pnctr rna fragment  (New England Biolabs)


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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 93 stars, based on 1251 article reviews
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    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 "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

    4) 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

    5) 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

    6) 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

    7) 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

    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 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

    9) 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:

    10) 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

    11) 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

    12) 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

    13) 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

    14) 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

    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. 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

    16) 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

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    Concentration Assay:

    Article Title: Transforming activity of an oncoprotein-encoding circular RNA from human papillomavirus
    Article Snippet: .. A concentration of 2 µg of total RNA was incubated with 5U RNase R (Lucigen, RNR07250), 10U murine ribonuclease inhibitor (New England Biolabs, M0314S), 0.5U DNase (Qiagen, 79254), and 1X RNase R buffer for 40 min at 37 °C and then placed on ice. ..

    Incubation:

    Article Title: Transforming activity of an oncoprotein-encoding circular RNA from human papillomavirus
    Article Snippet: .. A concentration of 2 µg of total RNA was incubated with 5U RNase R (Lucigen, RNR07250), 10U murine ribonuclease inhibitor (New England Biolabs, M0314S), 0.5U DNase (Qiagen, 79254), and 1X RNase R buffer for 40 min at 37 °C and then placed on ice. ..

    Ligation:

    Article Title: nextPARS: parallel probing of RNA structures in Illumina
    Article Snippet: .. Samples were then put on ice, and 2 µL of 5× HM Ligation Buffer, 1 µL of RNase inhibitor and 1 µL of T4 RNA Ligase 2, truncated (New England BioLabs) were added. .. After 1 h incubation at 28°C, we added 1 µL of stop solution, gently pipetted up and down, incubated for 15 min more at 28°C and placed the samples on ice.

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    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 <t>RNAse</t> A (50 μg/plug), as described in (A). Data are means of three to six independent measurements ± SEM.
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    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

    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

    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.

    Journal: Journal of Advanced Research

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

    doi: 10.1016/j.jare.2019.12.005

    Figure Lengend 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.

    Article Snippet: The reverse transcription was carried out in 50 µl reaction at 37 °C for 60 min as follows: to the 20 µl annealed probes/rRNA mix, added 1 µl of RNase Inhibitor, 5 µl of 10x RT buffer (NEB), 5ul of 10 mM dNTPs, 1 µl of M−MuLV reverse transcriptase, and 18 µl RNase-free ddH2 O.

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

    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 ).

    Journal: RNA

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

    doi: 10.1261/rna.063073.117

    Figure Lengend 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 ).

    Article Snippet: Samples were then put on ice, and 2 µL of 5× HM Ligation Buffer, 1 µL of RNase inhibitor and 1 µL of T4 RNA Ligase 2, truncated (New England BioLabs) were added.

    Techniques: 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.

    Journal: RNA

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

    doi: 10.1261/rna.063073.117

    Figure Lengend 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.

    Article Snippet: Samples were then put on ice, and 2 µL of 5× HM Ligation Buffer, 1 µL of RNase inhibitor and 1 µL of T4 RNA Ligase 2, truncated (New England BioLabs) were added.

    Techniques: