalized data fit to a reciprocal semi-log response curve (n = 3, error bars represent 1 standard deviation).

    Techniques Used: Inhibition, Activity Assay, Standard Deviation

    12) Product Images from "RNA binding to CBP stimulates histone acetylation and transcription"

    Article Title: RNA binding to CBP stimulates histone acetylation and transcription

    Journal: Cell

    doi: 10.1016/j.cell.2016.12.020

    CBP interacts with RNA in vivo A) Native RNA-IP of CBP. Top, RNA immunprecipitated with CBP. Bottom, CBP western blot. B) PAR-CLIP protocol. 4-Thiouridine (4-SU). C) CBP PAR-CLIP required 4-SU: top, autoradiography; bottom, CBP western blot.. D) Quantification of CBP PAR-CLIP. Error bars represent mean +/− s.e.m; n=4. E) CBP PAR-CLIP signal was sensitive to RNAse. 1× RNAse cocktail contained: RNAse A (0.01mU/ul) + RNase T1 (0.4mU/ul). F) Quantification of RNase titration. Error bars represent mean +/− s.e.m; n=4; P -values from two-tailed Student’s t-test: *P
    Figure Legend Snippet: CBP interacts with RNA in vivo A) Native RNA-IP of CBP. Top, RNA immunprecipitated with CBP. Bottom, CBP western blot. B) PAR-CLIP protocol. 4-Thiouridine (4-SU). C) CBP PAR-CLIP required 4-SU: top, autoradiography; bottom, CBP western blot.. D) Quantification of CBP PAR-CLIP. Error bars represent mean +/− s.e.m; n=4. E) CBP PAR-CLIP signal was sensitive to RNAse. 1× RNAse cocktail contained: RNAse A (0.01mU/ul) + RNase T1 (0.4mU/ul). F) Quantification of RNase titration. Error bars represent mean +/− s.e.m; n=4; P -values from two-tailed Student’s t-test: *P

    Techniques Used: In Vivo, Western Blot, Cross-linking Immunoprecipitation, Autoradiography, Titration, Two Tailed Test

    13) Product Images from "A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries"

    Article Title: A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries

    Journal: Journal of Advanced Research

    doi: 10.1016/j.jare.2019.12.005

    The schematic representation of the workflow for the reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy. Total RNA (usually 0.5–1.0 µg) is incubated and hybridized with a panel of 30 (human mouse) rRNA-specific DNA oligo probes ( a ), followed by reverse transcription (RT) ( b ). After the removal of excess oligo probes with Exonuclease I ( c ), the resultant RT products are subjected to RNase H digestion to degrade the rRNA portions of the RNA:DNA hybrid ( d ), and then the DNA components are degraded by DNase I ( e ). The intact mRNAs and noncoding RNAs are subsequently purified by ethanol precipitation ( f ) and subjected to RNA-seq library construction ( g ). The locations and sequences of individual rRNA-specific probes are shown in Suppl. Fig. S2 and Suppl. Table S1.
    Figure Legend Snippet: The schematic representation of the workflow for the reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy. Total RNA (usually 0.5–1.0 µg) is incubated and hybridized with a panel of 30 (human mouse) rRNA-specific DNA oligo probes ( a ), followed by reverse transcription (RT) ( b ). After the removal of excess oligo probes with Exonuclease I ( c ), the resultant RT products are subjected to RNase H digestion to degrade the rRNA portions of the RNA:DNA hybrid ( d ), and then the DNA components are degraded by DNase I ( e ). The intact mRNAs and noncoding RNAs are subsequently purified by ethanol precipitation ( f ) and subjected to RNA-seq library construction ( g ). The locations and sequences of individual rRNA-specific probes are shown in Suppl. Fig. S2 and Suppl. Table S1.

    Techniques Used: Incubation, Purification, Ethanol Precipitation, RNA Sequencing Assay

    14) Product Images from "RNA promotes the formation of spatial compartments in the nucleus"

    Article Title: RNA promotes the formation of spatial compartments in the nucleus

    Journal: bioRxiv

    doi: 10.1101/2020.08.25.267435

    RNA localization on DNA and within the nucleus for RNAs within each RNA hub. (A) Weighted genomic DNA localization heatmap of each individual RNA. RNAs are organized by their RNA hub occupancy (shown in Figure 2A ). Contacts are normalized from 0 to 1 to account for expression levels of each RNA. (B) Pearson correlation of RNA-DNA unweighted contact frequencies across the genome for individual RNAs within the nuclear hubs (nucleolar, centromeric, spliceosomal, and scaRNA hubs). Red represents high correlation and blue represents low correlation. (C) RNA FISH of various non-coding RNAs within the same hub in the nucleus. Spliceosomal hub (top): Malat1 lncRNA and 7SK RNA and (bottom): U6 and U1 spliceosomal RNAs. Nucleolar hub (top): snora26 snoRNA and 45S pre-rRNA ITS2 and (bottom): RNase MRP (Rmrp) and 45S pre-rRNA ITS1. Each panel is shown individually (left and middle) and overlaid (right). Dashed lines demarcate the nuclear boundary identified with DAPI. Scalebar is 10μm. (D) RNA FISH (left) along with nucleolin immunofluorescence (middle) and DAPI (right) of specific ncRNAs. 7SK RNA (top), ITS1 regions of 45S pre-rRNA (middle) and tRNAs (bottom). tRNAs are visualized using pooled RNA FISH probes (see Methods). Scalebar is 10μm.
    Figure Legend Snippet: RNA localization on DNA and within the nucleus for RNAs within each RNA hub. (A) Weighted genomic DNA localization heatmap of each individual RNA. RNAs are organized by their RNA hub occupancy (shown in Figure 2A ). Contacts are normalized from 0 to 1 to account for expression levels of each RNA. (B) Pearson correlation of RNA-DNA unweighted contact frequencies across the genome for individual RNAs within the nuclear hubs (nucleolar, centromeric, spliceosomal, and scaRNA hubs). Red represents high correlation and blue represents low correlation. (C) RNA FISH of various non-coding RNAs within the same hub in the nucleus. Spliceosomal hub (top): Malat1 lncRNA and 7SK RNA and (bottom): U6 and U1 spliceosomal RNAs. Nucleolar hub (top): snora26 snoRNA and 45S pre-rRNA ITS2 and (bottom): RNase MRP (Rmrp) and 45S pre-rRNA ITS1. Each panel is shown individually (left and middle) and overlaid (right). Dashed lines demarcate the nuclear boundary identified with DAPI. Scalebar is 10μm. (D) RNA FISH (left) along with nucleolin immunofluorescence (middle) and DAPI (right) of specific ncRNAs. 7SK RNA (top), ITS1 regions of 45S pre-rRNA (middle) and tRNAs (bottom). tRNAs are visualized using pooled RNA FISH probes (see Methods). Scalebar is 10μm.

    Techniques Used: Expressing, Fluorescence In Situ Hybridization, Immunofluorescence

    15) Product Images from "A Short Tandem Repeat-Enriched RNA Assembles a Nuclear Compartment to Control Alternative Splicing and Promote Cell Survival"

    Article Title: A Short Tandem Repeat-Enriched RNA Assembles a Nuclear Compartment to Control Alternative Splicing and Promote Cell Survival

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2018.08.041

    PNCTR Knockdown Promotes Programmed Cell Death (A) HeLa cells were transfected with 400 nM gmControl or gmPNCTR and plated at the densities indicated. Note dramatically reduced numbers of colonies in gmPNCTR-treated wells compared to gmControl. (B) Colony confluency in (A) quantified from 3 independent transfection experiments and shown as mean ± SD. p values are calculated using a two-tailed t test. (C) Growth curves of HeLa cells transfected with 400 nM gmControl or gmPNCTR show that gmPNCTR leads to a visible decline in cell viability between 24 and 72 hpt. Data are averaged from 6 transfection experiments ± SD and compared by a two-tailed t test. (D) Time-resolved qRT-PCR analyses showing that gmPNCTR reaches a maximal downregulation effect by 12 hpt. Data are averaged from 3 experiments ± SD. (E) gmPNCTR, but not gmControl, induces expression of the apoptotic marker cleaved caspase-3 (CC3) at 12–24 hpt. (F) Dampening PNCTR levels often leads to extensive activation of caspase-3 in HeLa cells (arrowheads). The close up in the top-right corner compares DAPI staining for a normal nucleus (nN) of a CC3-negative cell and a pyknotic nucleus (pN) of a cell undergoing apoptosis. Scale bars, 10 μm. (G) CC3 induction by gmPNCTR is less efficient in HeLa cells expressing a gmPNCTR-resistant PNCTR fragment containing (UC)n repeats compared to the corresponding empty vector control. In (E) and (G), GAPDH is used as a lane-loading control. (H) GAPDH-normalized CC3 expression levels in (G) averaged from 3 experiments ± SD and compared by a two-tailed t test. .
    Figure Legend Snippet: PNCTR Knockdown Promotes Programmed Cell Death (A) HeLa cells were transfected with 400 nM gmControl or gmPNCTR and plated at the densities indicated. Note dramatically reduced numbers of colonies in gmPNCTR-treated wells compared to gmControl. (B) Colony confluency in (A) quantified from 3 independent transfection experiments and shown as mean ± SD. p values are calculated using a two-tailed t test. (C) Growth curves of HeLa cells transfected with 400 nM gmControl or gmPNCTR show that gmPNCTR leads to a visible decline in cell viability between 24 and 72 hpt. Data are averaged from 6 transfection experiments ± SD and compared by a two-tailed t test. (D) Time-resolved qRT-PCR analyses showing that gmPNCTR reaches a maximal downregulation effect by 12 hpt. Data are averaged from 3 experiments ± SD. (E) gmPNCTR, but not gmControl, induces expression of the apoptotic marker cleaved caspase-3 (CC3) at 12–24 hpt. (F) Dampening PNCTR levels often leads to extensive activation of caspase-3 in HeLa cells (arrowheads). The close up in the top-right corner compares DAPI staining for a normal nucleus (nN) of a CC3-negative cell and a pyknotic nucleus (pN) of a cell undergoing apoptosis. Scale bars, 10 μm. (G) CC3 induction by gmPNCTR is less efficient in HeLa cells expressing a gmPNCTR-resistant PNCTR fragment containing (UC)n repeats compared to the corresponding empty vector control. In (E) and (G), GAPDH is used as a lane-loading control. (H) GAPDH-normalized CC3 expression levels in (G) averaged from 3 experiments ± SD and compared by a two-tailed t test. .

    Techniques Used: Transfection, Two Tailed Test, Quantitative RT-PCR, Expressing, Marker, Activation Assay, Staining, Plasmid Preparation

    PNCTR Is a pol-I Transcript Interacting with Multiple Copies of PTBP1 Protein (A) Diagram of the predicted PNCTR locus also showing an adjacent 47S/45S rRNA gene and probes used in this study. Mapping to chr21 should be considered provisional since different IGS sequences share extensive regions of homology, and not all parts of human rDNA have been sequenced. (B) Top: northern blot analysis of PNCTR expression in HeLa cells using the probe introduced in (A). Bottom: methylene-blue-stained membrane showing that the lanes were loaded equally. (C) RIP carried out with a PTBP1-specific antibody or a non-immune IgG control. Immunoprecipitated RNAs were analyzed by qRT-PCR using primers specific to PNCTR, PTBP2 pre-mRNA (positive control), or U6 snRNA (negative control). Data are averaged from three experiments ± SD and compared by a two-tailed t test. (D) EMSA with purified PTBP1 protein and a PNCTR-specific RNA probe (sequence on the top). Bottom right: multivalent complexes assemble on incubating the probe with increasing amounts of PTBP1. Bottom left: no band shifts are detected when PTBP1 is substituted with BSA. (E) The PTBP1-PNCTR interaction in (D) is specific since it can be disrupted by increasing amounts of unlabeled PNCTR probe (bottom left), but not a control competitor (top, control RNA sequence; bottom right, the EMSA result). (F) IF-FISH staining of HeLa cells showing that PNCTR co-localizes with PTBP1 in the perinucleolar compartment (PNC). FBL, nucleolar marker fibrillarin. Scale bar, 10 μm. .
    Figure Legend Snippet: PNCTR Is a pol-I Transcript Interacting with Multiple Copies of PTBP1 Protein (A) Diagram of the predicted PNCTR locus also showing an adjacent 47S/45S rRNA gene and probes used in this study. Mapping to chr21 should be considered provisional since different IGS sequences share extensive regions of homology, and not all parts of human rDNA have been sequenced. (B) Top: northern blot analysis of PNCTR expression in HeLa cells using the probe introduced in (A). Bottom: methylene-blue-stained membrane showing that the lanes were loaded equally. (C) RIP carried out with a PTBP1-specific antibody or a non-immune IgG control. Immunoprecipitated RNAs were analyzed by qRT-PCR using primers specific to PNCTR, PTBP2 pre-mRNA (positive control), or U6 snRNA (negative control). Data are averaged from three experiments ± SD and compared by a two-tailed t test. (D) EMSA with purified PTBP1 protein and a PNCTR-specific RNA probe (sequence on the top). Bottom right: multivalent complexes assemble on incubating the probe with increasing amounts of PTBP1. Bottom left: no band shifts are detected when PTBP1 is substituted with BSA. (E) The PTBP1-PNCTR interaction in (D) is specific since it can be disrupted by increasing amounts of unlabeled PNCTR probe (bottom left), but not a control competitor (top, control RNA sequence; bottom right, the EMSA result). (F) IF-FISH staining of HeLa cells showing that PNCTR co-localizes with PTBP1 in the perinucleolar compartment (PNC). FBL, nucleolar marker fibrillarin. Scale bar, 10 μm. .

    Techniques Used: Northern Blot, Expressing, Staining, Immunoprecipitation, Quantitative RT-PCR, Positive Control, Negative Control, Two Tailed Test, Purification, Sequencing, Fluorescence In Situ Hybridization, Marker

    PNCTR Is Often Upregulated in Cancer Cells (A) qRT-PCR analyses showing that PNCTR expression is orders of magnitude higher in transformed cells (HeLa, HCT116, SW620, MCF7, and the SV40-transformed clone VA-13 of the normal lung fibroblast line WI-38) than in their non-transformed counterparts (ARPE-19 and WI-38). The data are averaged from 3 assays ± SD and the expression levels in WI-38 cells were set to 1. (B) qRT-PCR analyses carried out with and without reverse transcriptase (RT) show that the PNCTR signals in ARPE-19 and WI-38 correspond to bona fide expression of this strRNA at low but detectable levels. ). Data were obtained using RqP1 primers, normalized to β-actin, averaged from 3 amplification experiments ± SD and compared by a two-tailed t test. (D) PNCTR-positive nuclear dots are readily detectable by RNA-FISH in a lymph node metastasis sample collected for the case (a) in (C), but not in the matching normal lung control. (E) A close up of the boxed area in (D). Scale bars in (D) and (E), 10 μm. .
    Figure Legend Snippet: PNCTR Is Often Upregulated in Cancer Cells (A) qRT-PCR analyses showing that PNCTR expression is orders of magnitude higher in transformed cells (HeLa, HCT116, SW620, MCF7, and the SV40-transformed clone VA-13 of the normal lung fibroblast line WI-38) than in their non-transformed counterparts (ARPE-19 and WI-38). The data are averaged from 3 assays ± SD and the expression levels in WI-38 cells were set to 1. (B) qRT-PCR analyses carried out with and without reverse transcriptase (RT) show that the PNCTR signals in ARPE-19 and WI-38 correspond to bona fide expression of this strRNA at low but detectable levels. ). Data were obtained using RqP1 primers, normalized to β-actin, averaged from 3 amplification experiments ± SD and compared by a two-tailed t test. (D) PNCTR-positive nuclear dots are readily detectable by RNA-FISH in a lymph node metastasis sample collected for the case (a) in (C), but not in the matching normal lung control. (E) A close up of the boxed area in (D). Scale bars in (D) and (E), 10 μm. .

    Techniques Used: Quantitative RT-PCR, Expressing, Transformation Assay, Amplification, Two Tailed Test, Fluorescence In Situ Hybridization

    Identification of strRNAs Enriched in RBP Interaction Motifs (A) Workflow used in this study. (B) Transcripts newly predicted by the pipeline in (A) (“new”) are significantly over-represented among RBP motif-enriched RNAs as compared to previously annotated (“known”) transcripts. (C) strRNAs have significantly shorter ORFs compared to annotated mRNAs and the entire transcriptome. (D) STR content of strRNAs substantially exceeds corresponding transcriptome and genome values. (E) qRT-PCR and RT-PCR validation of five newly identified strRNAs using samples prepared without reverse transcriptase (RT) as negative controls. Data are shown as mean ± SD. .
    Figure Legend Snippet: Identification of strRNAs Enriched in RBP Interaction Motifs (A) Workflow used in this study. (B) Transcripts newly predicted by the pipeline in (A) (“new”) are significantly over-represented among RBP motif-enriched RNAs as compared to previously annotated (“known”) transcripts. (C) strRNAs have significantly shorter ORFs compared to annotated mRNAs and the entire transcriptome. (D) STR content of strRNAs substantially exceeds corresponding transcriptome and genome values. (E) qRT-PCR and RT-PCR validation of five newly identified strRNAs using samples prepared without reverse transcriptase (RT) as negative controls. Data are shown as mean ± SD. .

    Techniques Used: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

    PNCTR Antagonizes Splicing Regulation Function of PTBP1 (A) RNA-seq analyses carried out to examine possible role of PNCTR in modulating PTBP1 activity as a regulator of alternative splicing. (B–E) Fisher’s exact tests showing that gmPNCTR-regulated alternative splicing events are significantly enriched among those regulated by (B) siPTBP1 or (C) siPTBP1/2, as compared to their occurrence in the entire list of alternative splicing events (Total) considered by ExpressionPlot. Note that alternative events controlled by both (D) gmPNCTR and siPTBP1 or (E) gmPNCTR and siPTBP1/2 are preferentially regulated in opposite directions (anti-regulated) rather than the same direction (co-regulated). (F) Regulation of CHEK2 exon 8 splicing by the PNCTR/PTBP1 circuitry. Left: the two alternative splicing possibilities. Right: RT-PCR analyses of HeLa cells showing that combined knockdown of PTBP1 and PTBP2 (siPTBP1/2) stimulates exon 8 inclusion, while knockdown of PNCTR (gmPNCTR, 400 nM) or overexpression of recombinant FLAG-tagged PTBP1 promotes its skipping. ) between experimental treatments and the corresponding controls. Positive ΔΨ values indicate an increase and negative, a decrease in exon 8 inclusion. Similar quantifications were also done for cells transfected with 25 and 100 nM gapmers. All data are averaged from 3 experimentally independent comparisons ± SD and analyzed by a paired t test. L and S6M. (I) CLIP/qRT-PCR experiment showing an increase in PTBP1 interaction efficiency with the CHEK2 exon 8 region (CLIP-CHEK2e8) in HeLa cells treated with gmPNCTR and a lack of this effect for an upstream (CLIP-CHEK2e2) and a downstream region (CLIP-CHEK2i8). PTBP2 pre-mRNA exon 10 region (CLIP-PTBP2e10) and ACTB mRNA are used as a positive and a negative control, respectively. Data are averaged from two triplicated CLIP/qRT-PCR experiments ± SD and compared by a two-tailed t test. (J) Left: immunoblot analysis showing a decrease in the CHEK2 protein levels in HeLa cells transfected for 24 hr with gmPNCTR compared to gmControl. CC3 is used as a sample identity marker and GAPDH as a lane-loading control. Right: immunoblot quantification showing GAPDH-normalized CHEK2 expression levels averaged from 3 experiments ± SD and compared by paired t test. (K) Left: HeLa cells treated with 50 nM of either siControl or siCHEK2 for 36 hr were post-transfected with 400 nM of gmPNCTR or gmControl for 12 hr and analyzed for CC3 expression. Note that the preemptive knockdown of CHEK2 facilitates induction of CC3 in the gmPNCTR samples. Right: GAPDH-normalized CC3 expression averaged from 6 experiments ± SD and compared by paired t test. (L) Expression of recombinant PTBP1 is sufficient to upregulate CC3 in HeLa cells. Left: immunoblot analysis of control and FLAG-PTBP1-transfected samples. Right: GAPDH-normalized CC3 expression averaged from 3 experiments ± SD and compared by paired t test. .
    Figure Legend Snippet: PNCTR Antagonizes Splicing Regulation Function of PTBP1 (A) RNA-seq analyses carried out to examine possible role of PNCTR in modulating PTBP1 activity as a regulator of alternative splicing. (B–E) Fisher’s exact tests showing that gmPNCTR-regulated alternative splicing events are significantly enriched among those regulated by (B) siPTBP1 or (C) siPTBP1/2, as compared to their occurrence in the entire list of alternative splicing events (Total) considered by ExpressionPlot. Note that alternative events controlled by both (D) gmPNCTR and siPTBP1 or (E) gmPNCTR and siPTBP1/2 are preferentially regulated in opposite directions (anti-regulated) rather than the same direction (co-regulated). (F) Regulation of CHEK2 exon 8 splicing by the PNCTR/PTBP1 circuitry. Left: the two alternative splicing possibilities. Right: RT-PCR analyses of HeLa cells showing that combined knockdown of PTBP1 and PTBP2 (siPTBP1/2) stimulates exon 8 inclusion, while knockdown of PNCTR (gmPNCTR, 400 nM) or overexpression of recombinant FLAG-tagged PTBP1 promotes its skipping. ) between experimental treatments and the corresponding controls. Positive ΔΨ values indicate an increase and negative, a decrease in exon 8 inclusion. Similar quantifications were also done for cells transfected with 25 and 100 nM gapmers. All data are averaged from 3 experimentally independent comparisons ± SD and analyzed by a paired t test. L and S6M. (I) CLIP/qRT-PCR experiment showing an increase in PTBP1 interaction efficiency with the CHEK2 exon 8 region (CLIP-CHEK2e8) in HeLa cells treated with gmPNCTR and a lack of this effect for an upstream (CLIP-CHEK2e2) and a downstream region (CLIP-CHEK2i8). PTBP2 pre-mRNA exon 10 region (CLIP-PTBP2e10) and ACTB mRNA are used as a positive and a negative control, respectively. Data are averaged from two triplicated CLIP/qRT-PCR experiments ± SD and compared by a two-tailed t test. (J) Left: immunoblot analysis showing a decrease in the CHEK2 protein levels in HeLa cells transfected for 24 hr with gmPNCTR compared to gmControl. CC3 is used as a sample identity marker and GAPDH as a lane-loading control. Right: immunoblot quantification showing GAPDH-normalized CHEK2 expression levels averaged from 3 experiments ± SD and compared by paired t test. (K) Left: HeLa cells treated with 50 nM of either siControl or siCHEK2 for 36 hr were post-transfected with 400 nM of gmPNCTR or gmControl for 12 hr and analyzed for CC3 expression. Note that the preemptive knockdown of CHEK2 facilitates induction of CC3 in the gmPNCTR samples. Right: GAPDH-normalized CC3 expression averaged from 6 experiments ± SD and compared by paired t test. (L) Expression of recombinant PTBP1 is sufficient to upregulate CC3 in HeLa cells. Left: immunoblot analysis of control and FLAG-PTBP1-transfected samples. Right: GAPDH-normalized CC3 expression averaged from 3 experiments ± SD and compared by paired t test. .

    Techniques Used: RNA Sequencing Assay, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Over Expression, Recombinant, Transfection, Cross-linking Immunoprecipitation, Quantitative RT-PCR, Negative Control, Two Tailed Test, Marker, Expressing

    16) Product Images from "nextPARS: parallel probing of RNA structures in Illumina"

    Article Title: nextPARS: parallel probing of RNA structures in Illumina

    Journal: RNA

    doi: 10.1261/rna.063073.117

    Summary of the different steps performed in the nextPARS protocol. From the cells or tissue of interest ( A ), total RNA is extracted ( B ) and then poly(A) + RNA is selected ( C ) to initially prepare the samples for nextPARS analyses. Once the quality and quantity of poly(A) + RNA samples is confirmed, RNA samples are denatured and in vitro folded to perform the enzymatic probing of the molecules with the corresponding concentrations of RNase V1 and S1 nuclease ( D ). For the library preparation using the Illumina TruSeq Small RNA Sample Preparation Kit, an initial phosphatase treatment of the 3′ends and a kinase treatment of the 5′ ends are required ( E ) to then ligate the corresponding 5′ and 3′ adapters at the ends of the RNA fragments ( F ). Then a reverse transcription of the RNA fragments and a PCR amplification are performed to obtain the library ( G ). The library is size-selected to get rid of primers and adapters dimers using an acrylamide gel and a final quality control is performed ( H ). Libraries are sequenced in single-reads with read lengths of 50 nucleotides (nt) using Illumina sequencing platforms ( I ) and computational analyses are done as described in the Materials and Methods section in order to map Illumina reads and determine the enzymatic cleavage points, using the first nucleotide in the 5′ end of the reads (which correspond to the 5′end of original RNA fragments) ( J ).
    Figure Legend Snippet: Summary of the different steps performed in the nextPARS protocol. From the cells or tissue of interest ( A ), total RNA is extracted ( B ) and then poly(A) + RNA is selected ( C ) to initially prepare the samples for nextPARS analyses. Once the quality and quantity of poly(A) + RNA samples is confirmed, RNA samples are denatured and in vitro folded to perform the enzymatic probing of the molecules with the corresponding concentrations of RNase V1 and S1 nuclease ( D ). For the library preparation using the Illumina TruSeq Small RNA Sample Preparation Kit, an initial phosphatase treatment of the 3′ends and a kinase treatment of the 5′ ends are required ( E ) to then ligate the corresponding 5′ and 3′ adapters at the ends of the RNA fragments ( F ). Then a reverse transcription of the RNA fragments and a PCR amplification are performed to obtain the library ( G ). The library is size-selected to get rid of primers and adapters dimers using an acrylamide gel and a final quality control is performed ( H ). Libraries are sequenced in single-reads with read lengths of 50 nucleotides (nt) using Illumina sequencing platforms ( I ) and computational analyses are done as described in the Materials and Methods section in order to map Illumina reads and determine the enzymatic cleavage points, using the first nucleotide in the 5′ end of the reads (which correspond to the 5′end of original RNA fragments) ( J ).

    Techniques Used: In Vitro, Sample Prep, Polymerase Chain Reaction, Amplification, Acrylamide Gel Assay, Sequencing

    Probing of RNA molecules with RNase A enzyme. Examples of the signals obtained in some RNA molecules when performing nextPARS using RNase A, an enzyme that cuts specifically in single-stranded cytosines (C) and uracils (U). Scores were calculated for each site by first capping all read counts for a given transcript at the 95th percentile and then normalizing to have a maximum of 1 (as done in the “Computation of nextPARS scores” of the Materials and Methods, but since Rnase A is the only enzyme in this case, there will be no subtraction performed, so all values will then fall in the range of 0 to 1). Cuts are considered for signals above a threshold of 0.8. ( A ]). In green, nucleotides with a cut signal above 0.8; green crosses (+) show cuts obtained in a C or U; pink asterisks (*) show cuts obtained in a G or A; and blue arrows (→) show cuts obtained in double-stranded positions. ( B ) Table summarizing the total number (N) and percentages (%) of cuts with a signal above 0.8 threshold obtained in five different RNA fragments with known secondary structure (TETp4p6, TETp9-9.1, SRA, B2, U1): first column, N and % of cuts with a signal above 0.8 in the molecules; second column, N and % of these cuts in C or U nucleotides; and third column, N and % of cuts in G or A nucleotides.
    Figure Legend Snippet: Probing of RNA molecules with RNase A enzyme. Examples of the signals obtained in some RNA molecules when performing nextPARS using RNase A, an enzyme that cuts specifically in single-stranded cytosines (C) and uracils (U). Scores were calculated for each site by first capping all read counts for a given transcript at the 95th percentile and then normalizing to have a maximum of 1 (as done in the “Computation of nextPARS scores” of the Materials and Methods, but since Rnase A is the only enzyme in this case, there will be no subtraction performed, so all values will then fall in the range of 0 to 1). Cuts are considered for signals above a threshold of 0.8. ( A ]). In green, nucleotides with a cut signal above 0.8; green crosses (+) show cuts obtained in a C or U; pink asterisks (*) show cuts obtained in a G or A; and blue arrows (→) show cuts obtained in double-stranded positions. ( B ) Table summarizing the total number (N) and percentages (%) of cuts with a signal above 0.8 threshold obtained in five different RNA fragments with known secondary structure (TETp4p6, TETp9-9.1, SRA, B2, U1): first column, N and % of cuts with a signal above 0.8 in the molecules; second column, N and % of these cuts in C or U nucleotides; and third column, N and % of cuts in G or A nucleotides.

    Techniques Used:

    17) Product Images from "Transforming activity of an oncoprotein-encoding circular RNA from human papillomavirus"

    Article Title: Transforming activity of an oncoprotein-encoding circular RNA from human papillomavirus

    Journal: Nature Communications

    doi: 10.1038/s41467-019-10246-5

    Biological functions of circE7 in tumors and episomal HPV. CaSki cells (4×10 6 ), which had been stably transduced with the indicated construct, were xenografted onto the flanks of NSG mice ( n = 8 per construct). Mice were given water with or without doxycycline (1 mg/mL) as indicated. a Image of representative CaSki tumor xenografts dissected from the indicated mice after 21 days (top). Weights of CaSki tumors with or without dox-induced circE7 sh1/2 expression (bottom). b Representative images of tumors formed by CaSki xenografts without (top) or with (bottom) doxycycline. Arrowhead indicates an area of invasive tumor. Arrows indicate mitotic figures and Ki-67-positive cells. Dashed box indicates area of detail. Scale bars, 200 μm. c TCGA RNA-Seq data (CESC, HNSC) was analyzed with vircircRNA and backsplices with ≥2 reads were tabulated. d RT-PCR from CaSki or HPV BP cells that possess integrated or episomal HPV16 genomes with or without RNase R reveals the presence of circE7 in both samples. e Human foreskin keratinocyte (HFK), keratinocytes infected with religated HPV31 (HFK + HPV31), or a HPV31 infected cell line derived from a grade II cervical biopsy (CIN612) were induced to differentiate with high calcium. Levels of HPV31 circE7 were assessed by RT-PCR (left) or RT-qPCR (right). Calcium-induced differentiation significantly decreased levels of HPV31 circE7. RT-PCR is representative of 4 independent experiments. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with two-tailed t test ( a , e )
    Figure Legend Snippet: Biological functions of circE7 in tumors and episomal HPV. CaSki cells (4×10 6 ), which had been stably transduced with the indicated construct, were xenografted onto the flanks of NSG mice ( n = 8 per construct). Mice were given water with or without doxycycline (1 mg/mL) as indicated. a Image of representative CaSki tumor xenografts dissected from the indicated mice after 21 days (top). Weights of CaSki tumors with or without dox-induced circE7 sh1/2 expression (bottom). b Representative images of tumors formed by CaSki xenografts without (top) or with (bottom) doxycycline. Arrowhead indicates an area of invasive tumor. Arrows indicate mitotic figures and Ki-67-positive cells. Dashed box indicates area of detail. Scale bars, 200 μm. c TCGA RNA-Seq data (CESC, HNSC) was analyzed with vircircRNA and backsplices with ≥2 reads were tabulated. d RT-PCR from CaSki or HPV BP cells that possess integrated or episomal HPV16 genomes with or without RNase R reveals the presence of circE7 in both samples. e Human foreskin keratinocyte (HFK), keratinocytes infected with religated HPV31 (HFK + HPV31), or a HPV31 infected cell line derived from a grade II cervical biopsy (CIN612) were induced to differentiate with high calcium. Levels of HPV31 circE7 were assessed by RT-PCR (left) or RT-qPCR (right). Calcium-induced differentiation significantly decreased levels of HPV31 circE7. RT-PCR is representative of 4 independent experiments. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with two-tailed t test ( a , e )

    Techniques Used: Stable Transfection, Transduction, Construct, Mouse Assay, Expressing, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Infection, Derivative Assay, Quantitative RT-PCR, Two Tailed Test

    Identification of HPV circRNAs. a A transcript map generated by vircircRNA summarizing the splicing events identified for HPV16 from the combined SRA datasets (Supplementary Fig. 1e ). Lines (top) indicate forward splicing events; arcs (bottom) indicate backsplicing; thickness = log 2 (read count); red arc highlights circE7. The lower panel represents a partial HPV16 genome with promoters (P, green arrowheads) and the early polyadenylation (A E , red line) indicated. Numbering from the NC_001526 reference sequence. b Alignment of sequencing reads spanning the circE7 backsplice junction from SRS2410540. Red indicates E7-E1 sequences, and blue indicates E6 sequence. c Predicted formation and size of HPV16 circE7. Arrows indicate primers used to detect linear E6/E7 and circE7. d RT-PCR of random hexamer primed total RNA from HPV16+ cancer cell lines. 2 μg of total RNA were treated with 5U of RNase R (or water for mock) in the presence of RNase inhibitor for 40 min prior to RT reaction. Results are representative of 4 independent experiments. e Sanger sequencing of PCR products from d confirmed the presence of the expected circE7 backsplice junction without the insertion of additional nucleotides. Sequencing traces were identical for 3 independent reactions from each cell line. f Northern blot of total RNA after mock (8 μg) or with RNase R treatment (20 μg) from the indicated HPV16+ cell line probed with HPV16 E7. Arrows indicates RNase resistant band with E7 sequence. Ethidium Bromide staining (bottom), RNase R treatment control. Results representative of 5 independent northerns
    Figure Legend Snippet: Identification of HPV circRNAs. a A transcript map generated by vircircRNA summarizing the splicing events identified for HPV16 from the combined SRA datasets (Supplementary Fig. 1e ). Lines (top) indicate forward splicing events; arcs (bottom) indicate backsplicing; thickness = log 2 (read count); red arc highlights circE7. The lower panel represents a partial HPV16 genome with promoters (P, green arrowheads) and the early polyadenylation (A E , red line) indicated. Numbering from the NC_001526 reference sequence. b Alignment of sequencing reads spanning the circE7 backsplice junction from SRS2410540. Red indicates E7-E1 sequences, and blue indicates E6 sequence. c Predicted formation and size of HPV16 circE7. Arrows indicate primers used to detect linear E6/E7 and circE7. d RT-PCR of random hexamer primed total RNA from HPV16+ cancer cell lines. 2 μg of total RNA were treated with 5U of RNase R (or water for mock) in the presence of RNase inhibitor for 40 min prior to RT reaction. Results are representative of 4 independent experiments. e Sanger sequencing of PCR products from d confirmed the presence of the expected circE7 backsplice junction without the insertion of additional nucleotides. Sequencing traces were identical for 3 independent reactions from each cell line. f Northern blot of total RNA after mock (8 μg) or with RNase R treatment (20 μg) from the indicated HPV16+ cell line probed with HPV16 E7. Arrows indicates RNase resistant band with E7 sequence. Ethidium Bromide staining (bottom), RNase R treatment control. Results representative of 5 independent northerns

    Techniques Used: Generated, Sequencing, Reverse Transcription Polymerase Chain Reaction, Random Hexamer Labeling, Polymerase Chain Reaction, Northern Blot, Staining

    Protein encoding circE7 is essential for CaSki cell growth. a CaSki cells were lentivirally transduced with doxycycline (dox)-inducible hairpins specific for the circE7 backsplice junction (circE7 sh1/2). RT-qPCR for levels of circE7 revealed that circE7 sh1/2 resulted in significant decreases of circE7 levels. ( n = 3 independent experiments, run in duplicate). b Northern blot of RNase R treated total RNA (30 μg) from CaSki cells with or without circE7 sh1/2 induction (2 days). Band density (bottom number) was quantitated and normalized to the uninduced control. c Western blots for E7 and E6 after circE7 sh1/2 induction (3 days). Western blots representative of 3 independent experiments. GAPDH, loading control. d A total of 6.0 × 10 4 CaSki cells were seeded in triplicate in six-well plates at day 0 and absolute cell number quantitated daily after day 2. CircE7 sh1/2 induction resulted in significantly slower growth of CaSki cells after day 4. Similar results were obtained in 3 independent experiments. e CaSki cells with or without circE7 sh1/2 induction (1 day) were plated in chamber slides and labeled with BrdU (10 μM for 1.5 h). Cells were stained with αBrdU and DAPI and scored as % of DAPI + cells. f 1.0 × 10 4 CaSki circE7 sh1/2 cells with or without induction (1 day) were seeded in triplicate in soft agar with or without dox (14 days). Average colonies per 35 mm. n = 4 independent transfections. g CaSki were doubly transduced with a shRNA resistant WT circE7 (circResist_WT) and circE7 sh1/2. MTT assay of circResist_WT cells with and without Dox induction. MTT values normalized to the uninduced (-Dox) condition. h CaSki were doubly transduced with a shRNA resistant circE7 with no start codons (circResist_noATG) and circE7 sh1/2. MTT assay of circResist_noATG cells with and without Dox induction. MTT values normalized to the uninduced (-Dox) condition. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with two-tailed t test ( d , g , h ) and one-way analysis of variance (ANOVA) with Holm–Sidak tests ( a , e , f ). Source data for b , c provided in Source Data file
    Figure Legend Snippet: Protein encoding circE7 is essential for CaSki cell growth. a CaSki cells were lentivirally transduced with doxycycline (dox)-inducible hairpins specific for the circE7 backsplice junction (circE7 sh1/2). RT-qPCR for levels of circE7 revealed that circE7 sh1/2 resulted in significant decreases of circE7 levels. ( n = 3 independent experiments, run in duplicate). b Northern blot of RNase R treated total RNA (30 μg) from CaSki cells with or without circE7 sh1/2 induction (2 days). Band density (bottom number) was quantitated and normalized to the uninduced control. c Western blots for E7 and E6 after circE7 sh1/2 induction (3 days). Western blots representative of 3 independent experiments. GAPDH, loading control. d A total of 6.0 × 10 4 CaSki cells were seeded in triplicate in six-well plates at day 0 and absolute cell number quantitated daily after day 2. CircE7 sh1/2 induction resulted in significantly slower growth of CaSki cells after day 4. Similar results were obtained in 3 independent experiments. e CaSki cells with or without circE7 sh1/2 induction (1 day) were plated in chamber slides and labeled with BrdU (10 μM for 1.5 h). Cells were stained with αBrdU and DAPI and scored as % of DAPI + cells. f 1.0 × 10 4 CaSki circE7 sh1/2 cells with or without induction (1 day) were seeded in triplicate in soft agar with or without dox (14 days). Average colonies per 35 mm. n = 4 independent transfections. g CaSki were doubly transduced with a shRNA resistant WT circE7 (circResist_WT) and circE7 sh1/2. MTT assay of circResist_WT cells with and without Dox induction. MTT values normalized to the uninduced (-Dox) condition. h CaSki were doubly transduced with a shRNA resistant circE7 with no start codons (circResist_noATG) and circE7 sh1/2. MTT assay of circResist_noATG cells with and without Dox induction. MTT values normalized to the uninduced (-Dox) condition. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with two-tailed t test ( d , g , h ) and one-way analysis of variance (ANOVA) with Holm–Sidak tests ( a , e , f ). Source data for b , c provided in Source Data file

    Techniques Used: Transduction, Quantitative RT-PCR, Northern Blot, Western Blot, Labeling, Staining, Transfection, shRNA, MTT Assay, Two Tailed Test

    Characterization of circE7. a CircE7-transfected cells were fractionated and indicated fractions analyzed by northern blot. Total RNA (4 μg) with mock or RNase R treatment of fractions from 293 T cells confirms that circE7 is enriched in the cytoplasm and is RNase R-resistant. MALAT1 and β-actin, fractionation controls. Band density (bottom) quantitated after normalization to the enriched fraction. Results are representative of 3 independent blots. b CircE7-transfected 293T (left) or untransfected CaSki (right) were fractionated and analyzed by RT-qPCR. MALAT1 and 18 S (top), fractionation controls. Values normalized to the enriched fraction. Results are representative of 3 independent fractionation experiments. c RT-qPCR of RNA IP (m 6 A or IgG control) after transfection with the indicated plasmid (24 h) ( n = 8 biological replicates from 4 transfections). SON, m 6 A RNA IP control. d Western blot for METTL3 from 293T co-transfected with control or METTL3 siRNA and circE7 construct (top). GAPDH, loading control. RT-qPCR of RNA IP (m 6 A or IgG control) from 293 T cells co-transfected with indicated siRNA and circE7 construct. RT-PCR is representative of 4 independent experiments. e Schematic of the DRACH consensus motifs for METTL3/14 and the sites mutated in the circE7_noDRACH construct (top). RT-qPCR for circE7 in cells transfected with the indicated construct. Loss of UTR DRACH motifs in circE7 results in a significant decrease in the abundance of circE7, but not linear E6/E7. ( n = 4 independent experiments). f Western blot for E7 from 293 T transfected with indicated circE7 construct. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with one-way analysis of variance (ANOVA) with Holm–Sidak tests. g Representative tracing of circE7-transfected cells after polysome enrichment assay with the monosome (M), light polysome (L), and heavy polysome (H) fractions indicated (left). Dashed lines indicate collected fraction. Detection of circE7 in polysome fraction by RT-PCR after transfection with circE7 or circE7_noATG (right). β-actin, control. Source data for a provided in Source Data file
    Figure Legend Snippet: Characterization of circE7. a CircE7-transfected cells were fractionated and indicated fractions analyzed by northern blot. Total RNA (4 μg) with mock or RNase R treatment of fractions from 293 T cells confirms that circE7 is enriched in the cytoplasm and is RNase R-resistant. MALAT1 and β-actin, fractionation controls. Band density (bottom) quantitated after normalization to the enriched fraction. Results are representative of 3 independent blots. b CircE7-transfected 293T (left) or untransfected CaSki (right) were fractionated and analyzed by RT-qPCR. MALAT1 and 18 S (top), fractionation controls. Values normalized to the enriched fraction. Results are representative of 3 independent fractionation experiments. c RT-qPCR of RNA IP (m 6 A or IgG control) after transfection with the indicated plasmid (24 h) ( n = 8 biological replicates from 4 transfections). SON, m 6 A RNA IP control. d Western blot for METTL3 from 293T co-transfected with control or METTL3 siRNA and circE7 construct (top). GAPDH, loading control. RT-qPCR of RNA IP (m 6 A or IgG control) from 293 T cells co-transfected with indicated siRNA and circE7 construct. RT-PCR is representative of 4 independent experiments. e Schematic of the DRACH consensus motifs for METTL3/14 and the sites mutated in the circE7_noDRACH construct (top). RT-qPCR for circE7 in cells transfected with the indicated construct. Loss of UTR DRACH motifs in circE7 results in a significant decrease in the abundance of circE7, but not linear E6/E7. ( n = 4 independent experiments). f Western blot for E7 from 293 T transfected with indicated circE7 construct. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with one-way analysis of variance (ANOVA) with Holm–Sidak tests. g Representative tracing of circE7-transfected cells after polysome enrichment assay with the monosome (M), light polysome (L), and heavy polysome (H) fractions indicated (left). Dashed lines indicate collected fraction. Detection of circE7 in polysome fraction by RT-PCR after transfection with circE7 or circE7_noATG (right). β-actin, control. Source data for a provided in Source Data file

    Techniques Used: Transfection, Northern Blot, Fractionation, Quantitative RT-PCR, Plasmid Preparation, Western Blot, Construct, Reverse Transcription Polymerase Chain Reaction

    18) 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

    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

    19) Product Images from "A novel CRISPR-based malaria diagnostic capable of Plasmodium detection, speciation, and drug-resistance genotyping"

    Article Title: A novel CRISPR-based malaria diagnostic capable of Plasmodium detection, speciation, and drug-resistance genotyping

    Journal: bioRxiv

    doi: 10.1101/2020.04.01.017962

    SHERLOCK reaction workflow. (A) DNA extracted from a patient with malaria or infected mosquito is subjected to (B) RPA including T7 promoter-tagged primers for amplification of the Plasmodium 18S rRNA or P. falciparum dhps genes, followed by (C) IVT and LwCas13a:crRNA complex binding to genus-, species-, or genotype-specific target RNA. This binding triggers collateral (D) activation of LwCas13a RNAse activity and cleavage of reporter RNA, separating the fluorescent reporter from its quencher and producing a signal. Abbreviations: RPA, recombinase polymerase amplification; IVT, in vitro transcription; R, reporter; Q, quencher.
    Figure Legend Snippet: SHERLOCK reaction workflow. (A) DNA extracted from a patient with malaria or infected mosquito is subjected to (B) RPA including T7 promoter-tagged primers for amplification of the Plasmodium 18S rRNA or P. falciparum dhps genes, followed by (C) IVT and LwCas13a:crRNA complex binding to genus-, species-, or genotype-specific target RNA. This binding triggers collateral (D) activation of LwCas13a RNAse activity and cleavage of reporter RNA, separating the fluorescent reporter from its quencher and producing a signal. Abbreviations: RPA, recombinase polymerase amplification; IVT, in vitro transcription; R, reporter; Q, quencher.

    Techniques Used: Infection, Recombinase Polymerase Amplification, Amplification, Binding Assay, Activation Assay, Activity Assay, In Vitro

    20) Product Images from "A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries"

    Article Title: A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries

    Journal: Journal of Advanced Research

    doi: 10.1016/j.jare.2019.12.005

    The schematic representation of the workflow for the reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy. Total RNA (usually 0.5–1.0 µg) is incubated and hybridized with a panel of 30 (human mouse) rRNA-specific DNA oligo probes ( a ), followed by reverse transcription (RT) ( b ). After the removal of excess oligo probes with Exonuclease I ( c ), the resultant RT products are subjected to RNase H digestion to degrade the rRNA portions of the RNA:DNA hybrid ( d ), and then the DNA components are degraded by DNase I ( e ). The intact mRNAs and noncoding RNAs are subsequently purified by ethanol precipitation ( f ) and subjected to RNA-seq library construction ( g ). The locations and sequences of individual rRNA-specific probes are shown in Suppl. Fig. S2 and Suppl. Table S1.
    Figure Legend Snippet: The schematic representation of the workflow for the reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy. Total RNA (usually 0.5–1.0 µg) is incubated and hybridized with a panel of 30 (human mouse) rRNA-specific DNA oligo probes ( a ), followed by reverse transcription (RT) ( b ). After the removal of excess oligo probes with Exonuclease I ( c ), the resultant RT products are subjected to RNase H digestion to degrade the rRNA portions of the RNA:DNA hybrid ( d ), and then the DNA components are degraded by DNase I ( e ). The intact mRNAs and noncoding RNAs are subsequently purified by ethanol precipitation ( f ) and subjected to RNA-seq library construction ( g ). The locations and sequences of individual rRNA-specific probes are shown in Suppl. Fig. S2 and Suppl. Table S1.

    Techniques Used: Incubation, Purification, Ethanol Precipitation, RNA Sequencing Assay

    21) Product Images from "Nucleoside analog studies indicate mechanistic differences between RNA-editing adenosine deaminases"

    Article Title: Nucleoside analog studies indicate mechanistic differences between RNA-editing adenosine deaminases

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks752

    ( A ) Plot of k obs as a function of ADAR1 concentration for the 84 nt RNA. Editing reaction carried out in 15 mM Tris–HCl, pH 7.5, 1.5 mM EDTA, 40 mM KCl, 26 mM NaCl, 5% glycerol, 0.003% Nonidet P-40, 0.5 mM DTT, 160 U/ml RNasin, 0.3 mM BME and 1.0 µg/ml yeast tRNA Phe . All data points reported are the average ± standard deviation for three experiments. ( B ) Duplex RNA substrates of ADAR1 used in this study. In the assays with analogs, N represents the site specifically labeled nucleotide, either A or one of the analogs listed in the table ( C ). The asterisks indicate the 32 P-containing phosphodiester. In the assays with in vitro transcribed RNA, N = A and is not radiolabeled.
    Figure Legend Snippet: ( A ) Plot of k obs as a function of ADAR1 concentration for the 84 nt RNA. Editing reaction carried out in 15 mM Tris–HCl, pH 7.5, 1.5 mM EDTA, 40 mM KCl, 26 mM NaCl, 5% glycerol, 0.003% Nonidet P-40, 0.5 mM DTT, 160 U/ml RNasin, 0.3 mM BME and 1.0 µg/ml yeast tRNA Phe . All data points reported are the average ± standard deviation for three experiments. ( B ) Duplex RNA substrates of ADAR1 used in this study. In the assays with analogs, N represents the site specifically labeled nucleotide, either A or one of the analogs listed in the table ( C ). The asterisks indicate the 32 P-containing phosphodiester. In the assays with in vitro transcribed RNA, N = A and is not radiolabeled.

    Techniques Used: Concentration Assay, Standard Deviation, Labeling, In Vitro

    22) Product Images from "RNA promotes the formation of spatial compartments in the nucleus"

    Article Title: RNA promotes the formation of spatial compartments in the nucleus

    Journal: bioRxiv

    doi: 10.1101/2020.08.25.267435

    RNA localization on DNA and within the nucleus for RNAs within each RNA hub. (A) Weighted genomic DNA localization heatmap of each individual RNA. RNAs are organized by their RNA hub occupancy (shown in Figure 2A ). Contacts are normalized from 0 to 1 to account for expression levels of each RNA. (B) Pearson correlation of RNA-DNA unweighted contact frequencies across the genome for individual RNAs within the nuclear hubs (nucleolar, centromeric, spliceosomal, and scaRNA hubs). Red represents high correlation and blue represents low correlation. (C) RNA FISH of various non-coding RNAs within the same hub in the nucleus. Spliceosomal hub (top): Malat1 lncRNA and 7SK RNA and (bottom): U6 and U1 spliceosomal RNAs. Nucleolar hub (top): snora26 snoRNA and 45S pre-rRNA ITS2 and (bottom): RNase MRP (Rmrp) and 45S pre-rRNA ITS1. Each panel is shown individually (left and middle) and overlaid (right). Dashed lines demarcate the nuclear boundary identified with DAPI. Scalebar is 10μm. (D) RNA FISH (left) along with nucleolin immunofluorescence (middle) and DAPI (right) of specific ncRNAs. 7SK RNA (top), ITS1 regions of 45S pre-rRNA (middle) and tRNAs (bottom). tRNAs are visualized using pooled RNA FISH probes (see Methods). Scalebar is 10μm.
    Figure Legend Snippet: RNA localization on DNA and within the nucleus for RNAs within each RNA hub. (A) Weighted genomic DNA localization heatmap of each individual RNA. RNAs are organized by their RNA hub occupancy (shown in Figure 2A ). Contacts are normalized from 0 to 1 to account for expression levels of each RNA. (B) Pearson correlation of RNA-DNA unweighted contact frequencies across the genome for individual RNAs within the nuclear hubs (nucleolar, centromeric, spliceosomal, and scaRNA hubs). Red represents high correlation and blue represents low correlation. (C) RNA FISH of various non-coding RNAs within the same hub in the nucleus. Spliceosomal hub (top): Malat1 lncRNA and 7SK RNA and (bottom): U6 and U1 spliceosomal RNAs. Nucleolar hub (top): snora26 snoRNA and 45S pre-rRNA ITS2 and (bottom): RNase MRP (Rmrp) and 45S pre-rRNA ITS1. Each panel is shown individually (left and middle) and overlaid (right). Dashed lines demarcate the nuclear boundary identified with DAPI. Scalebar is 10μm. (D) RNA FISH (left) along with nucleolin immunofluorescence (middle) and DAPI (right) of specific ncRNAs. 7SK RNA (top), ITS1 regions of 45S pre-rRNA (middle) and tRNAs (bottom). tRNAs are visualized using pooled RNA FISH probes (see Methods). Scalebar is 10μm.

    Techniques Used: Expressing, Fluorescence In Situ Hybridization, Immunofluorescence

    23) Product Images from "A long non-coding RNA is required for targeting centromeric protein A to the human centromere"

    Article Title: A long non-coding RNA is required for targeting centromeric protein A to the human centromere

    Journal: eLife

    doi: 10.7554/eLife.03254

    Centromeric transcripts are 1.3 kb in length. ( A ) To determine the size of the centromeric α-satellite transcripts, the graph of the distance (in y) between the border of the Northern blot and each band of the molecular weight as a function of the number of bases was made. The distance of the centromeric α-satellite transcript band was analyzed using the standard curve from this graph to deduce its size. ( B ) Total RNAs treated with RNase A were separated on a denaturing gel, and revealed by Northern blot with radiolabeled centromeric α-satellite probes. ( C ) eG1-synchronized cells were treated, or not, with α-amanitin (2 hr). RNAs were processed and analyzed on Northern blot as in ( B ) to examine whether trace DNA contamination could yield the same band as in ( A ). DOI: http://dx.doi.org/10.7554/eLife.03254.010
    Figure Legend Snippet: Centromeric transcripts are 1.3 kb in length. ( A ) To determine the size of the centromeric α-satellite transcripts, the graph of the distance (in y) between the border of the Northern blot and each band of the molecular weight as a function of the number of bases was made. The distance of the centromeric α-satellite transcript band was analyzed using the standard curve from this graph to deduce its size. ( B ) Total RNAs treated with RNase A were separated on a denaturing gel, and revealed by Northern blot with radiolabeled centromeric α-satellite probes. ( C ) eG1-synchronized cells were treated, or not, with α-amanitin (2 hr). RNAs were processed and analyzed on Northern blot as in ( B ) to examine whether trace DNA contamination could yield the same band as in ( A ). DOI: http://dx.doi.org/10.7554/eLife.03254.010

    Techniques Used: Northern Blot, Molecular Weight

    24) Product Images from "Transcriptome-Wide Analyses of 5?-Ends in RNase J Mutants of a Gram-Positive Pathogen Reveal a Role in RNA Maturation, Regulation and Degradation"

    Article Title: Transcriptome-Wide Analyses of 5?-Ends in RNase J Mutants of a Gram-Positive Pathogen Reveal a Role in RNA Maturation, Regulation and Degradation

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004207

    Both maturation and inactivation of RNase P RNA is carried out by RNase J. A) Histogram showing the percentage of reads mapping to a given position, out of the total number of reads mapping to the putative SA1279- rnpB operon in each strain (shown in parentheses). Only positions of interest are included, but the full data-set can be found in Table S6 . +1: The putative transcription start site of SA1279. +452 to +477: The RNase J mutants accumulate RNA with 5′-ends in this region. +485: The putative transcription start site of rnpB , a major detected RNA species in the WT, ΔY and ΔcshA, but very reduced in the RNase J mutants. +499: A major detected RNA species in the WT, ΔY and ΔcshA, however it is absent from the RNase J1 mutants and reduced in the ΔJ2 strain. B) The layout of the region around SA1279 and rnpB . DNA is represented as a wavy line, and RNA transcripts as straight black lines. (PP)P indicates a mix of tri- and mono-phosphorylated RNA, generated by pyrophosphohydrolases. Small blue arrows indicate the PCR-primers used to amplify circularised RnpB and SA1279-RnpB for mapping the 5′ and 3′-ends. R1 indicates the probe used for the Northern blot shown in Figure S2 . C) A blow-up of the region from +420 to +540, showing the proposed model for converting the +1 transcript into mature RnpB. P indicates mono-phosphorylation. D) Predicted secondary structures of RnpB, generated using mfold with default settings [30] , and based on the crystal structures of RNase P RNA [27] , [31] . Within the RNase P structure, the thin dotted arrows indicate the path of the RNA through the secondary and tertiary structure of RNase P, the RBS and start codon of SA1278 are in bold, and the region where the anti-sense RNA can hybridise is indicated with a thick black line. E) The difference in average length of RnpB in WT, ΔJ1, and ΔJ1ΔJ2 strains, revealed by the length of the PCR-product generated across the 5′/3′ junction. Results of the cloned and sequenced PCR-products are shown in Table 6 . M: Marker.
    Figure Legend Snippet: Both maturation and inactivation of RNase P RNA is carried out by RNase J. A) Histogram showing the percentage of reads mapping to a given position, out of the total number of reads mapping to the putative SA1279- rnpB operon in each strain (shown in parentheses). Only positions of interest are included, but the full data-set can be found in Table S6 . +1: The putative transcription start site of SA1279. +452 to +477: The RNase J mutants accumulate RNA with 5′-ends in this region. +485: The putative transcription start site of rnpB , a major detected RNA species in the WT, ΔY and ΔcshA, but very reduced in the RNase J mutants. +499: A major detected RNA species in the WT, ΔY and ΔcshA, however it is absent from the RNase J1 mutants and reduced in the ΔJ2 strain. B) The layout of the region around SA1279 and rnpB . DNA is represented as a wavy line, and RNA transcripts as straight black lines. (PP)P indicates a mix of tri- and mono-phosphorylated RNA, generated by pyrophosphohydrolases. Small blue arrows indicate the PCR-primers used to amplify circularised RnpB and SA1279-RnpB for mapping the 5′ and 3′-ends. R1 indicates the probe used for the Northern blot shown in Figure S2 . C) A blow-up of the region from +420 to +540, showing the proposed model for converting the +1 transcript into mature RnpB. P indicates mono-phosphorylation. D) Predicted secondary structures of RnpB, generated using mfold with default settings [30] , and based on the crystal structures of RNase P RNA [27] , [31] . Within the RNase P structure, the thin dotted arrows indicate the path of the RNA through the secondary and tertiary structure of RNase P, the RBS and start codon of SA1278 are in bold, and the region where the anti-sense RNA can hybridise is indicated with a thick black line. E) The difference in average length of RnpB in WT, ΔJ1, and ΔJ1ΔJ2 strains, revealed by the length of the PCR-product generated across the 5′/3′ junction. Results of the cloned and sequenced PCR-products are shown in Table 6 . M: Marker.

    Techniques Used: Generated, Polymerase Chain Reaction, Northern Blot, Clone Assay, Marker

    SA1075 mRNA inactivation by RNase J competes with translation initiation. A) Histogram showing the percentage of reads mapping to a given position, out of the total number of reads mapping to the SA1075 transcript in each strain (shown in parentheses). Only positions of interest are included, but the full data-set can be found in Table S8 . +1: The putative transcription start site. +16: The major detected RNA species in the WT, ΔY and ΔcshA. +45: Position where ΔJ1ΔJ2 differs from ΔJ1, ΔJ2 and J1 AGA , and appears similar to WT. B) Important positions indicated on the SA1075 gene. +1: Transcription Start Site. RBS: Ribosome Binding Site. R2 indicates the probe used for the Northern blot in panel D. C) Hairpin structure predicted by the mfold algorithm, which sequesters the RBS and start-codon (shown in bold). No secondary structure was predicted for the 50 nucleotides downstream of position +45. D) Northern blot of the SA1075 transcript, using probe R2. The +1 and +16 RNA species are not resolved, and can be seen as a single band, however the +45 species is clearly visible in the ΔJ1, ΔJ2, and J1 AGA strains. The marker was stained with methylene blue and photographed. As a loading control, the Northern blot was stripped and re-probed to detect the 5S rRNA. E) The proposed model for determining the fate of SA1075 mRNA via competition between RNase J, ribosomes and the nuclease that cleaves at position +16. (PP)P indicates a mix of tri- and mono-phosphorylated RNA, generated by pyrophosphohydrolases. Nascent SA1075 mRNA can either be occupied by ribosomes, binding to the RBS, or form Hairpin I which sequesters the RBS. Ribosomes will shield position +45 from RNase J, but the hairpin will not. If cleavage at position +16 occurs before RNase J has cleaved at position +45, then the RBS will be liberated from Hairpin I, and ribosomes can initiate translation. If ever the +45 cut is made by RNase J, then the mRNA, which no longer has RBS or start-codon, is immediately degraded (possibly by the RNase J1+J2 complex). Either RNase J1 or RNase J2 can perform a cleavage at position +45. The loss of both RNases prevents the +45 RNA species from being generated, thus explaining why the WT and the ΔJ1ΔJ2 strains appear similar in panel A (see discussion for details and other potential explanations).
    Figure Legend Snippet: SA1075 mRNA inactivation by RNase J competes with translation initiation. A) Histogram showing the percentage of reads mapping to a given position, out of the total number of reads mapping to the SA1075 transcript in each strain (shown in parentheses). Only positions of interest are included, but the full data-set can be found in Table S8 . +1: The putative transcription start site. +16: The major detected RNA species in the WT, ΔY and ΔcshA. +45: Position where ΔJ1ΔJ2 differs from ΔJ1, ΔJ2 and J1 AGA , and appears similar to WT. B) Important positions indicated on the SA1075 gene. +1: Transcription Start Site. RBS: Ribosome Binding Site. R2 indicates the probe used for the Northern blot in panel D. C) Hairpin structure predicted by the mfold algorithm, which sequesters the RBS and start-codon (shown in bold). No secondary structure was predicted for the 50 nucleotides downstream of position +45. D) Northern blot of the SA1075 transcript, using probe R2. The +1 and +16 RNA species are not resolved, and can be seen as a single band, however the +45 species is clearly visible in the ΔJ1, ΔJ2, and J1 AGA strains. The marker was stained with methylene blue and photographed. As a loading control, the Northern blot was stripped and re-probed to detect the 5S rRNA. E) The proposed model for determining the fate of SA1075 mRNA via competition between RNase J, ribosomes and the nuclease that cleaves at position +16. (PP)P indicates a mix of tri- and mono-phosphorylated RNA, generated by pyrophosphohydrolases. Nascent SA1075 mRNA can either be occupied by ribosomes, binding to the RBS, or form Hairpin I which sequesters the RBS. Ribosomes will shield position +45 from RNase J, but the hairpin will not. If cleavage at position +16 occurs before RNase J has cleaved at position +45, then the RBS will be liberated from Hairpin I, and ribosomes can initiate translation. If ever the +45 cut is made by RNase J, then the mRNA, which no longer has RBS or start-codon, is immediately degraded (possibly by the RNase J1+J2 complex). Either RNase J1 or RNase J2 can perform a cleavage at position +45. The loss of both RNases prevents the +45 RNA species from being generated, thus explaining why the WT and the ΔJ1ΔJ2 strains appear similar in panel A (see discussion for details and other potential explanations).

    Techniques Used: Binding Assay, Northern Blot, Marker, Staining, Generated

    mRNA maturation by RNase J reveals a potential regulation of translation. A) Histogram showing the percentage of reads mapping to a given position, out of the total number of reads mapping to the SA2322 transcript in each strain (shown in parentheses). Only positions of interest are included, but the full data-set can be found in Table S7 . +1 and +2: The putative transcription start sites. +52: A major detected RNA species in the WT, ΔY and ΔcshA, however it is absent from the RNase J1 mutants and strongly reduced in the ΔJ2 strain. B) The SA2322 locus with important positions indicated. C) A schematic view of the fate of SA2322 transcripts. A newly formed transcript can form a secondary structure, shown in panel D, which partially sequesters the ribosome binding site (RBS). RNase J can shorten the transcript by 51 nt, and is presumably blocked from further exonucleolytic digestion by ribosomes binding to the RBS. (PP)P indicates a mix of tri- and mono-phosphorylated RNA, generated by pyrophosphohydrolases. D) Predicted secondary structure at the 5′-end of the SA2322 transcript. ΔG values predicted by the mfold algorithm are in kcal/mol. RBS and start codon are indicated in bold.
    Figure Legend Snippet: mRNA maturation by RNase J reveals a potential regulation of translation. A) Histogram showing the percentage of reads mapping to a given position, out of the total number of reads mapping to the SA2322 transcript in each strain (shown in parentheses). Only positions of interest are included, but the full data-set can be found in Table S7 . +1 and +2: The putative transcription start sites. +52: A major detected RNA species in the WT, ΔY and ΔcshA, however it is absent from the RNase J1 mutants and strongly reduced in the ΔJ2 strain. B) The SA2322 locus with important positions indicated. C) A schematic view of the fate of SA2322 transcripts. A newly formed transcript can form a secondary structure, shown in panel D, which partially sequesters the ribosome binding site (RBS). RNase J can shorten the transcript by 51 nt, and is presumably blocked from further exonucleolytic digestion by ribosomes binding to the RBS. (PP)P indicates a mix of tri- and mono-phosphorylated RNA, generated by pyrophosphohydrolases. D) Predicted secondary structure at the 5′-end of the SA2322 transcript. ΔG values predicted by the mfold algorithm are in kcal/mol. RBS and start codon are indicated in bold.

    Techniques Used: Binding Assay, Generated

    25) Product Images from "Synthesis of low immunogenicity RNA with high-temperature in vitro transcription"

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    Journal: RNA

    doi: 10.1261/rna.073858.119

    High-temperature IVT does not affect antisense dsRNA by-product formation. ( A ) Native gel electrophoresis analysis of IVT reactions on 512B DNA template using wild-type T7 (37°C) with/without RNase III treatment. ( B ) dsRNA immunoblot with J2 antibody on IVT reactions (crude and purified) with 512B template. ( C ) Native gel electrophoresis analyses and dsRNA immunoblot analysis of 512B IVT reactions conducted with TsT7-1 at 37°C versus 50°C.
    Figure Legend Snippet: High-temperature IVT does not affect antisense dsRNA by-product formation. ( A ) Native gel electrophoresis analysis of IVT reactions on 512B DNA template using wild-type T7 (37°C) with/without RNase III treatment. ( B ) dsRNA immunoblot with J2 antibody on IVT reactions (crude and purified) with 512B template. ( C ) Native gel electrophoresis analyses and dsRNA immunoblot analysis of 512B IVT reactions conducted with TsT7-1 at 37°C versus 50°C.

    Techniques Used: Nucleic Acid Electrophoresis, Purification

    26) Product Images from "Streamlined inactivation, amplification, and Cas13-based detection of SARS-CoV-2"

    Article Title: Streamlined inactivation, amplification, and Cas13-based detection of SARS-CoV-2

    Journal: Nature Communications

    doi: 10.1038/s41467-020-19097-x

    SARS-CoV-2 detection from unextracted samples using SHINE. a Schematic of SHINE, which streamlines SARS-CoV-2 detection by using HUDSON to inactivate samples and single-step SHERLOCK to detect viral RNA with an in-tube fluorescent or colorimetric readout. Times suggested incubation times, C control line, T test line. b Measurement of RNase activity using RNaseAlert after 30 min at room temperature from treated or untreated universal viral transport medium (UTM), saliva, and phosphate-buffered saline (PBS). c SARS-CoV-2 RNA detection in UTM using SHINE with the in-tube fluorescence readout after 1 h. d SARS-CoV-2 RNA detection in saliva using SHINE with the in-tube fluorescence readout after 1 h. e Schematic of the companion smartphone application for quantitatively analyzing in-tube fluorescence and reporting binary outcomes of SARS-CoV-2 detection. f Colorimetric detection of SARS-CoV-2 RNA in unextracted patient NP swabs using SHINE after 1 h. g SARS-CoV-2 detection from 50 unextracted patient samples using SHINE and smartphone application quantification of in-tube fluorescence after 40 min. Threshold line plotted as mean readout value for controls plus 3 standard deviations. h Concordance table between SHINE and RT-qPCR for 50 patient samples. For b , center = mean for 2 technical replicates. For b , g , source data are provided as a Source data file.
    Figure Legend Snippet: SARS-CoV-2 detection from unextracted samples using SHINE. a Schematic of SHINE, which streamlines SARS-CoV-2 detection by using HUDSON to inactivate samples and single-step SHERLOCK to detect viral RNA with an in-tube fluorescent or colorimetric readout. Times suggested incubation times, C control line, T test line. b Measurement of RNase activity using RNaseAlert after 30 min at room temperature from treated or untreated universal viral transport medium (UTM), saliva, and phosphate-buffered saline (PBS). c SARS-CoV-2 RNA detection in UTM using SHINE with the in-tube fluorescence readout after 1 h. d SARS-CoV-2 RNA detection in saliva using SHINE with the in-tube fluorescence readout after 1 h. e Schematic of the companion smartphone application for quantitatively analyzing in-tube fluorescence and reporting binary outcomes of SARS-CoV-2 detection. f Colorimetric detection of SARS-CoV-2 RNA in unextracted patient NP swabs using SHINE after 1 h. g SARS-CoV-2 detection from 50 unextracted patient samples using SHINE and smartphone application quantification of in-tube fluorescence after 40 min. Threshold line plotted as mean readout value for controls plus 3 standard deviations. h Concordance table between SHINE and RT-qPCR for 50 patient samples. For b , center = mean for 2 technical replicates. For b , g , source data are provided as a Source data file.

    Techniques Used: Incubation, Activity Assay, RNA Detection, Fluorescence, Quantitative RT-PCR

    27) Product Images from "A Short Tandem Repeat-Enriched RNA Assembles a Nuclear Compartment to Control Alternative Splicing and Promote Cell Survival"

    Article Title: A Short Tandem Repeat-Enriched RNA Assembles a Nuclear Compartment to Control Alternative Splicing and Promote Cell Survival

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2018.08.041

    PNCTR Knockdown Promotes Programmed Cell Death (A) HeLa cells were transfected with 400 nM gmControl or gmPNCTR and plated at the densities indicated. Note dramatically reduced numbers of colonies in gmPNCTR-treated wells compared to gmControl. (B) Colony confluency in (A) quantified from 3 independent transfection experiments and shown as mean ± SD. p values are calculated using a two-tailed t test. (C) Growth curves of HeLa cells transfected with 400 nM gmControl or gmPNCTR show that gmPNCTR leads to a visible decline in cell viability between 24 and 72 hpt. Data are averaged from 6 transfection experiments ± SD and compared by a two-tailed t test. (D) Time-resolved qRT-PCR analyses showing that gmPNCTR reaches a maximal downregulation effect by 12 hpt. Data are averaged from 3 experiments ± SD. (E) gmPNCTR, but not gmControl, induces expression of the apoptotic marker cleaved caspase-3 (CC3) at 12–24 hpt. (F) Dampening PNCTR levels often leads to extensive activation of caspase-3 in HeLa cells (arrowheads). The close up in the top-right corner compares DAPI staining for a normal nucleus (nN) of a CC3-negative cell and a pyknotic nucleus (pN) of a cell undergoing apoptosis. Scale bars, 10 μm. (G) CC3 induction by gmPNCTR is less efficient in HeLa cells expressing a gmPNCTR-resistant PNCTR fragment containing (UC)n repeats compared to the corresponding empty vector control. In (E) and (G), GAPDH is used as a lane-loading control. (H) GAPDH-normalized CC3 expression levels in (G) averaged from 3 experiments ± SD and compared by a two-tailed t test. See also Figure S5 .
    Figure Legend Snippet: PNCTR Knockdown Promotes Programmed Cell Death (A) HeLa cells were transfected with 400 nM gmControl or gmPNCTR and plated at the densities indicated. Note dramatically reduced numbers of colonies in gmPNCTR-treated wells compared to gmControl. (B) Colony confluency in (A) quantified from 3 independent transfection experiments and shown as mean ± SD. p values are calculated using a two-tailed t test. (C) Growth curves of HeLa cells transfected with 400 nM gmControl or gmPNCTR show that gmPNCTR leads to a visible decline in cell viability between 24 and 72 hpt. Data are averaged from 6 transfection experiments ± SD and compared by a two-tailed t test. (D) Time-resolved qRT-PCR analyses showing that gmPNCTR reaches a maximal downregulation effect by 12 hpt. Data are averaged from 3 experiments ± SD. (E) gmPNCTR, but not gmControl, induces expression of the apoptotic marker cleaved caspase-3 (CC3) at 12–24 hpt. (F) Dampening PNCTR levels often leads to extensive activation of caspase-3 in HeLa cells (arrowheads). The close up in the top-right corner compares DAPI staining for a normal nucleus (nN) of a CC3-negative cell and a pyknotic nucleus (pN) of a cell undergoing apoptosis. Scale bars, 10 μm. (G) CC3 induction by gmPNCTR is less efficient in HeLa cells expressing a gmPNCTR-resistant PNCTR fragment containing (UC)n repeats compared to the corresponding empty vector control. In (E) and (G), GAPDH is used as a lane-loading control. (H) GAPDH-normalized CC3 expression levels in (G) averaged from 3 experiments ± SD and compared by a two-tailed t test. See also Figure S5 .

    Techniques Used: Transfection, Two Tailed Test, Quantitative RT-PCR, Expressing, Marker, Activation Assay, Staining, Plasmid Preparation

    PNCTR Is a pol-I Transcript Interacting with Multiple Copies of PTBP1 Protein (A) Diagram of the predicted PNCTR locus also showing an adjacent 47S/45S rRNA gene and probes used in this study. Mapping to chr21 should be considered provisional since different IGS sequences share extensive regions of homology, and not all parts of human rDNA have been sequenced. (B) Top: northern blot analysis of PNCTR expression in HeLa cells using the probe introduced in (A). Bottom: methylene-blue-stained membrane showing that the lanes were loaded equally. (C) RIP carried out with a PTBP1-specific antibody or a non-immune IgG control. Immunoprecipitated RNAs were analyzed by qRT-PCR using primers specific to PNCTR, PTBP2 pre-mRNA (positive control), or U6 snRNA (negative control). Data are averaged from three experiments ± SD and compared by a two-tailed t test. (D) EMSA with purified PTBP1 protein and a PNCTR-specific RNA probe (sequence on the top). Bottom right: multivalent complexes assemble on incubating the probe with increasing amounts of PTBP1. Bottom left: no band shifts are detected when PTBP1 is substituted with BSA. (E) The PTBP1-PNCTR interaction in (D) is specific since it can be disrupted by increasing amounts of unlabeled PNCTR probe (bottom left), but not a control competitor (top, control RNA sequence; bottom right, the EMSA result). (F) IF-FISH staining of HeLa cells showing that PNCTR co-localizes with PTBP1 in the perinucleolar compartment (PNC). FBL, nucleolar marker fibrillarin. Scale bar, 10 μm. See also Figure S2 .
    Figure Legend Snippet: PNCTR Is a pol-I Transcript Interacting with Multiple Copies of PTBP1 Protein (A) Diagram of the predicted PNCTR locus also showing an adjacent 47S/45S rRNA gene and probes used in this study. Mapping to chr21 should be considered provisional since different IGS sequences share extensive regions of homology, and not all parts of human rDNA have been sequenced. (B) Top: northern blot analysis of PNCTR expression in HeLa cells using the probe introduced in (A). Bottom: methylene-blue-stained membrane showing that the lanes were loaded equally. (C) RIP carried out with a PTBP1-specific antibody or a non-immune IgG control. Immunoprecipitated RNAs were analyzed by qRT-PCR using primers specific to PNCTR, PTBP2 pre-mRNA (positive control), or U6 snRNA (negative control). Data are averaged from three experiments ± SD and compared by a two-tailed t test. (D) EMSA with purified PTBP1 protein and a PNCTR-specific RNA probe (sequence on the top). Bottom right: multivalent complexes assemble on incubating the probe with increasing amounts of PTBP1. Bottom left: no band shifts are detected when PTBP1 is substituted with BSA. (E) The PTBP1-PNCTR interaction in (D) is specific since it can be disrupted by increasing amounts of unlabeled PNCTR probe (bottom left), but not a control competitor (top, control RNA sequence; bottom right, the EMSA result). (F) IF-FISH staining of HeLa cells showing that PNCTR co-localizes with PTBP1 in the perinucleolar compartment (PNC). FBL, nucleolar marker fibrillarin. Scale bar, 10 μm. See also Figure S2 .

    Techniques Used: Northern Blot, Expressing, Staining, Immunoprecipitation, Quantitative RT-PCR, Positive Control, Negative Control, Two Tailed Test, Purification, Sequencing, Fluorescence In Situ Hybridization, Marker

    PNCTR Is Often Upregulated in Cancer Cells (A) qRT-PCR analyses showing that PNCTR expression is orders of magnitude higher in transformed cells (HeLa, HCT116, SW620, MCF7, and the SV40-transformed clone VA-13 of the normal lung fibroblast line WI-38) than in their non-transformed counterparts (ARPE-19 and WI-38). The data are averaged from 3 assays ± SD and the expression levels in WI-38 cells were set to 1. (B) qRT-PCR analyses carried out with and without reverse transcriptase (RT) show that the PNCTR signals in ARPE-19 and WI-38 correspond to bona fide expression of this strRNA at low but detectable levels. (C) qRT-PCR comparison of PNCTR expression in five invasive lung cancers and patient-matched normal lung samples ( Table S3 ). Data were obtained using RqP1 primers, normalized to β-actin, averaged from 3 amplification experiments ± SD and compared by a two-tailed t test. (D) PNCTR-positive nuclear dots are readily detectable by RNA-FISH in a lymph node metastasis sample collected for the case (a) in (C), but not in the matching normal lung control. (E) A close up of the boxed area in (D). Scale bars in (D) and (E), 10 μm. See also Figure S7 .
    Figure Legend Snippet: PNCTR Is Often Upregulated in Cancer Cells (A) qRT-PCR analyses showing that PNCTR expression is orders of magnitude higher in transformed cells (HeLa, HCT116, SW620, MCF7, and the SV40-transformed clone VA-13 of the normal lung fibroblast line WI-38) than in their non-transformed counterparts (ARPE-19 and WI-38). The data are averaged from 3 assays ± SD and the expression levels in WI-38 cells were set to 1. (B) qRT-PCR analyses carried out with and without reverse transcriptase (RT) show that the PNCTR signals in ARPE-19 and WI-38 correspond to bona fide expression of this strRNA at low but detectable levels. (C) qRT-PCR comparison of PNCTR expression in five invasive lung cancers and patient-matched normal lung samples ( Table S3 ). Data were obtained using RqP1 primers, normalized to β-actin, averaged from 3 amplification experiments ± SD and compared by a two-tailed t test. (D) PNCTR-positive nuclear dots are readily detectable by RNA-FISH in a lymph node metastasis sample collected for the case (a) in (C), but not in the matching normal lung control. (E) A close up of the boxed area in (D). Scale bars in (D) and (E), 10 μm. See also Figure S7 .

    Techniques Used: Quantitative RT-PCR, Expressing, Transformation Assay, Amplification, Two Tailed Test, Fluorescence In Situ Hybridization

    Identification of strRNAs Enriched in RBP Interaction Motifs (A) Workflow used in this study. (B) Transcripts newly predicted by the pipeline in (A) (“new”) are significantly over-represented among RBP motif-enriched RNAs as compared to previously annotated (“known”) transcripts. (C) strRNAs have significantly shorter ORFs compared to annotated mRNAs and the entire transcriptome. (D) STR content of strRNAs substantially exceeds corresponding transcriptome and genome values. (E) qRT-PCR and RT-PCR validation of five newly identified strRNAs using samples prepared without reverse transcriptase (RT) as negative controls. Data are shown as mean ± SD. See also Figure S1 and Table S1 .
    Figure Legend Snippet: Identification of strRNAs Enriched in RBP Interaction Motifs (A) Workflow used in this study. (B) Transcripts newly predicted by the pipeline in (A) (“new”) are significantly over-represented among RBP motif-enriched RNAs as compared to previously annotated (“known”) transcripts. (C) strRNAs have significantly shorter ORFs compared to annotated mRNAs and the entire transcriptome. (D) STR content of strRNAs substantially exceeds corresponding transcriptome and genome values. (E) qRT-PCR and RT-PCR validation of five newly identified strRNAs using samples prepared without reverse transcriptase (RT) as negative controls. Data are shown as mean ± SD. See also Figure S1 and Table S1 .

    Techniques Used: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

    PNCTR Antagonizes Splicing Regulation Function of PTBP1 (A) RNA-seq analyses carried out to examine possible role of PNCTR in modulating PTBP1 activity as a regulator of alternative splicing. (B–E) Fisher’s exact tests showing that gmPNCTR-regulated alternative splicing events are significantly enriched among those regulated by (B) siPTBP1 or (C) siPTBP1/2, as compared to their occurrence in the entire list of alternative splicing events (Total) considered by ExpressionPlot. Note that alternative events controlled by both (D) gmPNCTR and siPTBP1 or (E) gmPNCTR and siPTBP1/2 are preferentially regulated in opposite directions (anti-regulated) rather than the same direction (co-regulated). (F) Regulation of CHEK2 exon 8 splicing by the PNCTR/PTBP1 circuitry. Left: the two alternative splicing possibilities. Right: RT-PCR analyses of HeLa cells showing that combined knockdown of PTBP1 and PTBP2 (siPTBP1/2) stimulates exon 8 inclusion, while knockdown of PNCTR (gmPNCTR, 400 nM) or overexpression of recombinant FLAG-tagged PTBP1 promotes its skipping. (G) Effects in (F) presented as differences in percent-spliced-in values (ΔΨ; Wang et al., 2008b ) between experimental treatments and the corresponding controls. Positive ΔΨ values indicate an increase and negative, a decrease in exon 8 inclusion. Similar quantifications were also done for cells transfected with 25 and 100 nM gapmers. All data are averaged from 3 experimentally independent comparisons ± SD and analyzed by a paired t test. (H) CLIP-seq and iCLIP analyses show that PTBP1 forms physical contacts with an extensive array of YUCUYY and YYUCUY motifs in front of CHEK2 exon 8. Functional significance of the PTBP1 interaction sequence highlighted in gray was validated in the minigene experiment in Figures S6 L and S6M. (I) CLIP/qRT-PCR experiment showing an increase in PTBP1 interaction efficiency with the CHEK2 exon 8 region (CLIP-CHEK2e8) in HeLa cells treated with gmPNCTR and a lack of this effect for an upstream (CLIP-CHEK2e2) and a downstream region (CLIP-CHEK2i8). PTBP2 pre-mRNA exon 10 region (CLIP-PTBP2e10) and ACTB mRNA are used as a positive and a negative control, respectively. Data are averaged from two triplicated CLIP/qRT-PCR experiments ± SD and compared by a two-tailed t test. (J) Left: immunoblot analysis showing a decrease in the CHEK2 protein levels in HeLa cells transfected for 24 hr with gmPNCTR compared to gmControl. CC3 is used as a sample identity marker and GAPDH as a lane-loading control. Right: immunoblot quantification showing GAPDH-normalized CHEK2 expression levels averaged from 3 experiments ± SD and compared by paired t test. (K) Left: HeLa cells treated with 50 nM of either siControl or siCHEK2 for 36 hr were post-transfected with 400 nM of gmPNCTR or gmControl for 12 hr and analyzed for CC3 expression. Note that the preemptive knockdown of CHEK2 facilitates induction of CC3 in the gmPNCTR samples. Right: GAPDH-normalized CC3 expression averaged from 6 experiments ± SD and compared by paired t test. (L) Expression of recombinant PTBP1 is sufficient to upregulate CC3 in HeLa cells. Left: immunoblot analysis of control and FLAG-PTBP1-transfected samples. Right: GAPDH-normalized CC3 expression averaged from 3 experiments ± SD and compared by paired t test. See also Figures S5 and S6 and Table S2 .
    Figure Legend Snippet: PNCTR Antagonizes Splicing Regulation Function of PTBP1 (A) RNA-seq analyses carried out to examine possible role of PNCTR in modulating PTBP1 activity as a regulator of alternative splicing. (B–E) Fisher’s exact tests showing that gmPNCTR-regulated alternative splicing events are significantly enriched among those regulated by (B) siPTBP1 or (C) siPTBP1/2, as compared to their occurrence in the entire list of alternative splicing events (Total) considered by ExpressionPlot. Note that alternative events controlled by both (D) gmPNCTR and siPTBP1 or (E) gmPNCTR and siPTBP1/2 are preferentially regulated in opposite directions (anti-regulated) rather than the same direction (co-regulated). (F) Regulation of CHEK2 exon 8 splicing by the PNCTR/PTBP1 circuitry. Left: the two alternative splicing possibilities. Right: RT-PCR analyses of HeLa cells showing that combined knockdown of PTBP1 and PTBP2 (siPTBP1/2) stimulates exon 8 inclusion, while knockdown of PNCTR (gmPNCTR, 400 nM) or overexpression of recombinant FLAG-tagged PTBP1 promotes its skipping. (G) Effects in (F) presented as differences in percent-spliced-in values (ΔΨ; Wang et al., 2008b ) between experimental treatments and the corresponding controls. Positive ΔΨ values indicate an increase and negative, a decrease in exon 8 inclusion. Similar quantifications were also done for cells transfected with 25 and 100 nM gapmers. All data are averaged from 3 experimentally independent comparisons ± SD and analyzed by a paired t test. (H) CLIP-seq and iCLIP analyses show that PTBP1 forms physical contacts with an extensive array of YUCUYY and YYUCUY motifs in front of CHEK2 exon 8. Functional significance of the PTBP1 interaction sequence highlighted in gray was validated in the minigene experiment in Figures S6 L and S6M. (I) CLIP/qRT-PCR experiment showing an increase in PTBP1 interaction efficiency with the CHEK2 exon 8 region (CLIP-CHEK2e8) in HeLa cells treated with gmPNCTR and a lack of this effect for an upstream (CLIP-CHEK2e2) and a downstream region (CLIP-CHEK2i8). PTBP2 pre-mRNA exon 10 region (CLIP-PTBP2e10) and ACTB mRNA are used as a positive and a negative control, respectively. Data are averaged from two triplicated CLIP/qRT-PCR experiments ± SD and compared by a two-tailed t test. (J) Left: immunoblot analysis showing a decrease in the CHEK2 protein levels in HeLa cells transfected for 24 hr with gmPNCTR compared to gmControl. CC3 is used as a sample identity marker and GAPDH as a lane-loading control. Right: immunoblot quantification showing GAPDH-normalized CHEK2 expression levels averaged from 3 experiments ± SD and compared by paired t test. (K) Left: HeLa cells treated with 50 nM of either siControl or siCHEK2 for 36 hr were post-transfected with 400 nM of gmPNCTR or gmControl for 12 hr and analyzed for CC3 expression. Note that the preemptive knockdown of CHEK2 facilitates induction of CC3 in the gmPNCTR samples. Right: GAPDH-normalized CC3 expression averaged from 6 experiments ± SD and compared by paired t test. (L) Expression of recombinant PTBP1 is sufficient to upregulate CC3 in HeLa cells. Left: immunoblot analysis of control and FLAG-PTBP1-transfected samples. Right: GAPDH-normalized CC3 expression averaged from 3 experiments ± SD and compared by paired t test. See also Figures S5 and S6 and Table S2 .

    Techniques Used: RNA Sequencing Assay, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Over Expression, Recombinant, Transfection, Cross-linking Immunoprecipitation, Functional Assay, Sequencing, Quantitative RT-PCR, Negative Control, Two Tailed Test, Marker, Expressing

    28) Product Images from "Dicer cleaves 5′-extended microRNA precursors originating from RNA polymerase II transcription start sites"

    Article Title: Dicer cleaves 5′-extended microRNA precursors originating from RNA polymerase II transcription start sites

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky306

    Processing of 5′-extended pre-miRNAs is independent of Dicer 5′ pocket. ( A ) In vitro transcribed 5′-extended pre-miR-HSUR4s were incubated with four different human Dicers purified by Flag IP. [WT: wild type Dicer; TN: transdominant negative mutation Dicer; 3′ mut: 3′ pocket mutation Dicer (Y926A); 5′ mut: 5′ pocket mutation Dicer (R778A/R780A/H982A)]. Northern blots were performed using probes targeting miR-HSUR4-3p. ( B ) Quantitations of relative mature miRNA levels (mature miRNA/pre-miRNA) compared to WT Dicer processing in (A) (mean ± standard deviation) were derived from three independent experiments. FC: fold change. ( C ) In vitro transcribed 5′-monophosphate pre-miR-HSUR4s without 5′ extension (–2 nt) but with 1 nt or 2 nt 3′ overhang were incubated with four different human Dicers purified by Flag IP. Schematics of pre-miR-HSUR4s with 1 nt or 2 nt 3′ overhang are given. ( D ) Quantitations of relative mature miRNA levels compared to WT Dicer processing in (C) were derived from three independent experiments. ( E ) In vitro transcribed 5′-monophosphate pre-let-7a-1 and m 7 G-capped +15 nt extended pre-let-7a-1 were incubated with four different human Dicers purified by Flag IP. Northern blots were performed using probes targeting let-7a-1 (5p). Schematic of +15 nt extended pre-let-7a-1 is shown, with extended sequence underlined. ( F ) Quantitations of relative mature miRNA levels compared to WT Dicer processing in (E) were derived from three independent experiments.
    Figure Legend Snippet: Processing of 5′-extended pre-miRNAs is independent of Dicer 5′ pocket. ( A ) In vitro transcribed 5′-extended pre-miR-HSUR4s were incubated with four different human Dicers purified by Flag IP. [WT: wild type Dicer; TN: transdominant negative mutation Dicer; 3′ mut: 3′ pocket mutation Dicer (Y926A); 5′ mut: 5′ pocket mutation Dicer (R778A/R780A/H982A)]. Northern blots were performed using probes targeting miR-HSUR4-3p. ( B ) Quantitations of relative mature miRNA levels (mature miRNA/pre-miRNA) compared to WT Dicer processing in (A) (mean ± standard deviation) were derived from three independent experiments. FC: fold change. ( C ) In vitro transcribed 5′-monophosphate pre-miR-HSUR4s without 5′ extension (–2 nt) but with 1 nt or 2 nt 3′ overhang were incubated with four different human Dicers purified by Flag IP. Schematics of pre-miR-HSUR4s with 1 nt or 2 nt 3′ overhang are given. ( D ) Quantitations of relative mature miRNA levels compared to WT Dicer processing in (C) were derived from three independent experiments. ( E ) In vitro transcribed 5′-monophosphate pre-let-7a-1 and m 7 G-capped +15 nt extended pre-let-7a-1 were incubated with four different human Dicers purified by Flag IP. Northern blots were performed using probes targeting let-7a-1 (5p). Schematic of +15 nt extended pre-let-7a-1 is shown, with extended sequence underlined. ( F ) Quantitations of relative mature miRNA levels compared to WT Dicer processing in (E) were derived from three independent experiments.

    Techniques Used: In Vitro, Incubation, Purification, Mutagenesis, Northern Blot, Standard Deviation, Derivative Assay, Sequencing

    29) Product Images from "SPRINT: a Cas13a-based platform for detection of small molecules"

    Article Title: SPRINT: a Cas13a-based platform for detection of small molecules

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa673

    Transcription and nuclease activities constitute SPRINT assays. ( A ) Certain allosteric transcription factors release their DNA binding site when they are bound by ligand, therefore enabling transcript elongation. ( B ) An ON-riboswitch allows transcript elongation by RNA polymerase when the riboswitch is bound to its cognate ligand. ( C ) Cas13a detects RNA transcripts that contain the target sequence (yellow). After binding its target, Cas13a collaterally cleaves RNA oligonucleotides and de-quenches fluorophores. ( D ) Workflow of a typical SPRINT experiment. A master mix containing components such as RNA polymerase, Cas13a and DNA template is incubated and then added to wells that contain compounds that regulate transcription. The transcriptional output is then measured via the fluorescence signal.
    Figure Legend Snippet: Transcription and nuclease activities constitute SPRINT assays. ( A ) Certain allosteric transcription factors release their DNA binding site when they are bound by ligand, therefore enabling transcript elongation. ( B ) An ON-riboswitch allows transcript elongation by RNA polymerase when the riboswitch is bound to its cognate ligand. ( C ) Cas13a detects RNA transcripts that contain the target sequence (yellow). After binding its target, Cas13a collaterally cleaves RNA oligonucleotides and de-quenches fluorophores. ( D ) Workflow of a typical SPRINT experiment. A master mix containing components such as RNA polymerase, Cas13a and DNA template is incubated and then added to wells that contain compounds that regulate transcription. The transcriptional output is then measured via the fluorescence signal.

    Techniques Used: Binding Assay, Sequencing, Incubation, Fluorescence

    SPRINT can be used for compound screens and enzyme-coupled assays. Fluorescent signal of the SPRINT reactions was background subtracted and normalized to 125 nM fluorescein. ( A ) The guanine riboswitch xpt/pbuE *6U regulated transcription in response to 30 different compounds. Signal with solvent only was subtracted from signal with ligand to correct for differences in solvents of ligands. Dots represent mean value, n = 3. ( B ) The effect of the compounds on transcription from a constitutive promoter was measured. Signal with solvent only was subtracted from signal with ligand to correct for differences in solvents of ligands. Dots represent one biological replicate. ( C ) SPRINT measured constitutive transcription of Cas13a target as transcription was inhibited at increasing concentrations of rifampicin. Bars indicate mean value, error bars indicate s.d. from the mean. n = 3. ( D ) Diagram of an enzyme-coupled assay. The enzyme hPNP converts inosine to hypoxanthine, which is detected by the guanine riboswitch xpt/pbuE *6U and triggers the SPRINT signal. ( E ) The enzyme-coupled assay was used to measure enzymatic activity of hPNP. Concentration of inosine was 1 mM, hPNP was added to an activity of 10 mU/μl, concentration of Immucillin-H was 10 μM. Bars indicate mean value, error bars indicate s.d. from the mean. n = 3. p -value was calculated using a two-tailed t -test. ( F ) The enzyme-coupled assay was used to measure concentration-dependent substrate conversion. hPNP was added to an activity of 1 mU/μl. Bars indicate mean value, error bars indicate s.d. from the mean. n = 3.
    Figure Legend Snippet: SPRINT can be used for compound screens and enzyme-coupled assays. Fluorescent signal of the SPRINT reactions was background subtracted and normalized to 125 nM fluorescein. ( A ) The guanine riboswitch xpt/pbuE *6U regulated transcription in response to 30 different compounds. Signal with solvent only was subtracted from signal with ligand to correct for differences in solvents of ligands. Dots represent mean value, n = 3. ( B ) The effect of the compounds on transcription from a constitutive promoter was measured. Signal with solvent only was subtracted from signal with ligand to correct for differences in solvents of ligands. Dots represent one biological replicate. ( C ) SPRINT measured constitutive transcription of Cas13a target as transcription was inhibited at increasing concentrations of rifampicin. Bars indicate mean value, error bars indicate s.d. from the mean. n = 3. ( D ) Diagram of an enzyme-coupled assay. The enzyme hPNP converts inosine to hypoxanthine, which is detected by the guanine riboswitch xpt/pbuE *6U and triggers the SPRINT signal. ( E ) The enzyme-coupled assay was used to measure enzymatic activity of hPNP. Concentration of inosine was 1 mM, hPNP was added to an activity of 10 mU/μl, concentration of Immucillin-H was 10 μM. Bars indicate mean value, error bars indicate s.d. from the mean. n = 3. p -value was calculated using a two-tailed t -test. ( F ) The enzyme-coupled assay was used to measure concentration-dependent substrate conversion. hPNP was added to an activity of 1 mU/μl. Bars indicate mean value, error bars indicate s.d. from the mean. n = 3.

    Techniques Used: Activity Assay, Concentration Assay, Two Tailed Test

    30) Product Images from "RNA promotes the formation of spatial compartments in the nucleus"

    Article Title: RNA promotes the formation of spatial compartments in the nucleus

    Journal: bioRxiv

    doi: 10.1101/2020.08.25.267435

    RNA localization on DNA and within the nucleus for RNAs within each RNA hub. (A) Weighted genomic DNA localization heatmap of each individual RNA. RNAs are organized by their RNA hub occupancy (shown in Figure 2A ). Contacts are normalized from 0 to 1 to account for expression levels of each RNA. (B) Pearson correlation of RNA-DNA unweighted contact frequencies across the genome for individual RNAs within the nuclear hubs (nucleolar, centromeric, spliceosomal, and scaRNA hubs). Red represents high correlation and blue represents low correlation. (C) RNA FISH of various non-coding RNAs within the same hub in the nucleus. Spliceosomal hub (top): Malat1 lncRNA and 7SK RNA and (bottom): U6 and U1 spliceosomal RNAs. Nucleolar hub (top): snora26 snoRNA and 45S pre-rRNA ITS2 and (bottom): RNase MRP (Rmrp) and 45S pre-rRNA ITS1. Each panel is shown individually (left and middle) and overlaid (right). Dashed lines demarcate the nuclear boundary identified with DAPI. Scalebar is 10μm. (D) RNA FISH (left) along with nucleolin immunofluorescence (middle) and DAPI (right) of specific ncRNAs. 7SK RNA (top), ITS1 regions of 45S pre-rRNA (middle) and tRNAs (bottom). tRNAs are visualized using pooled RNA FISH probes (see Methods). Scalebar is 10μm.
    Figure Legend Snippet: RNA localization on DNA and within the nucleus for RNAs within each RNA hub. (A) Weighted genomic DNA localization heatmap of each individual RNA. RNAs are organized by their RNA hub occupancy (shown in Figure 2A ). Contacts are normalized from 0 to 1 to account for expression levels of each RNA. (B) Pearson correlation of RNA-DNA unweighted contact frequencies across the genome for individual RNAs within the nuclear hubs (nucleolar, centromeric, spliceosomal, and scaRNA hubs). Red represents high correlation and blue represents low correlation. (C) RNA FISH of various non-coding RNAs within the same hub in the nucleus. Spliceosomal hub (top): Malat1 lncRNA and 7SK RNA and (bottom): U6 and U1 spliceosomal RNAs. Nucleolar hub (top): snora26 snoRNA and 45S pre-rRNA ITS2 and (bottom): RNase MRP (Rmrp) and 45S pre-rRNA ITS1. Each panel is shown individually (left and middle) and overlaid (right). Dashed lines demarcate the nuclear boundary identified with DAPI. Scalebar is 10μm. (D) RNA FISH (left) along with nucleolin immunofluorescence (middle) and DAPI (right) of specific ncRNAs. 7SK RNA (top), ITS1 regions of 45S pre-rRNA (middle) and tRNAs (bottom). tRNAs are visualized using pooled RNA FISH probes (see Methods). Scalebar is 10μm.

    Techniques Used: Expressing, Fluorescence In Situ Hybridization, Immunofluorescence

    31) Product Images from "RNA promotes the formation of spatial compartments in the nucleus"

    Article Title: RNA promotes the formation of spatial compartments in the nucleus

    Journal: bioRxiv

    doi: 10.1101/2020.08.25.267435

    RNA localization on DNA and within the nucleus for RNAs within each RNA hub. (A) Weighted genomic DNA localization heatmap of each individual RNA. RNAs are organized by their RNA hub occupancy (shown in Figure 2A ). Contacts are normalized from 0 to 1 to account for expression levels of each RNA. (B) Pearson correlation of RNA-DNA unweighted contact frequencies across the genome for individual RNAs within the nuclear hubs (nucleolar, centromeric, spliceosomal, and scaRNA hubs). Red represents high correlation and blue represents low correlation. (C) RNA FISH of various non-coding RNAs within the same hub in the nucleus. Spliceosomal hub (top): Malat1 lncRNA and 7SK RNA and (bottom): U6 and U1 spliceosomal RNAs. Nucleolar hub (top): snora26 snoRNA and 45S pre-rRNA ITS2 and (bottom): RNase MRP (Rmrp) and 45S pre-rRNA ITS1. Each panel is shown individually (left and middle) and overlaid (right). Dashed lines demarcate the nuclear boundary identified with DAPI. Scalebar is 10μm. (D) RNA FISH (left) along with nucleolin immunofluorescence (middle) and DAPI (right) of specific ncRNAs. 7SK RNA (top), ITS1 regions of 45S pre-rRNA (middle) and tRNAs (bottom). tRNAs are visualized using pooled RNA FISH probes (see Methods). Scalebar is 10μm.
    Figure Legend Snippet: RNA localization on DNA and within the nucleus for RNAs within each RNA hub. (A) Weighted genomic DNA localization heatmap of each individual RNA. RNAs are organized by their RNA hub occupancy (shown in Figure 2A ). Contacts are normalized from 0 to 1 to account for expression levels of each RNA. (B) Pearson correlation of RNA-DNA unweighted contact frequencies across the genome for individual RNAs within the nuclear hubs (nucleolar, centromeric, spliceosomal, and scaRNA hubs). Red represents high correlation and blue represents low correlation. (C) RNA FISH of various non-coding RNAs within the same hub in the nucleus. Spliceosomal hub (top): Malat1 lncRNA and 7SK RNA and (bottom): U6 and U1 spliceosomal RNAs. Nucleolar hub (top): snora26 snoRNA and 45S pre-rRNA ITS2 and (bottom): RNase MRP (Rmrp) and 45S pre-rRNA ITS1. Each panel is shown individually (left and middle) and overlaid (right). Dashed lines demarcate the nuclear boundary identified with DAPI. Scalebar is 10μm. (D) RNA FISH (left) along with nucleolin immunofluorescence (middle) and DAPI (right) of specific ncRNAs. 7SK RNA (top), ITS1 regions of 45S pre-rRNA (middle) and tRNAs (bottom). tRNAs are visualized using pooled RNA FISH probes (see Methods). Scalebar is 10μm.

    Techniques Used: Expressing, Fluorescence In Situ Hybridization, Immunofluorescence

    32) Product Images from "Streamlined inactivation, amplification, and Cas13-based detection of SARS-CoV-2"

    Article Title: Streamlined inactivation, amplification, and Cas13-based detection of SARS-CoV-2

    Journal: Nature Communications

    doi: 10.1038/s41467-020-19097-x

    SARS-CoV-2 detection from unextracted samples using SHINE. a Schematic of SHINE, which streamlines SARS-CoV-2 detection by using HUDSON to inactivate samples and single-step SHERLOCK to detect viral RNA with an in-tube fluorescent or colorimetric readout. Times suggested incubation times, C control line, T test line. b Measurement of RNase activity using RNaseAlert after 30 min at room temperature from treated or untreated universal viral transport medium (UTM), saliva, and phosphate-buffered saline (PBS). c SARS-CoV-2 RNA detection in UTM using SHINE with the in-tube fluorescence readout after 1 h. d SARS-CoV-2 RNA detection in saliva using SHINE with the in-tube fluorescence readout after 1 h. e Schematic of the companion smartphone application for quantitatively analyzing in-tube fluorescence and reporting binary outcomes of SARS-CoV-2 detection. f Colorimetric detection of SARS-CoV-2 RNA in unextracted patient NP swabs using SHINE after 1 h. g SARS-CoV-2 detection from 50 unextracted patient samples using SHINE and smartphone application quantification of in-tube fluorescence after 40 min. Threshold line plotted as mean readout value for controls plus 3 standard deviations. h Concordance table between SHINE and RT-qPCR for 50 patient samples. For b , center = mean for 2 technical replicates. For b , g , source data are provided as a Source data file.
    Figure Legend Snippet: SARS-CoV-2 detection from unextracted samples using SHINE. a Schematic of SHINE, which streamlines SARS-CoV-2 detection by using HUDSON to inactivate samples and single-step SHERLOCK to detect viral RNA with an in-tube fluorescent or colorimetric readout. Times suggested incubation times, C control line, T test line. b Measurement of RNase activity using RNaseAlert after 30 min at room temperature from treated or untreated universal viral transport medium (UTM), saliva, and phosphate-buffered saline (PBS). c SARS-CoV-2 RNA detection in UTM using SHINE with the in-tube fluorescence readout after 1 h. d SARS-CoV-2 RNA detection in saliva using SHINE with the in-tube fluorescence readout after 1 h. e Schematic of the companion smartphone application for quantitatively analyzing in-tube fluorescence and reporting binary outcomes of SARS-CoV-2 detection. f Colorimetric detection of SARS-CoV-2 RNA in unextracted patient NP swabs using SHINE after 1 h. g SARS-CoV-2 detection from 50 unextracted patient samples using SHINE and smartphone application quantification of in-tube fluorescence after 40 min. Threshold line plotted as mean readout value for controls plus 3 standard deviations. h Concordance table between SHINE and RT-qPCR for 50 patient samples. For b , center = mean for 2 technical replicates. For b , g , source data are provided as a Source data file.

    Techniques Used: Incubation, Activity Assay, RNA Detection, Fluorescence, Quantitative RT-PCR

    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

    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

    Related Articles

    In Vitro:

    Article Title: eIF3d is an mRNA cap-binding protein required for specialized translation initiation
    Article Snippet: .. Each translation reaction contained 50% in vitro translation lysate and buffer to make the final reaction with 0.84 mM ATP, 0.21 mM GTP, 21 mM creatine phosphate (Roche), 45 U ml-1 creatine phosphokinase (Roche), 10 mM HEPES-KOH pH 7.6, 2 mM DTT, 8 mM amino acids (Promega), 255 mM spermidine, 1 U ml-1 murine RNase inhibitor (NEB), and mRNA-specific concentrations of Mg(OAc)2 and KOAc. ..

    Protease Inhibitor:

    Article Title: RNA binding to CBP stimulates histone acetylation and transcription
    Article Snippet: .. Reactions contained 1× HAT assay buffer (50mM Tris-HCl pH 7.5 (RT), 5% glycerol, 0.1mM EDTA, 50mM KCl), 1mM DTT, 10mM Na-Butyrate, 1× Complete EDTA protease inhibitor cocktail (Roche), 0.4U/ul murine RNAse inhibitor (NEB), 0.1mg/ml BSA (NEB), 0–200nM H31–21 peptide (Anaspec) and the required concentration of RNA probe. .. 3 H labeled Acetyl Co-enzyme A (3 H-acetyl CoA) (specific activity = 3.95 Ci/mmol, 0.1uCi/ul, Perkin Elmer) was diluted in a 1:1 ratio with unlabeled acetyl CoA (Sigma) to give a final concentration of 100uM and 0.3uCi per reaction.

    Purification:

    Article Title: RNA targeting with CRISPR-Cas13a
    Article Snippet: .. Briefly, reactions consisted of 45 nM purified LwaCas13a, 22.5 nM crRNA, 125 nM quenched fluorescent RNA reporter (RNAse Alert v2, Thermo Scientific), 2 μL murine RNase inhibitor (New England Biolabs), 100 ng of background total human RNA (purified from HEK293FT culture), and varying amounts of input nucleic acid target, unless otherwise indicated, in nuclease assay buffer (40 mM Tris-HCl, 60 mM NaCl, 6 mM MgCl2, pH 7.3). .. Reactions were allowed to proceed for 1-3 hr at 37°C (unless otherwise indicated) on a fluorescent plate reader (BioTek) with fluorescent kinetics measured every 5 min.

    Concentration Assay:

    Article Title: RNA binding to CBP stimulates histone acetylation and transcription
    Article Snippet: .. Reactions contained 1× HAT assay buffer (50mM Tris-HCl pH 7.5 (RT), 5% glycerol, 0.1mM EDTA, 50mM KCl), 1mM DTT, 10mM Na-Butyrate, 1× Complete EDTA protease inhibitor cocktail (Roche), 0.4U/ul murine RNAse inhibitor (NEB), 0.1mg/ml BSA (NEB), 0–200nM H31–21 peptide (Anaspec) and the required concentration of RNA probe. .. 3 H labeled Acetyl Co-enzyme A (3 H-acetyl CoA) (specific activity = 3.95 Ci/mmol, 0.1uCi/ul, Perkin Elmer) was diluted in a 1:1 ratio with unlabeled acetyl CoA (Sigma) to give a final concentration of 100uM and 0.3uCi per reaction.

    Article Title: Polyvinylsulfonic acid: A Low-cost RNase inhibitor for enhanced RNA preservation and cell-free protein translation
    Article Snippet: .. For reference, using 10 mg/mL PVSA in place of the manufacturer's recommended concentration of Murine RNase inhibitor® or Recombinant RNase inhibitor reduces the cost of IVT reagents by more than 95%. .. Further, we compared the costs per mass of protein produced in a decoupled protein transcription and translation system with adding 10 mg/mL PVSA and without adding PVSA during transcription ( ).

    HAT Assay:

    Article Title: RNA binding to CBP stimulates histone acetylation and transcription
    Article Snippet: .. Reactions contained 1× HAT assay buffer (50mM Tris-HCl pH 7.5 (RT), 5% glycerol, 0.1mM EDTA, 50mM KCl), 1mM DTT, 10mM Na-Butyrate, 1× Complete EDTA protease inhibitor cocktail (Roche), 0.4U/ul murine RNAse inhibitor (NEB), 0.1mg/ml BSA (NEB), 0–200nM H31–21 peptide (Anaspec) and the required concentration of RNA probe. .. 3 H labeled Acetyl Co-enzyme A (3 H-acetyl CoA) (specific activity = 3.95 Ci/mmol, 0.1uCi/ul, Perkin Elmer) was diluted in a 1:1 ratio with unlabeled acetyl CoA (Sigma) to give a final concentration of 100uM and 0.3uCi per reaction.

    Nuclease Assay:

    Article Title: RNA targeting with CRISPR-Cas13a
    Article Snippet: .. Briefly, reactions consisted of 45 nM purified LwaCas13a, 22.5 nM crRNA, 125 nM quenched fluorescent RNA reporter (RNAse Alert v2, Thermo Scientific), 2 μL murine RNase inhibitor (New England Biolabs), 100 ng of background total human RNA (purified from HEK293FT culture), and varying amounts of input nucleic acid target, unless otherwise indicated, in nuclease assay buffer (40 mM Tris-HCl, 60 mM NaCl, 6 mM MgCl2, pH 7.3). .. Reactions were allowed to proceed for 1-3 hr at 37°C (unless otherwise indicated) on a fluorescent plate reader (BioTek) with fluorescent kinetics measured every 5 min.

    Recombinant:

    Article Title: Polyvinylsulfonic acid: A Low-cost RNase inhibitor for enhanced RNA preservation and cell-free protein translation
    Article Snippet: .. For reference, using 10 mg/mL PVSA in place of the manufacturer's recommended concentration of Murine RNase inhibitor® or Recombinant RNase inhibitor reduces the cost of IVT reagents by more than 95%. .. Further, we compared the costs per mass of protein produced in a decoupled protein transcription and translation system with adding 10 mg/mL PVSA and without adding PVSA during transcription ( ).

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    New England Biolabs murine rnase inhibitor
    Biochemical characterization of LwaCas13a <t>RNA</t> cleavage activity a, LwaCas13a has more active <t>RNAse</t> activity than LshCas13a. b, Gel electrophoresis of ssRNA1 after incubation with LwaCas13a and with and without crRNA 1 for varying amounts of times. c, Gel electrophoresis of ssRNA1 after incubation with varying amounts of LwaCas13a-crRNA complex. d, Sequence and structure of ssRNA 4 and ssRNA 5. crRNA spacer sequence is highlighted in blue. e, Gel electrophoresis of ssRNA 4 and ssRNA 5 after incubation with LwaCas13a and crRNA 1. f, Sequence and structure of ssRNA 4 with sites of poly-x modifications highlighted in red. crRNA spacer sequence is highlighted in blue. g, Gel electrophoresis of ssRNA 4 with each of 4 possible poly-x modifications incubated with LwaCas13a and crRNA 1. h, LwaCas13a can process pre-crRNA from the L. wadei CRISPR-Cas locus. i, Cleavage efficiency of ssRNA 1 for crRNA spacer truncations after incubation with LwaCas13a.
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    Biochemical characterization of LwaCas13a RNA cleavage activity a, LwaCas13a has more active RNAse activity than LshCas13a. b, Gel electrophoresis of ssRNA1 after incubation with LwaCas13a and with and without crRNA 1 for varying amounts of times. c, Gel electrophoresis of ssRNA1 after incubation with varying amounts of LwaCas13a-crRNA complex. d, Sequence and structure of ssRNA 4 and ssRNA 5. crRNA spacer sequence is highlighted in blue. e, Gel electrophoresis of ssRNA 4 and ssRNA 5 after incubation with LwaCas13a and crRNA 1. f, Sequence and structure of ssRNA 4 with sites of poly-x modifications highlighted in red. crRNA spacer sequence is highlighted in blue. g, Gel electrophoresis of ssRNA 4 with each of 4 possible poly-x modifications incubated with LwaCas13a and crRNA 1. h, LwaCas13a can process pre-crRNA from the L. wadei CRISPR-Cas locus. i, Cleavage efficiency of ssRNA 1 for crRNA spacer truncations after incubation with LwaCas13a.

    Journal: Nature

    Article Title: RNA targeting with CRISPR-Cas13a

    doi: 10.1038/nature24049

    Figure Lengend Snippet: Biochemical characterization of LwaCas13a RNA cleavage activity a, LwaCas13a has more active RNAse activity than LshCas13a. b, Gel electrophoresis of ssRNA1 after incubation with LwaCas13a and with and without crRNA 1 for varying amounts of times. c, Gel electrophoresis of ssRNA1 after incubation with varying amounts of LwaCas13a-crRNA complex. d, Sequence and structure of ssRNA 4 and ssRNA 5. crRNA spacer sequence is highlighted in blue. e, Gel electrophoresis of ssRNA 4 and ssRNA 5 after incubation with LwaCas13a and crRNA 1. f, Sequence and structure of ssRNA 4 with sites of poly-x modifications highlighted in red. crRNA spacer sequence is highlighted in blue. g, Gel electrophoresis of ssRNA 4 with each of 4 possible poly-x modifications incubated with LwaCas13a and crRNA 1. h, LwaCas13a can process pre-crRNA from the L. wadei CRISPR-Cas locus. i, Cleavage efficiency of ssRNA 1 for crRNA spacer truncations after incubation with LwaCas13a.

    Article Snippet: Briefly, reactions consisted of 45 nM purified LwaCas13a, 22.5 nM crRNA, 125 nM quenched fluorescent RNA reporter (RNAse Alert v2, Thermo Scientific), 2 μL murine RNase inhibitor (New England Biolabs), 100 ng of background total human RNA (purified from HEK293FT culture), and varying amounts of input nucleic acid target, unless otherwise indicated, in nuclease assay buffer (40 mM Tris-HCl, 60 mM NaCl, 6 mM MgCl2, pH 7.3).

    Techniques: Activity Assay, Nucleic Acid Electrophoresis, Incubation, Sequencing, CRISPR

    Effect of the growth parameters on RiCF. (A) Schematics of a hypothetical scenario when RNA inhibits NAPs that could potentially cleave DNA. During lysis, quick RNA degradation removes the inhibition resulting in breakage of chromosomes. (B) Growth phase dependence of RiCF. AB1157 was grown at 37°C with periodic OD measurements, and samples for plugs were withdrawn at various times. The cells were made into plugs using lysis agarose and RNase (50 μg/plug) and the plugs were lysed and electrophoresed under standard conditions. Data points are means of at least three independent assays ± SEM. (C) Effect of translation and transcription inhibition on RiCF. Cells were grown till OD 0.5–0.6, split into three parts and chloramphenicol (40 μg/ml) or rifampicin (150 μg/ml) were added to two samples. All sample were shaken for another 2–3 hours at 37°C before making plugs as described in (B). Data points are means of four independent assays ± SEM. (D) Growth in minimal medium reduces RiCF. Cells were grown in LB or MOPS till the OD reached 0.6 and made into plugs using standard conditions. The values presented are means of six independent assays ± SEM. (E) Effect of growth temperature on RNase-induced chromosomal fragmentation. Cultures of AB1157 were grown at 20°C, 30°C, 37°C, 42°C or 45°C to same cell densities (A 600 = 0.6), and plugs were made in lysis agarose with RNAse A (50 μg/plug), as described in (A). Data are means of three to six independent measurements ± SEM.

    Journal: PLoS ONE

    Article Title: Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome

    doi: 10.1371/journal.pone.0190177

    Figure Lengend Snippet: Effect of the growth parameters on RiCF. (A) Schematics of a hypothetical scenario when RNA inhibits NAPs that could potentially cleave DNA. During lysis, quick RNA degradation removes the inhibition resulting in breakage of chromosomes. (B) Growth phase dependence of RiCF. AB1157 was grown at 37°C with periodic OD measurements, and samples for plugs were withdrawn at various times. The cells were made into plugs using lysis agarose and RNase (50 μg/plug) and the plugs were lysed and electrophoresed under standard conditions. Data points are means of at least three independent assays ± SEM. (C) Effect of translation and transcription inhibition on RiCF. Cells were grown till OD 0.5–0.6, split into three parts and chloramphenicol (40 μg/ml) or rifampicin (150 μg/ml) were added to two samples. All sample were shaken for another 2–3 hours at 37°C before making plugs as described in (B). Data points are means of four independent assays ± SEM. (D) Growth in minimal medium reduces RiCF. Cells were grown in LB or MOPS till the OD reached 0.6 and made into plugs using standard conditions. The values presented are means of six independent assays ± SEM. (E) Effect of growth temperature on RNase-induced chromosomal fragmentation. Cultures of AB1157 were grown at 20°C, 30°C, 37°C, 42°C or 45°C to same cell densities (A 600 = 0.6), and plugs were made in lysis agarose with RNAse A (50 μg/plug), as described in (A). Data are means of three to six independent measurements ± SEM.

    Article Snippet: XRN-I, RNase If , Exonuclease T (Exo T), EcoRI and RNase A inhibitor were all from New England Biolabs.

    Techniques: Lysis, Inhibition

    RNA degradation causes chromosomal fragmentation. (A)  Schematics of a hypothetical scenario when RNA makes the central core of nucleoids, and its degradation results in collapse of the nucleoid structure, causing chromosomal fragmentation.  (B)  Radiogram of a pulsed field gel showing chromosomal fragmentation in AB1157 when cells were embedded in agarose plugs in the presence and absence of proteinase K (25 μg/plug) and/or RNase (50 μg/plug) and lysed overnight at 62°C.  (C)  Radiogram showing DNase I sensitivity of the signal entering the gel. Plugs were lysed at 62°C, washed extensively to remove traces of lysis buffer and then treated with DNase I at 37°C before PFGE.  (D)  A representative gel showing that RNA degradation by different enzymes causes chromosomal fragmentation. Plugs were made in the absence of proteinase K in 1x restriction enzyme buffer (NEBuffer 3 for RNase A, XRN-1 and RNAse I f  and NEBuffer 4 for Exo T). The concentrations of the enzymes used were, RNase, 50 μg/plug; XRN-1, 5 U/plug; RNAse I f , 100 U/plug and Exo T, 20 U/plug.  (E)  Quantification of the chromosomal fragmentation when plugs were made in the presence of various RNA degrading enzymes. The values presented are means of four independent assays ± SEM. CZ, compression zone.

    Journal: PLoS ONE

    Article Title: Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome

    doi: 10.1371/journal.pone.0190177

    Figure Lengend Snippet: RNA degradation causes chromosomal fragmentation. (A) Schematics of a hypothetical scenario when RNA makes the central core of nucleoids, and its degradation results in collapse of the nucleoid structure, causing chromosomal fragmentation. (B) Radiogram of a pulsed field gel showing chromosomal fragmentation in AB1157 when cells were embedded in agarose plugs in the presence and absence of proteinase K (25 μg/plug) and/or RNase (50 μg/plug) and lysed overnight at 62°C. (C) Radiogram showing DNase I sensitivity of the signal entering the gel. Plugs were lysed at 62°C, washed extensively to remove traces of lysis buffer and then treated with DNase I at 37°C before PFGE. (D) A representative gel showing that RNA degradation by different enzymes causes chromosomal fragmentation. Plugs were made in the absence of proteinase K in 1x restriction enzyme buffer (NEBuffer 3 for RNase A, XRN-1 and RNAse I f and NEBuffer 4 for Exo T). The concentrations of the enzymes used were, RNase, 50 μg/plug; XRN-1, 5 U/plug; RNAse I f , 100 U/plug and Exo T, 20 U/plug. (E) Quantification of the chromosomal fragmentation when plugs were made in the presence of various RNA degrading enzymes. The values presented are means of four independent assays ± SEM. CZ, compression zone.

    Article Snippet: XRN-I, RNase If , Exonuclease T (Exo T), EcoRI and RNase A inhibitor were all from New England Biolabs.

    Techniques: Pulsed-Field Gel, Lysis

    eIF3d cap-binding activity is required for efficient 48S initiation complex formation on specific mRNAs a , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to eIF3 in the presence of competitor ligands. b , Electrostatic surface view of the eIF3d cap-binding domain colored by charge, with a zoomed view of single stranded RNA (ssRNA) and cap analog modeled according to their positions bound to DXO 15 . Positive charge is colored blue and negative charge is in red, and the RNA gate is removed for clarity. c , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to wild type or helix α5 or helix α11-mutant eIF3. eIF3d-helix α5 mutant (D249Q/V262I/Y263A), helix α11 mutant (T317E/N320E/H321A). WT, wild type. d , Incorporation of c-Jun and ACTB mRNA into initiation complexes by wild type, helix α5, or helix α11-mutant eIF3d as measured by quantitative RT-PCR. mRNA-ribosome association is expressed as the ratio between the quantity of mRNA transcripts to 18S rRNA and normalized to the wild type sample. The results are representative of three independent experiments and given as the mean ± s.d. from a representative quantitative RT-PCR experiment performed in duplicate.

    Journal: Nature

    Article Title: eIF3d is an mRNA cap-binding protein required for specialized translation initiation

    doi: 10.1038/nature18954

    Figure Lengend Snippet: eIF3d cap-binding activity is required for efficient 48S initiation complex formation on specific mRNAs a , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to eIF3 in the presence of competitor ligands. b , Electrostatic surface view of the eIF3d cap-binding domain colored by charge, with a zoomed view of single stranded RNA (ssRNA) and cap analog modeled according to their positions bound to DXO 15 . Positive charge is colored blue and negative charge is in red, and the RNA gate is removed for clarity. c , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to wild type or helix α5 or helix α11-mutant eIF3. eIF3d-helix α5 mutant (D249Q/V262I/Y263A), helix α11 mutant (T317E/N320E/H321A). WT, wild type. d , Incorporation of c-Jun and ACTB mRNA into initiation complexes by wild type, helix α5, or helix α11-mutant eIF3d as measured by quantitative RT-PCR. mRNA-ribosome association is expressed as the ratio between the quantity of mRNA transcripts to 18S rRNA and normalized to the wild type sample. The results are representative of three independent experiments and given as the mean ± s.d. from a representative quantitative RT-PCR experiment performed in duplicate.

    Article Snippet: Each translation reaction contained 50% in vitro translation lysate and buffer to make the final reaction with 0.84 mM ATP, 0.21 mM GTP, 21 mM creatine phosphate (Roche), 45 U ml-1 creatine phosphokinase (Roche), 10 mM HEPES-KOH pH 7.6, 2 mM DTT, 8 mM amino acids (Promega), 255 mM spermidine, 1 U ml-1 murine RNase inhibitor (NEB), and mRNA-specific concentrations of Mg(OAc)2 and KOAc.

    Techniques: Binding Assay, Activity Assay, SDS-Gel, Labeling, Mutagenesis, Quantitative RT-PCR

    eIF4E recognizes the 5′ end of the c-Jun mRNA less efficiently than ACTB mRNA a , Coomassie blue stained SDS gel of recombinant human eIF4E expressed in E. coli. b , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled ACTB or c-Jun 5′ UTR RNA crosslinked to eIF4E. The result is representative of three independent experiments. For gel source data, see Supplementary Figure 1 .

    Journal: Nature

    Article Title: eIF3d is an mRNA cap-binding protein required for specialized translation initiation

    doi: 10.1038/nature18954

    Figure Lengend Snippet: eIF4E recognizes the 5′ end of the c-Jun mRNA less efficiently than ACTB mRNA a , Coomassie blue stained SDS gel of recombinant human eIF4E expressed in E. coli. b , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled ACTB or c-Jun 5′ UTR RNA crosslinked to eIF4E. The result is representative of three independent experiments. For gel source data, see Supplementary Figure 1 .

    Article Snippet: Each translation reaction contained 50% in vitro translation lysate and buffer to make the final reaction with 0.84 mM ATP, 0.21 mM GTP, 21 mM creatine phosphate (Roche), 45 U ml-1 creatine phosphokinase (Roche), 10 mM HEPES-KOH pH 7.6, 2 mM DTT, 8 mM amino acids (Promega), 255 mM spermidine, 1 U ml-1 murine RNase inhibitor (NEB), and mRNA-specific concentrations of Mg(OAc)2 and KOAc.

    Techniques: Staining, SDS-Gel, Recombinant, Labeling

    5' end recognition of c-Jun mRNA is eIF4F-independent a , Distribution of c-Jun or ACTB mRNA-containing initiation complexes in programmed 293T cell in vitro translation extracts. The mRNA abundance (black line) is expressed as the fraction of total recovered transcripts. The results are given as the mean ± standard deviation (s.d.) of a representative quantitative RT-PCR experiment performed in duplicate. The polysome profile (gray line) is plotted as relative absorbance at 254 nm versus elution fractions. b , Western blot analysis of initiation factors in 48S translation complexes formed on c-Jun and ACTB mRNAs. 293T, total protein from 293T in vitro translation extracts. For gel source data, see Supplementary Figure 1 . c , Phosphorimage of SDS gel resolving RNase-protected 32 P-internal or 32 P-cap-labeled c-Jun 5' UTR RNA crosslinked to eIF3 subunits. Recombinant eIF3a migrates at ~100 kDa due to a C-terminal truncation 26 . The results of a - c are representative of three independent experiments.

    Journal: Nature

    Article Title: eIF3d is an mRNA cap-binding protein required for specialized translation initiation

    doi: 10.1038/nature18954

    Figure Lengend Snippet: 5' end recognition of c-Jun mRNA is eIF4F-independent a , Distribution of c-Jun or ACTB mRNA-containing initiation complexes in programmed 293T cell in vitro translation extracts. The mRNA abundance (black line) is expressed as the fraction of total recovered transcripts. The results are given as the mean ± standard deviation (s.d.) of a representative quantitative RT-PCR experiment performed in duplicate. The polysome profile (gray line) is plotted as relative absorbance at 254 nm versus elution fractions. b , Western blot analysis of initiation factors in 48S translation complexes formed on c-Jun and ACTB mRNAs. 293T, total protein from 293T in vitro translation extracts. For gel source data, see Supplementary Figure 1 . c , Phosphorimage of SDS gel resolving RNase-protected 32 P-internal or 32 P-cap-labeled c-Jun 5' UTR RNA crosslinked to eIF3 subunits. Recombinant eIF3a migrates at ~100 kDa due to a C-terminal truncation 26 . The results of a - c are representative of three independent experiments.

    Article Snippet: Each translation reaction contained 50% in vitro translation lysate and buffer to make the final reaction with 0.84 mM ATP, 0.21 mM GTP, 21 mM creatine phosphate (Roche), 45 U ml-1 creatine phosphokinase (Roche), 10 mM HEPES-KOH pH 7.6, 2 mM DTT, 8 mM amino acids (Promega), 255 mM spermidine, 1 U ml-1 murine RNase inhibitor (NEB), and mRNA-specific concentrations of Mg(OAc)2 and KOAc.

    Techniques: In Vitro, Standard Deviation, Quantitative RT-PCR, Western Blot, SDS-Gel, Labeling, Recombinant

    High-temperature IVT does not affect antisense dsRNA by-product formation. ( A ) Native gel electrophoresis analysis of IVT reactions on 512B DNA template using wild-type T7 (37°C) with/without RNase III treatment. ( B ) dsRNA immunoblot with J2 antibody on IVT reactions (crude and purified) with 512B template. ( C ) Native gel electrophoresis analyses and dsRNA immunoblot analysis of 512B IVT reactions conducted with TsT7-1 at 37°C versus 50°C.

    Journal: RNA

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    doi: 10.1261/rna.073858.119

    Figure Lengend Snippet: High-temperature IVT does not affect antisense dsRNA by-product formation. ( A ) Native gel electrophoresis analysis of IVT reactions on 512B DNA template using wild-type T7 (37°C) with/without RNase III treatment. ( B ) dsRNA immunoblot with J2 antibody on IVT reactions (crude and purified) with 512B template. ( C ) Native gel electrophoresis analyses and dsRNA immunoblot analysis of 512B IVT reactions conducted with TsT7-1 at 37°C versus 50°C.

    Article Snippet: Reactions with wild-type T7 RNAP were carried out in 40 mM Tris-HCl pH 7.9, 19 mM MgCl2 , 5 mM DTT, 1 mM spermidine and supplemented with RNase inhibitor (1000 units/mL) and inorganic pyrophosphatase (4.15 units/mL) (New England Biolabs) at 37°C for 1 h. Reactions with thermostable T7 RNAP (TsT7-1—Hi-T7 RNAP; New England Biolabs and TsT7-2—Thermostable T7 RNAP; Toyobo Life Sciences) were carried out in a buffer containing 40 mM Tris-HCl pH 7.5, 19 mM MgCl2 , 50 mM NaCl, 5 mM DTT, 1 mM spermidine at varying temperatures (37°C to 60°C) for 1 h. Reactions were supplemented with RNase inhibitor (1000 units/mL) and inorganic pyrophosphatase (4.15 units/mL) (New England Biolabs).

    Techniques: Nucleic Acid Electrophoresis, Purification