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  • 99
    New England Biolabs rnase inhibitor
    In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including <t>[α-</t> 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with <t>RNase</t> (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.
    Rnase Inhibitor, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2221 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rnase inhibitor
    Ribonuclease activity of the recombinant <t>TcPR-4b</t> on tomato ( Solanum lycopersicum var. Micro-Tom) total RNA (5 μg). The incubation with TcPR-4b was carried out for 30 min at 25°C. The boiling conditions were 10 min at 95°C. The <t>RNase</t> inhibitor was the RiboLock (40 U; Thermo Scientific). The incubation conditions of the RNase A (Thermo Scientific) were 10 min at 25°C.
    Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 23393 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega rnase inhibitor
    Regulation of IL-8 mRNA expression in immortalized human gingival keratinocytes. (A) A representative storage phosphor scan demonstrates that, by using quantitative <t>RNase</t> protection assay, moderate IL-8 mRNA expression was detected in 8 μg of total <t>RNA</t> from immortalized keratinocytes under normal control conditions as seen at time zero. Following treatment of separate flasks of the same culture over 24 h with 50 ng of PMA per ml, IL-8 mRNA expression dramatically increases over a 6-h period, returning to nearly constitutive levels by 12 h. Test lanes, hours 0 to 24, show the protected probes after binding to IL-8 (374 bp) and GAPDH (220 bp) mRNA, respectively, within the sample, followed by RNase treatment. An increase in the band intensity of GAPDH at 2 to 6 h reflects an increase in the total amount of mRNA, as expected with PMA stimulation. This did not affect quantitation (see panel B, below), since the ratio of GAPDH to IL-8 was still proportionally constant. Control lanes (C1 and C2) show the two probes GAPDH (C1, 266 bp) and IL-8 (C2, 420 bp) to which no RNase has been added. The results are representative of three experiments. (B) Quantitation of IL-8 mRNA in immortalized keratinocytes following induction with PMA. From panel A the signal intensity determined for the IL-8 protected fragment was normalized to the abundance of the internal control GAPDH at each time point. The data shown are the mean ± the standard deviation ( n = 3).
    Rnase Inhibitor, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 10413 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher ribolock rnase inhibitor
    <t>RNase</t> protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7 , 8–11 , 12–15 , 16–19 , 20–23 , respectively) for 6 h at 37 °C, followed by 1 μL (40 U) <t>RiboLock</t> RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1 , after incubation with heparin ( lane 2 ) and 0.35 μg RNase A per 1 μg siRNA ( lane 3 ).
    Ribolock Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5157 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher superase• in rnase inhibitor
    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total <t>RNA</t> was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by <t>RT-PCR.</t> Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p
    Superase• In Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1031 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim rnasin
    Elevated polyadenylation activity of the dark extract. Polyadenylation was assayed under the conditions described for the in vitro processing assays except that 2 mM <t>ATP</t> and 20 U <t>RNasin</t> were added to inactivate the endogenous nucleases. After the indicated times the reaction was stopped and the RNA products analyzed by electrophoresis and autoradiography. Control assays were incubated for 40 min in the presence of 4 mM cordycepin (Sigma; lanes 40′ + Cord.).
    Rnasin, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 207 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Toyobo rnase inhibitor
    Products of influenza virus replication factor (IREF-2)-dependent unprimed RNA synthesis. ( A ) <t>RNase</t> T2 protection assay. Radioactively labeled vRNA products were synthesized in the cell-free viral RNA synthesis system with micrococcal nuclease-treated vRNP (mnRNP) and the v53 model template in the presence of ApG (lanes 1–3), the c53 model template in the presence of ApG (lanes 4–6), and c53 in the presence of the IREF-2 fraction and in the absence of ApG (lanes 7–9). Viral RNA products were hybridized with excess amounts of nonlabeled v53 (lanes 2, 5, and 8) or c53 (lanes 3, 6, and 9), which was followed by digestion with RNase T2. Hybridized and digested RNA samples were extracted, collected, and subjected to 10% Urea-PAGE followed by autoradiography visualization. The closed arrowhead indicates 53-nt-long RNAs. ( B ) Analysis of the 5'-terminal structure of IREF-2-dependent unprimed vRNA products. [α- 32 P] <t>GTP-labeled</t> IREF-2-dependent unprimed vRNA products were prepared in the cell-free viral RNA synthesis system. The radioactively labeled 53-nt-long vRNA products were isolated from 10% Urea-PAGE, which was followed by excision and elution from the gel. A portion of the isolated 53-nt-long products was treated with alkaline phosphatase (lanes 2 and 4). Both nontreated and alkaline phosphatase-treated [α- 32 P] GTP-labeled unprimed vRNA products were digested with RNase T2 (lanes 1–4) or snake venom phosphodiesterase (lane 5). The digested materials were spotted onto a polyethylenimine (PEI)-cellulose thin layer and developed with 1 N acetic acid-4 M LiCl (4:1, v/v) (left panel; lanes 1 and 2) or 1.6 M LiCl (right panel; lanes 3–6) and visualized by autoradiography. For mobility standards, nonradiolabeled AMP, ADP, and ATP were also subjected to thin-layer chromatography and are indicated on the left side of each panel. For a marker of pppAp, [γ- 32 P] ATP-labeled v53 synthesized using T7 RNA polymerase was also subjected to RNase T2 digestion and then to thin-layer chromatography (lane 6). The expected nucleotide positions are indicated on the right side of each panel by closed arrowheads. DOI: http://dx.doi.org/10.7554/eLife.08939.004
    Rnase Inhibitor, supplied by Toyobo, used in various techniques. Bioz Stars score: 92/100, based on 706 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega recombinant rnasin ribonuclease inhibitor
    Products of influenza virus replication factor (IREF-2)-dependent unprimed RNA synthesis. ( A ) <t>RNase</t> T2 protection assay. Radioactively labeled vRNA products were synthesized in the cell-free viral RNA synthesis system with micrococcal nuclease-treated vRNP (mnRNP) and the v53 model template in the presence of ApG (lanes 1–3), the c53 model template in the presence of ApG (lanes 4–6), and c53 in the presence of the IREF-2 fraction and in the absence of ApG (lanes 7–9). Viral RNA products were hybridized with excess amounts of nonlabeled v53 (lanes 2, 5, and 8) or c53 (lanes 3, 6, and 9), which was followed by digestion with RNase T2. Hybridized and digested RNA samples were extracted, collected, and subjected to 10% Urea-PAGE followed by autoradiography visualization. The closed arrowhead indicates 53-nt-long RNAs. ( B ) Analysis of the 5'-terminal structure of IREF-2-dependent unprimed vRNA products. [α- 32 P] <t>GTP-labeled</t> IREF-2-dependent unprimed vRNA products were prepared in the cell-free viral RNA synthesis system. The radioactively labeled 53-nt-long vRNA products were isolated from 10% Urea-PAGE, which was followed by excision and elution from the gel. A portion of the isolated 53-nt-long products was treated with alkaline phosphatase (lanes 2 and 4). Both nontreated and alkaline phosphatase-treated [α- 32 P] GTP-labeled unprimed vRNA products were digested with RNase T2 (lanes 1–4) or snake venom phosphodiesterase (lane 5). The digested materials were spotted onto a polyethylenimine (PEI)-cellulose thin layer and developed with 1 N acetic acid-4 M LiCl (4:1, v/v) (left panel; lanes 1 and 2) or 1.6 M LiCl (right panel; lanes 3–6) and visualized by autoradiography. For mobility standards, nonradiolabeled AMP, ADP, and ATP were also subjected to thin-layer chromatography and are indicated on the left side of each panel. For a marker of pppAp, [γ- 32 P] ATP-labeled v53 synthesized using T7 RNA polymerase was also subjected to RNase T2 digestion and then to thin-layer chromatography (lane 6). The expected nucleotide positions are indicated on the right side of each panel by closed arrowheads. DOI: http://dx.doi.org/10.7554/eLife.08939.004
    Recombinant Rnasin Ribonuclease Inhibitor, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 1166 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa recombinant rnase inhibitor
    Fluorometric detection. (A) Principle of the fluorometric detection based on the fluorescence resonance energy transfer [ 34 ]. The fluorescent of 6-FAM is typically quenched by BHQ-1, but it emits a bright signal when the oligonucleotide is cleaved. (B-F) Investigation on the <t>MazFpp</t> cleavage site. One hundred nanograms of <t>RNase</t> A (blue) or MazFpp (green) was incubated with each oligonucleotide. The yellow plot represents a control reaction in which no enzyme was added.
    Recombinant Rnase Inhibitor, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 608 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen rnase inhibitor
    (a) SDS-PAGE of <t>NLP.</t> Lane 1, protein molecular mass marker; lane 2, untreated VSV NLP; lane 3, empty NLP after <t>RNase</t> A treatment; lane 4, reconstituted VSV NLP containing poly(rA) RNA. (b) Image of crystals of NLP with encapsidated poly(rG) RNA.
    Rnase Inhibitor, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 814 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Promega recombinant rnasin
    2′-F modified <t>siRNA</t> inhibits gene expression in living mice. Representative bioluminescence images of light emitted from living mice transfected, 2 d earlier, with the luciferase expression plasmids pGL3-control. Mice ( N = 4–5 mice per group) received pGL3-control alone ( A ) or were cotransfected with ( B ) 2′-OH GL3 siRNA, ( C ) 2′-OH GL3 siRNA and the RNase inhibitor, <t>RNasin,</t> ( D ) 2′-F GL3 siRNA, ( E ) 2′-F inverted GL2 siRNA. The mouse in panel F received a low pressure i.v. injection of 2′-F GL3 siRNA on day 1 to test if stabilized siRNAs could be taken up by the liver without hydrodynamic transfection. Mice in panels B , C , and D show significant reductions in emitted light as a result of RNA. Hydrodynamic transfection with 2′-F inverted GL2 siRNA and low pressure i.v. transfection of 2′-F GL3 siRNA did not result in significant RNAi.
    Recombinant Rnasin, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 598 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare rnase inhibitor
    2′-F modified <t>siRNA</t> inhibits gene expression in living mice. Representative bioluminescence images of light emitted from living mice transfected, 2 d earlier, with the luciferase expression plasmids pGL3-control. Mice ( N = 4–5 mice per group) received pGL3-control alone ( A ) or were cotransfected with ( B ) 2′-OH GL3 siRNA, ( C ) 2′-OH GL3 siRNA and the RNase inhibitor, <t>RNasin,</t> ( D ) 2′-F GL3 siRNA, ( E ) 2′-F inverted GL2 siRNA. The mouse in panel F received a low pressure i.v. injection of 2′-F GL3 siRNA on day 1 to test if stabilized siRNAs could be taken up by the liver without hydrodynamic transfection. Mice in panels B , C , and D show significant reductions in emitted light as a result of RNA. Hydrodynamic transfection with 2′-F inverted GL2 siRNA and low pressure i.v. transfection of 2′-F GL3 siRNA did not result in significant RNAi.
    Rnase Inhibitor, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 672 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rnase inhibitor
    m 6 A alters RNA structure to recruit <t>HNRNPG.</t> ( A ) Sequence logo of the most enriched motif within HNRNPG PAR-CLIP peaks. ( B ) Left: secondary structure of the MALAT1 hairpin, showing the A-2,515-to-G/C/U mutations that were introduced at the m 6 A site. Right: quantification of relative HNRNPG pull-down with the original (2,515-A) and mutated (2,515-G/C/U) MALAT1 hairpins, normalized to pulled-down HIST1H1C. Data shown as mean; error bar = standard deviation; n = 3 biological replicates. ( C ) Left: structural probing of the unmethylated and methylated MALAT1 hairpins. The orange lines indicate regions with marked differences between the unmethylated and methylated hairpins. The location of the m 6 A residue is indicated by a red dot. Ctrl, no nuclease added; V1; <t>RNase</t> V1 digestion; S1, S1 nuclease digestion; T1, RNase T1 digestion; G-L, G-ladder; AH, alkaline hydrolysis. Right: secondary structure of the unmethylated and methylated MALAT1 hairpins, marked at their S1 nuclease (red lines) and V1 nuclease (green lines) cleavage sites. ( D ) Model showing that m 6 A disrupts RNA structure, exposes a motif that includes the m 6 A site, and recruits an RNA binding protein.
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    New England Biolabs rnase inhibitor murine
    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 <t>RNA-Seq</t> 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 <t>RNase</t> 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 )
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    Image Search Results


    In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.

    Article Snippet: Reverse transcription reactions with HIV-1 RT were carried out as above, except in 10 μl and containing 10 units RNase inhibitor (NEB), 0.1 μCi/μl [α-32 P]dCTP, 30 ng/μl RNA template, 1 μM PG117 primer ( ) and 2 units of HIV-1 RT (Worthington Biochemical), and the reaction was carried out for 30 min.

    Techniques: In Vitro, Binding Assay, Sequencing, Incubation, Polyacrylamide Gel Electrophoresis, Labeling, Marker, Activity Assay, Produced

    Core DGR RNA. ( A ) Schematic of core DGR RNA. ( B ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the core DGR RNA as template. Prior to the reverse transcription reaction, the RNA template was untreated (-Per) or treated with periodate (+Per). Products from the reaction were untreated (U) or treated with RNase (+R), and resolved by 6% denaturing PAGE. Lane T corresponds to internally-labeled core DGR RNA as a marker for the size of the template. Red arrowheads indicate radiolabeled product bands that migrate at the same position or slower than the core DGR RNA, and green arrowheads ones that migrate faster. The positions of the 120 and 90 nt cDNA bands are indicated. The two panels are from the same gel, with the black line indicating that intermediate lanes were removed. ( C ) Internally-labeled core DGR RNA was not incubated (–), or incubated with bRT-Avd alone or bRT-Avd with 100 μM standard dNTPs (+dNTP), 100 μM dCTP (+CTP), 100 μM dNTPs excluding dCTP (+d(A,T,G)TP), or 100 μM nonhydrolyzeable analog of dCTP (+N-dCTP) for 2 h. Incubation products were resolved by denaturing PAGE. The band corresponding to the 5′ fragment of the cleaved core RNA containing either a deoxycytidine alone (5′+dC) or cDNA (5′+cDNA), and the band corresponding to the 3′ fragment of the RNA are indicated. ( D ) The core DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The core DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( E ) Radiolabeled products resulting from bRT-Avd activity for 12 h with core, hybrid core dA56, or hybrid core A56 DGR RNA as template. Products were untreated (U) or treated with RNase (+R), and resolved by denaturing PAGE. Separate samples of core dA56 and A56 were 5′ 32 P-labeled for visualization of inputs (I). The positions of the 120 and 90 nt cDNAs are indicated.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: Core DGR RNA. ( A ) Schematic of core DGR RNA. ( B ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the core DGR RNA as template. Prior to the reverse transcription reaction, the RNA template was untreated (-Per) or treated with periodate (+Per). Products from the reaction were untreated (U) or treated with RNase (+R), and resolved by 6% denaturing PAGE. Lane T corresponds to internally-labeled core DGR RNA as a marker for the size of the template. Red arrowheads indicate radiolabeled product bands that migrate at the same position or slower than the core DGR RNA, and green arrowheads ones that migrate faster. The positions of the 120 and 90 nt cDNA bands are indicated. The two panels are from the same gel, with the black line indicating that intermediate lanes were removed. ( C ) Internally-labeled core DGR RNA was not incubated (–), or incubated with bRT-Avd alone or bRT-Avd with 100 μM standard dNTPs (+dNTP), 100 μM dCTP (+CTP), 100 μM dNTPs excluding dCTP (+d(A,T,G)TP), or 100 μM nonhydrolyzeable analog of dCTP (+N-dCTP) for 2 h. Incubation products were resolved by denaturing PAGE. The band corresponding to the 5′ fragment of the cleaved core RNA containing either a deoxycytidine alone (5′+dC) or cDNA (5′+cDNA), and the band corresponding to the 3′ fragment of the RNA are indicated. ( D ) The core DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The core DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( E ) Radiolabeled products resulting from bRT-Avd activity for 12 h with core, hybrid core dA56, or hybrid core A56 DGR RNA as template. Products were untreated (U) or treated with RNase (+R), and resolved by denaturing PAGE. Separate samples of core dA56 and A56 were 5′ 32 P-labeled for visualization of inputs (I). The positions of the 120 and 90 nt cDNAs are indicated.

    Article Snippet: Reverse transcription reactions with HIV-1 RT were carried out as above, except in 10 μl and containing 10 units RNase inhibitor (NEB), 0.1 μCi/μl [α-32 P]dCTP, 30 ng/μl RNA template, 1 μM PG117 primer ( ) and 2 units of HIV-1 RT (Worthington Biochemical), and the reaction was carried out for 30 min.

    Techniques: Activity Assay, Polyacrylamide Gel Electrophoresis, Labeling, Marker, Incubation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.

    Article Snippet: Reverse transcription reactions with HIV-1 RT were carried out as above, except in 10 μl and containing 10 units RNase inhibitor (NEB), 0.1 μCi/μl [α-32 P]dCTP, 30 ng/μl RNA template, 1 μM PG117 primer ( ) and 2 units of HIV-1 RT (Worthington Biochemical), and the reaction was carried out for 30 min.

    Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Amplification, Produced, Sequencing, Activity Assay, Polyacrylamide Gel Electrophoresis

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

    Journal: Nature

    Article Title: RNA targeting with CRISPR-Cas13a

    doi: 10.1038/nature24049

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

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

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

    Ribonuclease activity of the recombinant TcPR-4b on tomato ( Solanum lycopersicum var. Micro-Tom) total RNA (5 μg). The incubation with TcPR-4b was carried out for 30 min at 25°C. The boiling conditions were 10 min at 95°C. The RNase inhibitor was the RiboLock (40 U; Thermo Scientific). The incubation conditions of the RNase A (Thermo Scientific) were 10 min at 25°C.

    Journal: BMC Plant Biology

    Article Title: The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa

    doi: 10.1186/1471-2229-14-161

    Figure Lengend Snippet: Ribonuclease activity of the recombinant TcPR-4b on tomato ( Solanum lycopersicum var. Micro-Tom) total RNA (5 μg). The incubation with TcPR-4b was carried out for 30 min at 25°C. The boiling conditions were 10 min at 95°C. The RNase inhibitor was the RiboLock (40 U; Thermo Scientific). The incubation conditions of the RNase A (Thermo Scientific) were 10 min at 25°C.

    Article Snippet: As observed in other works [ , , ], the RNase activity of TcPR-4b was inhibited by heating and in the presence of RNase inhibitor (RiboLock, Thermo Scientific) which is able to annul the activity of type A, B and C RNases.

    Techniques: Activity Assay, Recombinant, Incubation

    Action of TcPR-4b on dikaryotic M. perniciosa survival in relation to RNase and DNase activity. A . Action of TcPR-4b on dikaryotic M. perniciosa survival in presence of RNase inhibitor. The following concentrations were used: 40 μg/ml of TcPR-4b and 800 U of RNase inhibitor. B . Action of TcPR-4b on dikaryotic M. perniciosa survival in presence of MgCl 2 . The following concentrations were used: 40 μg/ml of TcPR-4b and 10 mM of MgCl 2 .

    Journal: BMC Plant Biology

    Article Title: The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa

    doi: 10.1186/1471-2229-14-161

    Figure Lengend Snippet: Action of TcPR-4b on dikaryotic M. perniciosa survival in relation to RNase and DNase activity. A . Action of TcPR-4b on dikaryotic M. perniciosa survival in presence of RNase inhibitor. The following concentrations were used: 40 μg/ml of TcPR-4b and 800 U of RNase inhibitor. B . Action of TcPR-4b on dikaryotic M. perniciosa survival in presence of MgCl 2 . The following concentrations were used: 40 μg/ml of TcPR-4b and 10 mM of MgCl 2 .

    Article Snippet: As observed in other works [ , , ], the RNase activity of TcPR-4b was inhibited by heating and in the presence of RNase inhibitor (RiboLock, Thermo Scientific) which is able to annul the activity of type A, B and C RNases.

    Techniques: Activity Assay

    Unique binding motifs in the N-terminus of SLBP interact with FEM1A, FEM1B, and FEM1C. (A) Diagram representing the domain structure of SLBP. The amino acid sequence and substrate receptor binding motifs representing the “degron hotspot” are shown. TAD, translational activation domain; NLS, nuclear localization sequence; RBD, RNA binding domain. (B) FEM1A, FEM1B, and FEM1C interact with amino acids 1–99 of SLBP. C-E Mapping the FEM1A, FEM1B and FEM1C binding regions in SLBP. HEK293T cells were transfected with either empty vector (EV) or FS-tagged SLBP constructs. MLN4924 was added to the cells for 4 hours before collection. Cell lysates were affinity precipitated with anti-STREP resin, and affinity precipitations were probed with the indicated antibodies. (F) The ligase-deficient SLBP(ABCdegron) mutant is unable to bind to CTIF. HEK293T cells were transfected with FLAG-tagged SLBP constructs. Cell lysates were supplemented with SUPERase-In™ RNase Inhibitor and immunoprecipitated with anti-FLAG resin. The immunoprecipitations were probed with the indicated antibodies.

    Journal: Cell Cycle

    Article Title: FEM1 proteins are ancient regulators of SLBP degradation

    doi: 10.1080/15384101.2017.1284715

    Figure Lengend Snippet: Unique binding motifs in the N-terminus of SLBP interact with FEM1A, FEM1B, and FEM1C. (A) Diagram representing the domain structure of SLBP. The amino acid sequence and substrate receptor binding motifs representing the “degron hotspot” are shown. TAD, translational activation domain; NLS, nuclear localization sequence; RBD, RNA binding domain. (B) FEM1A, FEM1B, and FEM1C interact with amino acids 1–99 of SLBP. C-E Mapping the FEM1A, FEM1B and FEM1C binding regions in SLBP. HEK293T cells were transfected with either empty vector (EV) or FS-tagged SLBP constructs. MLN4924 was added to the cells for 4 hours before collection. Cell lysates were affinity precipitated with anti-STREP resin, and affinity precipitations were probed with the indicated antibodies. (F) The ligase-deficient SLBP(ABCdegron) mutant is unable to bind to CTIF. HEK293T cells were transfected with FLAG-tagged SLBP constructs. Cell lysates were supplemented with SUPERase-In™ RNase Inhibitor and immunoprecipitated with anti-FLAG resin. The immunoprecipitations were probed with the indicated antibodies.

    Article Snippet: SUPERase-In™ RNase Inhibitor (Thermo Fisher Scientific) was used at 1U/μL where indicated.

    Techniques: Binding Assay, Sequencing, Activation Assay, RNA Binding Assay, Transfection, Plasmid Preparation, Construct, Mutagenesis, Immunoprecipitation

    TRIM3 is present in mRNP particles but is not essential for mRNP particle trafficking. (A) TRIM3 interacts with PURA in an RNA-dependent manner. TRIM3 was immunoprecipitated from hippocampal synapse-enriched fractions and samples were immunoblotted (IB) and stained for TRIM3 and PURA. PURA was detected in RNase inhibitor–treated samples, but not in RNase-treated samples. Samples prepared from Trim3 −/− mice served as negative control for the immunoprecipitation. (B) TRIM3 and PURA do not interact when expressed in HEK293 cells. HEK293 cells were cotransfected with full-length TRIM3 and GFP-PURA. TRIM3 and PURA were immunoprecipitated from lysates, immunoblotted and stained for TRIM3 and GFP (PURA). PURA did not coimmunoprecipitate with TRIM3, and TRIM3 did not coimmunoprecipiate with PURA. (C and D) PURA levels are not altered in Trim3 −/− mice. Hippocampal synapse-enriched fractions were prepared from wild-type and Trim3 −/− mice under control conditions (home cage) and 2 h after contextual fear conditioning (shock). Samples were immunoblotted and stained for PURA. Normalized PURA levels did not differ between conditions (means ± SEM, n = 4 per genotype). (E) TRIM3 does not alter PURA levels in HEK293 cells. HEK293 cells were cotransfected with PURA and TRIM3, PURA and ΔRBCC-TRIM3, or PURA alone. No differences in PURA levels were observed at any time point after transfection. (F) TRIM3 is not essential for PURA trafficking. Example time-lapse images of a mobile GFP-PURA cluster (red arrow) over a timespan of 90 s are shown. Bars, 2 µm. (G and H) Trim3 −/− neurons and wild-type control neurons expressed equal amounts of PURA clusters (G) and had equal fractions of mobile clusters (H). (I) Trim3 −/− neurons and wild-type control neurons expressed equal amounts of short-distance and long-distance clusters. (J and K) Long-distance PURA clusters show slightly increased kinetics in Trim3 −/− neurons. The total distance moved (J) did not change, but the maximum velocity (K) and the maximum distance reached from origin (L) were significantly increased in Trim3 −/− neurons (means ± SEM, two-tailed t test, *, P

    Journal: The Journal of Cell Biology

    Article Title: Ubiquitin ligase TRIM3 controls hippocampal plasticity and learning by regulating synaptic γ-actin levels

    doi: 10.1083/jcb.201506048

    Figure Lengend Snippet: TRIM3 is present in mRNP particles but is not essential for mRNP particle trafficking. (A) TRIM3 interacts with PURA in an RNA-dependent manner. TRIM3 was immunoprecipitated from hippocampal synapse-enriched fractions and samples were immunoblotted (IB) and stained for TRIM3 and PURA. PURA was detected in RNase inhibitor–treated samples, but not in RNase-treated samples. Samples prepared from Trim3 −/− mice served as negative control for the immunoprecipitation. (B) TRIM3 and PURA do not interact when expressed in HEK293 cells. HEK293 cells were cotransfected with full-length TRIM3 and GFP-PURA. TRIM3 and PURA were immunoprecipitated from lysates, immunoblotted and stained for TRIM3 and GFP (PURA). PURA did not coimmunoprecipitate with TRIM3, and TRIM3 did not coimmunoprecipiate with PURA. (C and D) PURA levels are not altered in Trim3 −/− mice. Hippocampal synapse-enriched fractions were prepared from wild-type and Trim3 −/− mice under control conditions (home cage) and 2 h after contextual fear conditioning (shock). Samples were immunoblotted and stained for PURA. Normalized PURA levels did not differ between conditions (means ± SEM, n = 4 per genotype). (E) TRIM3 does not alter PURA levels in HEK293 cells. HEK293 cells were cotransfected with PURA and TRIM3, PURA and ΔRBCC-TRIM3, or PURA alone. No differences in PURA levels were observed at any time point after transfection. (F) TRIM3 is not essential for PURA trafficking. Example time-lapse images of a mobile GFP-PURA cluster (red arrow) over a timespan of 90 s are shown. Bars, 2 µm. (G and H) Trim3 −/− neurons and wild-type control neurons expressed equal amounts of PURA clusters (G) and had equal fractions of mobile clusters (H). (I) Trim3 −/− neurons and wild-type control neurons expressed equal amounts of short-distance and long-distance clusters. (J and K) Long-distance PURA clusters show slightly increased kinetics in Trim3 −/− neurons. The total distance moved (J) did not change, but the maximum velocity (K) and the maximum distance reached from origin (L) were significantly increased in Trim3 −/− neurons (means ± SEM, two-tailed t test, *, P

    Article Snippet: Quantitative real-time PCR Immunoprecipitations were performed using antibodies against TRIM3 or PURA in the presence of RNase inhibitors (40 U/ml; Thermo Fisher Scientific).

    Techniques: Immunoprecipitation, Staining, Mouse Assay, Negative Control, Transfection, Two Tailed Test

    Regulation of IL-8 mRNA expression in immortalized human gingival keratinocytes. (A) A representative storage phosphor scan demonstrates that, by using quantitative RNase protection assay, moderate IL-8 mRNA expression was detected in 8 μg of total RNA from immortalized keratinocytes under normal control conditions as seen at time zero. Following treatment of separate flasks of the same culture over 24 h with 50 ng of PMA per ml, IL-8 mRNA expression dramatically increases over a 6-h period, returning to nearly constitutive levels by 12 h. Test lanes, hours 0 to 24, show the protected probes after binding to IL-8 (374 bp) and GAPDH (220 bp) mRNA, respectively, within the sample, followed by RNase treatment. An increase in the band intensity of GAPDH at 2 to 6 h reflects an increase in the total amount of mRNA, as expected with PMA stimulation. This did not affect quantitation (see panel B, below), since the ratio of GAPDH to IL-8 was still proportionally constant. Control lanes (C1 and C2) show the two probes GAPDH (C1, 266 bp) and IL-8 (C2, 420 bp) to which no RNase has been added. The results are representative of three experiments. (B) Quantitation of IL-8 mRNA in immortalized keratinocytes following induction with PMA. From panel A the signal intensity determined for the IL-8 protected fragment was normalized to the abundance of the internal control GAPDH at each time point. The data shown are the mean ± the standard deviation ( n = 3).

    Journal: Infection and Immunity

    Article Title: Calprotectin Expression by Gingival Epithelial Cells

    doi: 10.1128/IAI.69.5.3248-3254.2001

    Figure Lengend Snippet: Regulation of IL-8 mRNA expression in immortalized human gingival keratinocytes. (A) A representative storage phosphor scan demonstrates that, by using quantitative RNase protection assay, moderate IL-8 mRNA expression was detected in 8 μg of total RNA from immortalized keratinocytes under normal control conditions as seen at time zero. Following treatment of separate flasks of the same culture over 24 h with 50 ng of PMA per ml, IL-8 mRNA expression dramatically increases over a 6-h period, returning to nearly constitutive levels by 12 h. Test lanes, hours 0 to 24, show the protected probes after binding to IL-8 (374 bp) and GAPDH (220 bp) mRNA, respectively, within the sample, followed by RNase treatment. An increase in the band intensity of GAPDH at 2 to 6 h reflects an increase in the total amount of mRNA, as expected with PMA stimulation. This did not affect quantitation (see panel B, below), since the ratio of GAPDH to IL-8 was still proportionally constant. Control lanes (C1 and C2) show the two probes GAPDH (C1, 266 bp) and IL-8 (C2, 420 bp) to which no RNase has been added. The results are representative of three experiments. (B) Quantitation of IL-8 mRNA in immortalized keratinocytes following induction with PMA. From panel A the signal intensity determined for the IL-8 protected fragment was normalized to the abundance of the internal control GAPDH at each time point. The data shown are the mean ± the standard deviation ( n = 3).

    Article Snippet: The radiolabeled probes were synthesized under the following reaction conditions: 500 μM concentrations each of rCTP, rGTP, and rATP, and 1 μM rUTP; 3 μM [α-32 P]UTP (800 Ci/mmol, 10 mCi/ml) (DuPont NEN Research Products, Boston, Mass.); 0.5 μl of PCR template; 1 U of T7 or T3 RNA polymerase (Stratagene); 2 μl of transcription buffer (Stratagene); 40 U of RNase inhibitor (Promega, Madison, Wis.); and distilled H2 O to a total volume of 10 μl.

    Techniques: Expressing, Rnase Protection Assay, Binding Assay, Quantitation Assay, Standard Deviation

    Ex-miRNA Are Stable and Protected from RNases in BALF (A) qPCR of synthetic miRNAs (“calibrators” Cal1 and Cal2) spiked into Trizol or BALF with or without an RNase inhibitor (RNAsin) (n = 3 from 1–2 independent experiments, 1-way ANOVA with Bonferroni’s multiple comparison test). (B) qPCR of analysis of miRNAs from BALF and RNA pellets treated with RNaseA (n = 3 from 3 independent experiments, 2-way ANOVA with Bonferroni’s multiple comparison test). Error bars are mean + SD; *p

    Journal: Cell reports

    Article Title: Increased Hematopoietic Extracellular RNAs and Vesicles in the Lung during Allergic Airway Responses

    doi: 10.1016/j.celrep.2019.01.002

    Figure Lengend Snippet: Ex-miRNA Are Stable and Protected from RNases in BALF (A) qPCR of synthetic miRNAs (“calibrators” Cal1 and Cal2) spiked into Trizol or BALF with or without an RNase inhibitor (RNAsin) (n = 3 from 1–2 independent experiments, 1-way ANOVA with Bonferroni’s multiple comparison test). (B) qPCR of analysis of miRNAs from BALF and RNA pellets treated with RNaseA (n = 3 from 3 independent experiments, 2-way ANOVA with Bonferroni’s multiple comparison test). Error bars are mean + SD; *p

    Article Snippet: To test whether synthetic miRNA-like oligoribonucleotide sequences were intrinsically stable in BALF, 22 nucleotide RNA calibrators (calibrators 1-4 ( )) were spiked into BALF and incubated for 5min at 37°C with or without RNAsin ribonuclease inhibitor (Promega) before RNA extraction.

    Techniques: Real-time Polymerase Chain Reaction

    Telomerase activity in kinetoplastid parasites. Lanes 1–6, T. brucei DEAE eluate; lanes 8–13, L. tarentolae DEAE eluate; lanes 15–20, L. major DEAE eluate. Telomerase products were fractionated in 10% sequencing gels to reveal the periodicity of banding pattern. In lanes 2, 9, and 16, extracts were pretreated with RNase A; lanes 3, 10, and 17, RNasin incubated with extract before addition of RNase; lanes 4, 11, and 18, RNase A was added after telomerase step incubation (+). nTS, reaction performed without the forward primer; nC, reaction performed in the absence of CX-ext; nE, reaction in which the extracts were omitted. ( A ) One-tube TRAP using primer CX-ext as reverse primer and semipurified extracts. ( B ) Two-tube modified TRAP using CX-ext reverse primer. The assays were performed with half the amount of DEAE fractions used in A .

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

    Article Title: Telomerase in kinetoplastid parasitic protozoa

    doi:

    Figure Lengend Snippet: Telomerase activity in kinetoplastid parasites. Lanes 1–6, T. brucei DEAE eluate; lanes 8–13, L. tarentolae DEAE eluate; lanes 15–20, L. major DEAE eluate. Telomerase products were fractionated in 10% sequencing gels to reveal the periodicity of banding pattern. In lanes 2, 9, and 16, extracts were pretreated with RNase A; lanes 3, 10, and 17, RNasin incubated with extract before addition of RNase; lanes 4, 11, and 18, RNase A was added after telomerase step incubation (+). nTS, reaction performed without the forward primer; nC, reaction performed in the absence of CX-ext; nE, reaction in which the extracts were omitted. ( A ) One-tube TRAP using primer CX-ext as reverse primer and semipurified extracts. ( B ) Two-tube modified TRAP using CX-ext reverse primer. The assays were performed with half the amount of DEAE fractions used in A .

    Article Snippet: Activity in the extracts was tested for RNase A sensitivity by incubation with 100 ng of RNase A (Sigma) for 5 min at 37°C before or after the telomerase reaction step and with or without 1 unit of RNase inhibitor RNasin (Promega) before addition of RNase A.

    Techniques: Activity Assay, Sequencing, Incubation, Modification

    T. brucei activity monitored directly by telomerase primer-extension assay. Reactions were performed with DEAE fraction and primer tel 2. Lane 1, standard reaction; lane 2, extract pretreated with 100 ng of RNase A; lane 3, extract incubated with RNasin before addition of RNase A; lane 4, RNase A treatment after telomerase reaction (+); lane 5, nP, no input primer, lane 6, nE, extract substituted by reaction buffer; lane M, terminal deoxynucleotidyltransferase used to label tel 6 with [α- 32 P]dCTP (19 indicates the position of the primer plus 1-nt molecular weight marker).

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

    Article Title: Telomerase in kinetoplastid parasitic protozoa

    doi:

    Figure Lengend Snippet: T. brucei activity monitored directly by telomerase primer-extension assay. Reactions were performed with DEAE fraction and primer tel 2. Lane 1, standard reaction; lane 2, extract pretreated with 100 ng of RNase A; lane 3, extract incubated with RNasin before addition of RNase A; lane 4, RNase A treatment after telomerase reaction (+); lane 5, nP, no input primer, lane 6, nE, extract substituted by reaction buffer; lane M, terminal deoxynucleotidyltransferase used to label tel 6 with [α- 32 P]dCTP (19 indicates the position of the primer plus 1-nt molecular weight marker).

    Article Snippet: Activity in the extracts was tested for RNase A sensitivity by incubation with 100 ng of RNase A (Sigma) for 5 min at 37°C before or after the telomerase reaction step and with or without 1 unit of RNase inhibitor RNasin (Promega) before addition of RNase A.

    Techniques: Activity Assay, Primer Extension Assay, Incubation, Molecular Weight, Marker

    FUS forms a complex with itself, PABP and RNA. ( A ) Co-immunoprecipitation of HA-FUS, Myc-FUS and PABP from HEK293T cells. Immunoprecipitation with a Myc (mouse anti-Myc, CST) antibody pulled down PABP (mouse anti-PABP, Sigma) along with anti-HA-FUS (rabbit HA, CST). RNasin was added to block all RNase activity. ( B ) The interaction between HA-FUS and Myc-FUS was not altered by the addition of RNase, while the co-immunoprecipitation of PABP was completely abolished. ( C ) Immunoprecipitation of FUS (mouse anti-FUS, Santa Cruz) from untransfected cells shows that endogenous FUS (rabbit anti-FUS, Novus Biologicals) also interacts with PABP (mouse anti-PABP, Sigma) and treatment with RNase shows that this interaction is also dependent on RNA. ( D ) Mock immunoprecipitation experiments with either untransfected cells or a single transfection of either HA-FUS WT or Myc-FUS WT with no antibody showed that neither endogenous nor tagged FUS binds to the beads. ( E ) Immunoprecipitation of single transfections with an antibody to the wrong tag showed that a Myc antibody does not pull down HA-FUS and a HA antibody does not precipitate Myc-FUS. FT indicates flow-through of proteins that did not bind to the beads.

    Journal: Human Molecular Genetics

    Article Title: ALS mutant FUS disrupts nuclear localization and sequesters wild-type FUS within cytoplasmic stress granules

    doi: 10.1093/hmg/ddt117

    Figure Lengend Snippet: FUS forms a complex with itself, PABP and RNA. ( A ) Co-immunoprecipitation of HA-FUS, Myc-FUS and PABP from HEK293T cells. Immunoprecipitation with a Myc (mouse anti-Myc, CST) antibody pulled down PABP (mouse anti-PABP, Sigma) along with anti-HA-FUS (rabbit HA, CST). RNasin was added to block all RNase activity. ( B ) The interaction between HA-FUS and Myc-FUS was not altered by the addition of RNase, while the co-immunoprecipitation of PABP was completely abolished. ( C ) Immunoprecipitation of FUS (mouse anti-FUS, Santa Cruz) from untransfected cells shows that endogenous FUS (rabbit anti-FUS, Novus Biologicals) also interacts with PABP (mouse anti-PABP, Sigma) and treatment with RNase shows that this interaction is also dependent on RNA. ( D ) Mock immunoprecipitation experiments with either untransfected cells or a single transfection of either HA-FUS WT or Myc-FUS WT with no antibody showed that neither endogenous nor tagged FUS binds to the beads. ( E ) Immunoprecipitation of single transfections with an antibody to the wrong tag showed that a Myc antibody does not pull down HA-FUS and a HA antibody does not precipitate Myc-FUS. FT indicates flow-through of proteins that did not bind to the beads.

    Article Snippet: 5 µl of an RNase A/T1 mix (Fermentas, Yorkshire, UK) or RNasin Plus RNase inhibitor (Promega, Southampton, UK) was added to the tubes and incubated at 37°C for 5 min, then placed back on ice.

    Techniques: Immunoprecipitation, Blocking Assay, Activity Assay, Transfection, Flow Cytometry

    Characterization of MRPP3 ΔMTS alone and in complex with MRPP1 ΔMTS and MRPP2. A , domain organization for MRPP3 ΔMTS indicating the N extension (residues 50–206), PPR domain (residues 207–329, red ), core domain (residues 330–361 and 542–583, yellow ), NYN domain (residues 362–541, brown ), and structural zinc ion bound in a Cys-Cys-His-Cys motif in the core domain. B , top , analytical SEC profiles for the mixture of MRPP1 ΔMTS , MRPP2, and MRPP3 ΔMTS ( green line ) and the mixture of MRPP1 ΔMTS , MRPP2, MRPP3 ΔMTS , and pre-(mt)tRNA Ile ( black line ) applied to a Superdex S200 column. A replicate of this experiment is shown in Fig. S5 . The elution volumes for individual MRPP1 ΔMTS (from Fig. 3 A ), MRPP2 (from Fig. 3 A ), and MRPP3 ΔMTS (from Fig. S4 F ) proteins, as well as the mixture of MRPP1 ΔMTS and MRPP2 (from Fig. 3 A ), are indicated with arrows above the chromatogram ( blue , orange , red , and pink , respectively). Bottom , SDS-PAGE ( first two gel panels ) and urea-PAGE ( last two gel panels ) analysis of eluted fractions to visualize proteins and pre-(mt)tRNA Ile , respectively. The black dashed box indicates lanes where the complex containing MRPP1 ΔMTS , MRPP2, MRPP3, and pre-(mt)tRNA Ile would be found on the SDS-PAGE. C , urea-polyacrylamide denaturing gels showing the RNase P reaction of pre-(mt)tRNA Ile set up, for mixtures of MRPP2 and MRPP3 ΔMTS with different MRPP1 truncated proteins (MRPP1 ΔMTS , MRPP1 MT+C(Δ202) , MRPP1 N ). All lanes shown are taken from one experimental gel only. A downward shift in band location of processed (mt)tRNA Ile relative to that of pre-(mt)tRNA Ile and the concomitant appearance of a band corresponding to the removed 5′-leader indicate RNase P activity. D , urea-polyacrylamide denaturing gels showing the RNase P reaction of pre-(mt)tRNA Ile set up for the complex of MRPP1 ΔMTS and MRPP2 without MRPP3 ( lane 2 ) and in the presence of different truncated MRPP3 proteins ( lanes 3–6 ). A negative control of MRPP1 ΔMTS , MRPP2, and MRPP3 ΔMTS mixed with EDTA ( lane 1 ) is included. M r RNA markers are shown ( lane M ). All lanes shown are taken from one experimental gel only. Reactions in C and D were run for 30 min at protein concentrations of 300 n m MRPP1–MRPP2 and 150 n m MRPP3.

    Journal: The Journal of Biological Chemistry

    Article Title: Structural insight into the human mitochondrial tRNA purine N1-methyltransferase and ribonuclease P complexes

    doi: 10.1074/jbc.RA117.001286

    Figure Lengend Snippet: Characterization of MRPP3 ΔMTS alone and in complex with MRPP1 ΔMTS and MRPP2. A , domain organization for MRPP3 ΔMTS indicating the N extension (residues 50–206), PPR domain (residues 207–329, red ), core domain (residues 330–361 and 542–583, yellow ), NYN domain (residues 362–541, brown ), and structural zinc ion bound in a Cys-Cys-His-Cys motif in the core domain. B , top , analytical SEC profiles for the mixture of MRPP1 ΔMTS , MRPP2, and MRPP3 ΔMTS ( green line ) and the mixture of MRPP1 ΔMTS , MRPP2, MRPP3 ΔMTS , and pre-(mt)tRNA Ile ( black line ) applied to a Superdex S200 column. A replicate of this experiment is shown in Fig. S5 . The elution volumes for individual MRPP1 ΔMTS (from Fig. 3 A ), MRPP2 (from Fig. 3 A ), and MRPP3 ΔMTS (from Fig. S4 F ) proteins, as well as the mixture of MRPP1 ΔMTS and MRPP2 (from Fig. 3 A ), are indicated with arrows above the chromatogram ( blue , orange , red , and pink , respectively). Bottom , SDS-PAGE ( first two gel panels ) and urea-PAGE ( last two gel panels ) analysis of eluted fractions to visualize proteins and pre-(mt)tRNA Ile , respectively. The black dashed box indicates lanes where the complex containing MRPP1 ΔMTS , MRPP2, MRPP3, and pre-(mt)tRNA Ile would be found on the SDS-PAGE. C , urea-polyacrylamide denaturing gels showing the RNase P reaction of pre-(mt)tRNA Ile set up, for mixtures of MRPP2 and MRPP3 ΔMTS with different MRPP1 truncated proteins (MRPP1 ΔMTS , MRPP1 MT+C(Δ202) , MRPP1 N ). All lanes shown are taken from one experimental gel only. A downward shift in band location of processed (mt)tRNA Ile relative to that of pre-(mt)tRNA Ile and the concomitant appearance of a band corresponding to the removed 5′-leader indicate RNase P activity. D , urea-polyacrylamide denaturing gels showing the RNase P reaction of pre-(mt)tRNA Ile set up for the complex of MRPP1 ΔMTS and MRPP2 without MRPP3 ( lane 2 ) and in the presence of different truncated MRPP3 proteins ( lanes 3–6 ). A negative control of MRPP1 ΔMTS , MRPP2, and MRPP3 ΔMTS mixed with EDTA ( lane 1 ) is included. M r RNA markers are shown ( lane M ). All lanes shown are taken from one experimental gel only. Reactions in C and D were run for 30 min at protein concentrations of 300 n m MRPP1–MRPP2 and 150 n m MRPP3.

    Article Snippet: RNase P activity assay RNase P cleavage was performed by mixing 300 nm MRPP1–MRPP2, 150 nm MRPP3, 10 units of RNase inhibitors (RNasin from Promega), and 400 nm in vitro transcribed pre-(mt) tRNAIle in a buffer of 30 mm Tris-HCl, pH 8, 40 mm NaCl, 4.5 mm MgCl2 , and 2 mm DTT to a total reaction volume of 8.25 μl.

    Techniques: Size-exclusion Chromatography, SDS Page, Polyacrylamide Gel Electrophoresis, Activity Assay, Negative Control

    RNase protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7 , 8–11 , 12–15 , 16–19 , 20–23 , respectively) for 6 h at 37 °C, followed by 1 μL (40 U) RiboLock RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1 , after incubation with heparin ( lane 2 ) and 0.35 μg RNase A per 1 μg siRNA ( lane 3 ).

    Journal: Molecular Pharmaceutics

    Article Title: Poly(amidoamine) Dendrimer Nanocarriers and Their Aerosol Formulations for siRNA Delivery to the Lung Epithelium

    doi: 10.1021/mp4006358

    Figure Lengend Snippet: RNase protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7 , 8–11 , 12–15 , 16–19 , 20–23 , respectively) for 6 h at 37 °C, followed by 1 μL (40 U) RiboLock RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1 , after incubation with heparin ( lane 2 ) and 0.35 μg RNase A per 1 μg siRNA ( lane 3 ).

    Article Snippet: RiboLock RNase Inhibitor (RI, EO0381, 40 U × μL–1 ) was purchased from Thermo Scientific (part of Thermo Fisher Scientific, Waltham, MA, U.S.A.).

    Techniques: Rnase Protection Assay, Agarose Gel Electrophoresis, Concentration Assay, Incubation, Blocking Assay, Activity Assay

    RNase protection assay (non-denaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes as a function of the N/P ratio. Dendriplexes incubated in the absence (−) or presence (+) of the treatments: RNase A (0.162 μg per 1 μg siRNA) for 6 h at 37 °C, followed by 1 μL (40 U) RiboLock RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (300 ng) before ( lane 1 ) and after ( lane 2 ) incubation with RNase A.

    Journal: Molecular Pharmaceutics

    Article Title: Poly(amidoamine) Dendrimer Nanocarriers and Their Aerosol Formulations for siRNA Delivery to the Lung Epithelium

    doi: 10.1021/mp4006358

    Figure Lengend Snippet: RNase protection assay (non-denaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes as a function of the N/P ratio. Dendriplexes incubated in the absence (−) or presence (+) of the treatments: RNase A (0.162 μg per 1 μg siRNA) for 6 h at 37 °C, followed by 1 μL (40 U) RiboLock RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (300 ng) before ( lane 1 ) and after ( lane 2 ) incubation with RNase A.

    Article Snippet: RiboLock RNase Inhibitor (RI, EO0381, 40 U × μL–1 ) was purchased from Thermo Scientific (part of Thermo Fisher Scientific, Waltham, MA, U.S.A.).

    Techniques: Rnase Protection Assay, Agarose Gel Electrophoresis, Incubation, Blocking Assay, Activity Assay

    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total RNA was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by RT-PCR. Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p

    Journal: PLoS Pathogens

    Article Title: KSHV induces immunoglobulin rearrangements in mature B lymphocytes

    doi: 10.1371/journal.ppat.1006967

    Figure Lengend Snippet: A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total RNA was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by RT-PCR. Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p

    Article Snippet: Single cells were harvested by flow sorting into 96-well PCR plates containing 4μl of RNA lysis buffer (0.5x PBS+10mM DTT+4U SUPERas-In (Thermo Cat #AM2694)).

    Techniques: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction

    Elevated polyadenylation activity of the dark extract. Polyadenylation was assayed under the conditions described for the in vitro processing assays except that 2 mM ATP and 20 U RNasin were added to inactivate the endogenous nucleases. After the indicated times the reaction was stopped and the RNA products analyzed by electrophoresis and autoradiography. Control assays were incubated for 40 min in the presence of 4 mM cordycepin (Sigma; lanes 40′ + Cord.).

    Journal: Nucleic Acids Research

    Article Title: Endonucleolytic activation directs dark-induced chloroplast mRNA degradation

    doi:

    Figure Lengend Snippet: Elevated polyadenylation activity of the dark extract. Polyadenylation was assayed under the conditions described for the in vitro processing assays except that 2 mM ATP and 20 U RNasin were added to inactivate the endogenous nucleases. After the indicated times the reaction was stopped and the RNA products analyzed by electrophoresis and autoradiography. Control assays were incubated for 40 min in the presence of 4 mM cordycepin (Sigma; lanes 40′ + Cord.).

    Article Snippet: The assay contained 40 mM Tris–HCl pH 8.0, 6 mM MgCl2 , 2 mM spermidin, 10 mM DTT, 0.5 mM of GTP, CTP and ATP, 0.1 mM UTP, 10 U RNasin (Boehringer-Mannheim/Roche), 10 U T7-RNA polymerase (Boehringer-Mannheim/Roche) and 100 µCi [α-32 P]UTP (20 µCi/µl, 800 Ci/mmol).

    Techniques: Activity Assay, In Vitro, Electrophoresis, Autoradiography, Incubation

    Products of influenza virus replication factor (IREF-2)-dependent unprimed RNA synthesis. ( A ) RNase T2 protection assay. Radioactively labeled vRNA products were synthesized in the cell-free viral RNA synthesis system with micrococcal nuclease-treated vRNP (mnRNP) and the v53 model template in the presence of ApG (lanes 1–3), the c53 model template in the presence of ApG (lanes 4–6), and c53 in the presence of the IREF-2 fraction and in the absence of ApG (lanes 7–9). Viral RNA products were hybridized with excess amounts of nonlabeled v53 (lanes 2, 5, and 8) or c53 (lanes 3, 6, and 9), which was followed by digestion with RNase T2. Hybridized and digested RNA samples were extracted, collected, and subjected to 10% Urea-PAGE followed by autoradiography visualization. The closed arrowhead indicates 53-nt-long RNAs. ( B ) Analysis of the 5'-terminal structure of IREF-2-dependent unprimed vRNA products. [α- 32 P] GTP-labeled IREF-2-dependent unprimed vRNA products were prepared in the cell-free viral RNA synthesis system. The radioactively labeled 53-nt-long vRNA products were isolated from 10% Urea-PAGE, which was followed by excision and elution from the gel. A portion of the isolated 53-nt-long products was treated with alkaline phosphatase (lanes 2 and 4). Both nontreated and alkaline phosphatase-treated [α- 32 P] GTP-labeled unprimed vRNA products were digested with RNase T2 (lanes 1–4) or snake venom phosphodiesterase (lane 5). The digested materials were spotted onto a polyethylenimine (PEI)-cellulose thin layer and developed with 1 N acetic acid-4 M LiCl (4:1, v/v) (left panel; lanes 1 and 2) or 1.6 M LiCl (right panel; lanes 3–6) and visualized by autoradiography. For mobility standards, nonradiolabeled AMP, ADP, and ATP were also subjected to thin-layer chromatography and are indicated on the left side of each panel. For a marker of pppAp, [γ- 32 P] ATP-labeled v53 synthesized using T7 RNA polymerase was also subjected to RNase T2 digestion and then to thin-layer chromatography (lane 6). The expected nucleotide positions are indicated on the right side of each panel by closed arrowheads. DOI: http://dx.doi.org/10.7554/eLife.08939.004

    Journal: eLife

    Article Title: pp32 and APRIL are host cell-derived regulators of influenza virus RNA synthesis from cRNA

    doi: 10.7554/eLife.08939

    Figure Lengend Snippet: Products of influenza virus replication factor (IREF-2)-dependent unprimed RNA synthesis. ( A ) RNase T2 protection assay. Radioactively labeled vRNA products were synthesized in the cell-free viral RNA synthesis system with micrococcal nuclease-treated vRNP (mnRNP) and the v53 model template in the presence of ApG (lanes 1–3), the c53 model template in the presence of ApG (lanes 4–6), and c53 in the presence of the IREF-2 fraction and in the absence of ApG (lanes 7–9). Viral RNA products were hybridized with excess amounts of nonlabeled v53 (lanes 2, 5, and 8) or c53 (lanes 3, 6, and 9), which was followed by digestion with RNase T2. Hybridized and digested RNA samples were extracted, collected, and subjected to 10% Urea-PAGE followed by autoradiography visualization. The closed arrowhead indicates 53-nt-long RNAs. ( B ) Analysis of the 5'-terminal structure of IREF-2-dependent unprimed vRNA products. [α- 32 P] GTP-labeled IREF-2-dependent unprimed vRNA products were prepared in the cell-free viral RNA synthesis system. The radioactively labeled 53-nt-long vRNA products were isolated from 10% Urea-PAGE, which was followed by excision and elution from the gel. A portion of the isolated 53-nt-long products was treated with alkaline phosphatase (lanes 2 and 4). Both nontreated and alkaline phosphatase-treated [α- 32 P] GTP-labeled unprimed vRNA products were digested with RNase T2 (lanes 1–4) or snake venom phosphodiesterase (lane 5). The digested materials were spotted onto a polyethylenimine (PEI)-cellulose thin layer and developed with 1 N acetic acid-4 M LiCl (4:1, v/v) (left panel; lanes 1 and 2) or 1.6 M LiCl (right panel; lanes 3–6) and visualized by autoradiography. For mobility standards, nonradiolabeled AMP, ADP, and ATP were also subjected to thin-layer chromatography and are indicated on the left side of each panel. For a marker of pppAp, [γ- 32 P] ATP-labeled v53 synthesized using T7 RNA polymerase was also subjected to RNase T2 digestion and then to thin-layer chromatography (lane 6). The expected nucleotide positions are indicated on the right side of each panel by closed arrowheads. DOI: http://dx.doi.org/10.7554/eLife.08939.004

    Article Snippet: Cell-free viral RNA synthesis was carried out at 30°C in a final volume of 20 μl or 25 μl in the presence of 50 mM HEPES-NaOH (pH 7.9), 3 mM MgCl2 , 50 mM KCl, 1 mM DTT, 500 μM each of ATP, UTP, and CTP and 25 μM GTP, 5 μCi of [α-32 P] GTP (3000 Ci/mmol), 8 U of RNase inhibitor from human placenta (Toyobo, Japan), 10 ng of a 53-nt-long model viral RNA template of negative or positive polarity (v53 and c53, respectively) and approximately 40 μg NP-equivalent, alternatively 5 ng PB1-equivalent of mnRNP as an enzyme source.

    Techniques: Labeling, Synthesized, Polyacrylamide Gel Electrophoresis, Autoradiography, Isolation, Thin Layer Chromatography, Marker

    Electrophoresis mobility shift assay for influenza virus replication factor-2 (IREF-2) and viral RNA. Radioactively labeled 53-nt-long model vRNA and complementary RNA(cRNA) probes (v53 and c53; 246.9 cpm/fmol) were synthesized by T7 RNA polymerase using [α- 32 P] GTP and isolated by gel excision. Each 500 pM (final concentration) of the labeled viral RNA probes, v53 (lanes 1–7) and c53 (lanes 8–14) was incubated with 10 nM or 50 nM of recombinant NP prepared using the Escherichia. coli expression system (lanes 2, 3, 9, and 10), recombinant pp32 (lanes 4, 5, 11, and 12), and recombinant APRIL (lanes 6, 7, 13, and 14) in 50 mM HEPES-NaOH (pH 7.9), 50 mM KCl, 0.5 U/μl of RNase inhibitor, and 15% (v/v) glycerol at 30°C for 30 min. After incubation, each binding mixture was loaded onto 0.6% agarose gel (buffered with TBE) and separated by electrophoresis (50 V for 3 hr). The gel was dried and visualized by autoradiography. DOI: http://dx.doi.org/10.7554/eLife.08939.008

    Journal: eLife

    Article Title: pp32 and APRIL are host cell-derived regulators of influenza virus RNA synthesis from cRNA

    doi: 10.7554/eLife.08939

    Figure Lengend Snippet: Electrophoresis mobility shift assay for influenza virus replication factor-2 (IREF-2) and viral RNA. Radioactively labeled 53-nt-long model vRNA and complementary RNA(cRNA) probes (v53 and c53; 246.9 cpm/fmol) were synthesized by T7 RNA polymerase using [α- 32 P] GTP and isolated by gel excision. Each 500 pM (final concentration) of the labeled viral RNA probes, v53 (lanes 1–7) and c53 (lanes 8–14) was incubated with 10 nM or 50 nM of recombinant NP prepared using the Escherichia. coli expression system (lanes 2, 3, 9, and 10), recombinant pp32 (lanes 4, 5, 11, and 12), and recombinant APRIL (lanes 6, 7, 13, and 14) in 50 mM HEPES-NaOH (pH 7.9), 50 mM KCl, 0.5 U/μl of RNase inhibitor, and 15% (v/v) glycerol at 30°C for 30 min. After incubation, each binding mixture was loaded onto 0.6% agarose gel (buffered with TBE) and separated by electrophoresis (50 V for 3 hr). The gel was dried and visualized by autoradiography. DOI: http://dx.doi.org/10.7554/eLife.08939.008

    Article Snippet: Cell-free viral RNA synthesis was carried out at 30°C in a final volume of 20 μl or 25 μl in the presence of 50 mM HEPES-NaOH (pH 7.9), 3 mM MgCl2 , 50 mM KCl, 1 mM DTT, 500 μM each of ATP, UTP, and CTP and 25 μM GTP, 5 μCi of [α-32 P] GTP (3000 Ci/mmol), 8 U of RNase inhibitor from human placenta (Toyobo, Japan), 10 ng of a 53-nt-long model viral RNA template of negative or positive polarity (v53 and c53, respectively) and approximately 40 μg NP-equivalent, alternatively 5 ng PB1-equivalent of mnRNP as an enzyme source.

    Techniques: Electrophoresis, Mobility Shift, Labeling, Synthesized, Isolation, Concentration Assay, Incubation, Recombinant, Expressing, Binding Assay, Agarose Gel Electrophoresis, Autoradiography

    Fluorometric detection. (A) Principle of the fluorometric detection based on the fluorescence resonance energy transfer [ 34 ]. The fluorescent of 6-FAM is typically quenched by BHQ-1, but it emits a bright signal when the oligonucleotide is cleaved. (B-F) Investigation on the MazFpp cleavage site. One hundred nanograms of RNase A (blue) or MazFpp (green) was incubated with each oligonucleotide. The yellow plot represents a control reaction in which no enzyme was added.

    Journal: PLoS ONE

    Article Title: Characterization of MazF-Mediated Sequence-Specific RNA Cleavage in Pseudomonas putida Using Massive Parallel Sequencing

    doi: 10.1371/journal.pone.0149494

    Figure Lengend Snippet: Fluorometric detection. (A) Principle of the fluorometric detection based on the fluorescence resonance energy transfer [ 34 ]. The fluorescent of 6-FAM is typically quenched by BHQ-1, but it emits a bright signal when the oligonucleotide is cleaved. (B-F) Investigation on the MazFpp cleavage site. One hundred nanograms of RNase A (blue) or MazFpp (green) was incubated with each oligonucleotide. The yellow plot represents a control reaction in which no enzyme was added.

    Article Snippet: This substrate was incubated at 37°C for 15 minutes with two distinct RNA interferases: 2 U of MazF (Takara) in MazF buffer (Takara) containing 4 U of recombinant RNase inhibitor (Takara) or 300 ng of MazFpp in MazFpp buffer (20 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, 0.01% tritonX-100, and 4 U of recombinant RNase inhibitor).

    Techniques: Fluorescence, Förster Resonance Energy Transfer, Incubation

    (a) SDS-PAGE of NLP. Lane 1, protein molecular mass marker; lane 2, untreated VSV NLP; lane 3, empty NLP after RNase A treatment; lane 4, reconstituted VSV NLP containing poly(rA) RNA. (b) Image of crystals of NLP with encapsidated poly(rG) RNA.

    Journal: Journal of Virology

    Article Title: Access to RNA Encapsidated in the Nucleocapsid of Vesicular Stomatitis Virus ▿

    doi: 10.1128/JVI.01927-10

    Figure Lengend Snippet: (a) SDS-PAGE of NLP. Lane 1, protein molecular mass marker; lane 2, untreated VSV NLP; lane 3, empty NLP after RNase A treatment; lane 4, reconstituted VSV NLP containing poly(rA) RNA. (b) Image of crystals of NLP with encapsidated poly(rG) RNA.

    Article Snippet: In the presence of RNase inhibitor (Qiagen), the empty NLP was incubated with poly(rA) (Midland) at a molar ratio of 1:5 for 15 min at 42°C.

    Techniques: SDS Page, Marker

    RNA analysis and electron microscopy of VSV nucleocapsid-like particles (NLP) and viral nucleocapsids digested with RNase A. (a) RNA electrophoresis. Purified VSV NLP treated with RNase A (1 mg/ml, final concentration) at room temperature (lane 3), 37°C

    Journal: Journal of Virology

    Article Title: Access to RNA Encapsidated in the Nucleocapsid of Vesicular Stomatitis Virus ▿

    doi: 10.1128/JVI.01927-10

    Figure Lengend Snippet: RNA analysis and electron microscopy of VSV nucleocapsid-like particles (NLP) and viral nucleocapsids digested with RNase A. (a) RNA electrophoresis. Purified VSV NLP treated with RNase A (1 mg/ml, final concentration) at room temperature (lane 3), 37°C

    Article Snippet: In the presence of RNase inhibitor (Qiagen), the empty NLP was incubated with poly(rA) (Midland) at a molar ratio of 1:5 for 15 min at 42°C.

    Techniques: Electron Microscopy, Electrophoresis, Purification, Concentration Assay

    2′-F modified siRNA inhibits gene expression in living mice. Representative bioluminescence images of light emitted from living mice transfected, 2 d earlier, with the luciferase expression plasmids pGL3-control. Mice ( N = 4–5 mice per group) received pGL3-control alone ( A ) or were cotransfected with ( B ) 2′-OH GL3 siRNA, ( C ) 2′-OH GL3 siRNA and the RNase inhibitor, RNasin, ( D ) 2′-F GL3 siRNA, ( E ) 2′-F inverted GL2 siRNA. The mouse in panel F received a low pressure i.v. injection of 2′-F GL3 siRNA on day 1 to test if stabilized siRNAs could be taken up by the liver without hydrodynamic transfection. Mice in panels B , C , and D show significant reductions in emitted light as a result of RNA. Hydrodynamic transfection with 2′-F inverted GL2 siRNA and low pressure i.v. transfection of 2′-F GL3 siRNA did not result in significant RNAi.

    Journal: RNA

    Article Title: In vivo activity of nuclease-resistant siRNAs

    doi: 10.1261/rna.5239604

    Figure Lengend Snippet: 2′-F modified siRNA inhibits gene expression in living mice. Representative bioluminescence images of light emitted from living mice transfected, 2 d earlier, with the luciferase expression plasmids pGL3-control. Mice ( N = 4–5 mice per group) received pGL3-control alone ( A ) or were cotransfected with ( B ) 2′-OH GL3 siRNA, ( C ) 2′-OH GL3 siRNA and the RNase inhibitor, RNasin, ( D ) 2′-F GL3 siRNA, ( E ) 2′-F inverted GL2 siRNA. The mouse in panel F received a low pressure i.v. injection of 2′-F GL3 siRNA on day 1 to test if stabilized siRNAs could be taken up by the liver without hydrodynamic transfection. Mice in panels B , C , and D show significant reductions in emitted light as a result of RNA. Hydrodynamic transfection with 2′-F inverted GL2 siRNA and low pressure i.v. transfection of 2′-F GL3 siRNA did not result in significant RNAi.

    Article Snippet: Briefly, 1.8 mL of DPBS (with no MgCl2 or CaCl2 ) was mixed with 3 μg of pGL3-Control (Promega), 3 μg of pThAAT , 10 μg of indicated siRNA, and if indicated, 20 μL of recombinant RNasin (Promega).

    Techniques: Modification, Expressing, Mouse Assay, Transfection, Luciferase, Injection

    m 6 A alters RNA structure to recruit HNRNPG. ( A ) Sequence logo of the most enriched motif within HNRNPG PAR-CLIP peaks. ( B ) Left: secondary structure of the MALAT1 hairpin, showing the A-2,515-to-G/C/U mutations that were introduced at the m 6 A site. Right: quantification of relative HNRNPG pull-down with the original (2,515-A) and mutated (2,515-G/C/U) MALAT1 hairpins, normalized to pulled-down HIST1H1C. Data shown as mean; error bar = standard deviation; n = 3 biological replicates. ( C ) Left: structural probing of the unmethylated and methylated MALAT1 hairpins. The orange lines indicate regions with marked differences between the unmethylated and methylated hairpins. The location of the m 6 A residue is indicated by a red dot. Ctrl, no nuclease added; V1; RNase V1 digestion; S1, S1 nuclease digestion; T1, RNase T1 digestion; G-L, G-ladder; AH, alkaline hydrolysis. Right: secondary structure of the unmethylated and methylated MALAT1 hairpins, marked at their S1 nuclease (red lines) and V1 nuclease (green lines) cleavage sites. ( D ) Model showing that m 6 A disrupts RNA structure, exposes a motif that includes the m 6 A site, and recruits an RNA binding protein.

    Journal: Nucleic Acids Research

    Article Title: N6-methyladenosine alters RNA structure to regulate binding of a low-complexity protein

    doi: 10.1093/nar/gkx141

    Figure Lengend Snippet: m 6 A alters RNA structure to recruit HNRNPG. ( A ) Sequence logo of the most enriched motif within HNRNPG PAR-CLIP peaks. ( B ) Left: secondary structure of the MALAT1 hairpin, showing the A-2,515-to-G/C/U mutations that were introduced at the m 6 A site. Right: quantification of relative HNRNPG pull-down with the original (2,515-A) and mutated (2,515-G/C/U) MALAT1 hairpins, normalized to pulled-down HIST1H1C. Data shown as mean; error bar = standard deviation; n = 3 biological replicates. ( C ) Left: structural probing of the unmethylated and methylated MALAT1 hairpins. The orange lines indicate regions with marked differences between the unmethylated and methylated hairpins. The location of the m 6 A residue is indicated by a red dot. Ctrl, no nuclease added; V1; RNase V1 digestion; S1, S1 nuclease digestion; T1, RNase T1 digestion; G-L, G-ladder; AH, alkaline hydrolysis. Right: secondary structure of the unmethylated and methylated MALAT1 hairpins, marked at their S1 nuclease (red lines) and V1 nuclease (green lines) cleavage sites. ( D ) Model showing that m 6 A disrupts RNA structure, exposes a motif that includes the m 6 A site, and recruits an RNA binding protein.

    Article Snippet: The HNRNPG PAR-CLIP RNA sample was incubated with m6 A-specific antibody (202003, SYSY), RNase inhibitor (80 units, Sigma-Aldrich), human placental RNase inhibitor (NEB) in 200 μl 1× IP buffer (50 mM Tris-Cl (pH 7.4), 750 mM NaCl and 0.5% (vol/vol) Igepal CA-630) at 4°C for 2 h under gentle shaking conditions.

    Techniques: Sequencing, Cross-linking Immunoprecipitation, Standard Deviation, Methylation, RNA Binding Assay

    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 )

    Journal: Nature Communications

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

    doi: 10.1038/s41467-019-10246-5

    Figure Lengend 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 )

    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.

    Techniques: 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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-019-10246-5

    Figure Lengend 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

    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.

    Techniques: 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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-019-10246-5

    Figure Lengend 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

    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.

    Techniques: 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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-019-10246-5

    Figure Lengend 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

    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.

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