Structured Review

Roche rnase a
Chromatographic analysis of TFIIIC-α–containing complexes. Nuclear proteins from tissue culture cells of C. tentans were fractionated on a gel filtration Superose HR6 column. (A) The chromatogram, showing the fractionation of some molecular mass standards, in kDa, used for calibration. V O : void volume. (B) Fractions were pooled two by two (for example, lane 12 contains fractions 12 and 13), separated by SDS-PAGE, and analyzed by immunoblotting using mAb 2D10. The mobilities of molecular mass standards in SDS-PAGE are shown on the left. (C) Nuclear extract was preincubated with 25 μg/ml <t>RNase</t> A for 20 min before chromatography and Western blot analysis as in B.
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Images

1) Product Images from "Evidence for a Posttranscriptional Role of a TFIIIC?-like Protein in Chironomus tentans"

Article Title: Evidence for a Posttranscriptional Role of a TFIIIC?-like Protein in Chironomus tentans

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.01-09-0436

Chromatographic analysis of TFIIIC-α–containing complexes. Nuclear proteins from tissue culture cells of C. tentans were fractionated on a gel filtration Superose HR6 column. (A) The chromatogram, showing the fractionation of some molecular mass standards, in kDa, used for calibration. V O : void volume. (B) Fractions were pooled two by two (for example, lane 12 contains fractions 12 and 13), separated by SDS-PAGE, and analyzed by immunoblotting using mAb 2D10. The mobilities of molecular mass standards in SDS-PAGE are shown on the left. (C) Nuclear extract was preincubated with 25 μg/ml RNase A for 20 min before chromatography and Western blot analysis as in B.
Figure Legend Snippet: Chromatographic analysis of TFIIIC-α–containing complexes. Nuclear proteins from tissue culture cells of C. tentans were fractionated on a gel filtration Superose HR6 column. (A) The chromatogram, showing the fractionation of some molecular mass standards, in kDa, used for calibration. V O : void volume. (B) Fractions were pooled two by two (for example, lane 12 contains fractions 12 and 13), separated by SDS-PAGE, and analyzed by immunoblotting using mAb 2D10. The mobilities of molecular mass standards in SDS-PAGE are shown on the left. (C) Nuclear extract was preincubated with 25 μg/ml RNase A for 20 min before chromatography and Western blot analysis as in B.

Techniques Used: Filtration, Fractionation, SDS Page, Chromatography, Western Blot

2) Product Images from "Newly Synthesized APOBEC3G Is Incorporated into HIV Virions, Inhibited by HIV RNA, and Subsequently Activated by RNase H"

Article Title: Newly Synthesized APOBEC3G Is Incorporated into HIV Virions, Inhibited by HIV RNA, and Subsequently Activated by RNase H

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.0030015

Virion-Incorporated HA-A3G Associates with Viral Genomic RNA (A) Viral genomic RNA, detected by RT-PCR, was detected in virions and virus-producing cells but not in lysates of uninfected cells. Genomic RNA was also detected in the IVAC derived from virions (fraction 7) and coimmunoprecipitated with HA-A3G from both virions and producer cell lysates. RT was performed using RNA derived from either whole lysates (L) or anti-HA immunoprecipitates (IP). Control reactions were performed in the absence of RT (–RT). Control PCRs were performed using proviral plasmid DNA, in the absence or presence of Taq, as indicated. (B) Viral genomic RNA, detected by RT-PCR, was assessed from size-fractionated virion lysates that lacked (HA) or contained HA-A3G. Amplicons generated probed across the TAR/Gag region or Pol/Vpu regions, as indicated. (C) Incorporation of HA-A3G into virions enhances the recruitment of NC into the IVAC. (D) HA-A3G from virus-producing cells is HMM and is converted to LMM form after RNase A treatment. “IB” indicates immunoblotting with the indicated antibody.
Figure Legend Snippet: Virion-Incorporated HA-A3G Associates with Viral Genomic RNA (A) Viral genomic RNA, detected by RT-PCR, was detected in virions and virus-producing cells but not in lysates of uninfected cells. Genomic RNA was also detected in the IVAC derived from virions (fraction 7) and coimmunoprecipitated with HA-A3G from both virions and producer cell lysates. RT was performed using RNA derived from either whole lysates (L) or anti-HA immunoprecipitates (IP). Control reactions were performed in the absence of RT (–RT). Control PCRs were performed using proviral plasmid DNA, in the absence or presence of Taq, as indicated. (B) Viral genomic RNA, detected by RT-PCR, was assessed from size-fractionated virion lysates that lacked (HA) or contained HA-A3G. Amplicons generated probed across the TAR/Gag region or Pol/Vpu regions, as indicated. (C) Incorporation of HA-A3G into virions enhances the recruitment of NC into the IVAC. (D) HA-A3G from virus-producing cells is HMM and is converted to LMM form after RNase A treatment. “IB” indicates immunoblotting with the indicated antibody.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Plasmid Preparation, Generated

Intravirion A3G Enzymatic Activity Is Negatively Regulated by Binding to Genomic HIV RNA (A) HA-A3G was immunoprecipitated from IVAC fraction 7 (F7) of virion lysates ( Figure 3 A) or from a lower fraction, F17, generated by treatment of the virion lysates with RNase A ( Figure 3 B). Immunoprecipitates (IPs) were tested for enzymatic activity in an in vitro deoxycytidine deaminase assay with or without RNase A addition and contained equivalent amounts of HA-A3G as shown in the corresponding immunoblot. The generation of a shorter cleavage product from the input ssDNA substrate reveals A3G deoxycytidine deaminase activity. Data shown are representative of multiple experiments. (B) Lysates of virions containing or lacking A3G were assessed in the deaminase assay, with or without RNase A treatment. (C) Lysates of virions containing increasing amounts of HA-A3G (as shown in the corresponding immunoblot) were assessed in the deaminase assay, with or without RNase A treatment. The asterisk marks bleed-through of marker loaded to the left of the samples. The triangles represent the increasing dose of A3G relative to provirus and correspond to the sample numbers presented in Figure 1 A. (A–C) All deaminase reactions were carried out in 50 mM Tris (pH 7.4) with (+) or without (−) RNase A, as indicated. (D) IPs of HMM or LMM HA-A3G from producer cell lysates were similarly assessed in the deaminase assay, with (+) or without (−) added RNase A. The IPs contained equivalent amounts of HA-A3G as shown in the corresponding immunoblot (IB).
Figure Legend Snippet: Intravirion A3G Enzymatic Activity Is Negatively Regulated by Binding to Genomic HIV RNA (A) HA-A3G was immunoprecipitated from IVAC fraction 7 (F7) of virion lysates ( Figure 3 A) or from a lower fraction, F17, generated by treatment of the virion lysates with RNase A ( Figure 3 B). Immunoprecipitates (IPs) were tested for enzymatic activity in an in vitro deoxycytidine deaminase assay with or without RNase A addition and contained equivalent amounts of HA-A3G as shown in the corresponding immunoblot. The generation of a shorter cleavage product from the input ssDNA substrate reveals A3G deoxycytidine deaminase activity. Data shown are representative of multiple experiments. (B) Lysates of virions containing or lacking A3G were assessed in the deaminase assay, with or without RNase A treatment. (C) Lysates of virions containing increasing amounts of HA-A3G (as shown in the corresponding immunoblot) were assessed in the deaminase assay, with or without RNase A treatment. The asterisk marks bleed-through of marker loaded to the left of the samples. The triangles represent the increasing dose of A3G relative to provirus and correspond to the sample numbers presented in Figure 1 A. (A–C) All deaminase reactions were carried out in 50 mM Tris (pH 7.4) with (+) or without (−) RNase A, as indicated. (D) IPs of HMM or LMM HA-A3G from producer cell lysates were similarly assessed in the deaminase assay, with (+) or without (−) added RNase A. The IPs contained equivalent amounts of HA-A3G as shown in the corresponding immunoblot (IB).

Techniques Used: Activity Assay, Binding Assay, Immunoprecipitation, Generated, In Vitro, Marker

Enzymatically Inactive Virion-Incorporated HA-A3G Is Activated by Viral RNase H (A) Recombinant RTs containing either a WT or mutant (E478Q) RNase H catalytic domain were assessed for RNase H activity in vitro in the absence or presence of the RNase H inhibitor Compound I (final concentration of 1, 10, or 100 μM). The RNA of an RNA–DNA hybrid remains intact unless RNase H digests the RNA into a smaller cleavage product that is distinguishable from the more complete cleavage product generated by RNase A. WT RNase H cannot digest ssDNA or DNA of an RNA–DNA hybrid, or RNA–RNA hybrids (data not shown). RNase H assays were performed in RNase H buffer (50 mM Tris [pH 8.0], 60 mM KCl) with (+) or without (−) 5 mM MgCl 2 or RNase A, as indicated. (B) Viruses bearing the RNase H E478Q mutation are compromised for in vitro RNase H activity. RNase H assays were performed in RNase H buffer with (+) or without (−) 5 mM MgCl 2 or RNase A, as indicated. (C) Virion lysates were subjected to endogenous reverse transcription (enRT) conditions with or without Compound I (final concentration of 0.1, 1, 10, or 100 μM), and A3G activity in these samples assessed in the in vitro deoxycytidine deaminase assay. Deaminase assays were performed in RNase H buffer either supplemented (enRT:+) or not (enRT:−) with 4 mM MgCl 2 and 1 mM dNTPs. (D) Compound I does not inhibit the intrinsic deoxycytidine deaminase activity of A3G. HA-A3G from RNase A–treated virion lysates was assessed for in vitro deaminase activity in the presence of increasing doses of Compound I (0.1, 1, 10, and 100 μM). Deaminase assay was performed in RNase H buffer supplemented with RNase A only. (E) Virions containing WT RNase H or the E478Q mutation in the RNase H catalytic domain were subjected to the enRT reaction followed by assessment of A3G enzymatic activity. Deaminase assays were performed in RNase H buffer either supplemented (enRT:+) or not (enRT:−) with 4 mM MgCl 2 and 1 mM dNTPs. (F) WT and RNase H–compromised ΔVif virions containing WT or mutant RNase H displayed equivalent A3G activity when RNase A was added to the virion lysate. Deaminase assay was performed in RNase H buffer with (+) or without (−) RNase A, as indicated. All data are representative of multiple experiments.
Figure Legend Snippet: Enzymatically Inactive Virion-Incorporated HA-A3G Is Activated by Viral RNase H (A) Recombinant RTs containing either a WT or mutant (E478Q) RNase H catalytic domain were assessed for RNase H activity in vitro in the absence or presence of the RNase H inhibitor Compound I (final concentration of 1, 10, or 100 μM). The RNA of an RNA–DNA hybrid remains intact unless RNase H digests the RNA into a smaller cleavage product that is distinguishable from the more complete cleavage product generated by RNase A. WT RNase H cannot digest ssDNA or DNA of an RNA–DNA hybrid, or RNA–RNA hybrids (data not shown). RNase H assays were performed in RNase H buffer (50 mM Tris [pH 8.0], 60 mM KCl) with (+) or without (−) 5 mM MgCl 2 or RNase A, as indicated. (B) Viruses bearing the RNase H E478Q mutation are compromised for in vitro RNase H activity. RNase H assays were performed in RNase H buffer with (+) or without (−) 5 mM MgCl 2 or RNase A, as indicated. (C) Virion lysates were subjected to endogenous reverse transcription (enRT) conditions with or without Compound I (final concentration of 0.1, 1, 10, or 100 μM), and A3G activity in these samples assessed in the in vitro deoxycytidine deaminase assay. Deaminase assays were performed in RNase H buffer either supplemented (enRT:+) or not (enRT:−) with 4 mM MgCl 2 and 1 mM dNTPs. (D) Compound I does not inhibit the intrinsic deoxycytidine deaminase activity of A3G. HA-A3G from RNase A–treated virion lysates was assessed for in vitro deaminase activity in the presence of increasing doses of Compound I (0.1, 1, 10, and 100 μM). Deaminase assay was performed in RNase H buffer supplemented with RNase A only. (E) Virions containing WT RNase H or the E478Q mutation in the RNase H catalytic domain were subjected to the enRT reaction followed by assessment of A3G enzymatic activity. Deaminase assays were performed in RNase H buffer either supplemented (enRT:+) or not (enRT:−) with 4 mM MgCl 2 and 1 mM dNTPs. (F) WT and RNase H–compromised ΔVif virions containing WT or mutant RNase H displayed equivalent A3G activity when RNase A was added to the virion lysate. Deaminase assay was performed in RNase H buffer with (+) or without (−) RNase A, as indicated. All data are representative of multiple experiments.

Techniques Used: Recombinant, Mutagenesis, Activity Assay, In Vitro, Concentration Assay, Generated

Virion-Incorporated HA-A3G Resides in a Large RNase A–Sensitive Complex and Biochemically Fractionates with Viral RNP Proteins (A) Virions collected from cells expressing HIV-1ΔVif contain HA-A3G that predominantly fractionates in a large complex (fractions 6 to 8) as assessed by gel filtration. (B) The IVAC is sensitive to RNase A treatment which shifts HA-A3G into lower fractions (fractions 15 to 19). (C) Virion cores obtained in Figure 1 were subjected to further biochemical fractionation to generate viral RNPs. Shown are the viral RNPs from virions either lacking or containing A3G, as indicated, and containing viral RT, IN, and NC but not p24-CA, as detected by immunoblotting (IB). The triangles represent the increasing dose of A3G relative to provirus and correspond exactly to the sample numbers in Figure 1 A.
Figure Legend Snippet: Virion-Incorporated HA-A3G Resides in a Large RNase A–Sensitive Complex and Biochemically Fractionates with Viral RNP Proteins (A) Virions collected from cells expressing HIV-1ΔVif contain HA-A3G that predominantly fractionates in a large complex (fractions 6 to 8) as assessed by gel filtration. (B) The IVAC is sensitive to RNase A treatment which shifts HA-A3G into lower fractions (fractions 15 to 19). (C) Virion cores obtained in Figure 1 were subjected to further biochemical fractionation to generate viral RNPs. Shown are the viral RNPs from virions either lacking or containing A3G, as indicated, and containing viral RT, IN, and NC but not p24-CA, as detected by immunoblotting (IB). The triangles represent the increasing dose of A3G relative to provirus and correspond exactly to the sample numbers in Figure 1 A.

Techniques Used: Expressing, Filtration, Fractionation

3) Product Images from "Identification of Neuronal RNA Targets of TDP-43-containing Ribonucleoprotein Complexes *"

Article Title: Identification of Neuronal RNA Targets of TDP-43-containing Ribonucleoprotein Complexes *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.190884

Genomic distribution of reads from TDP-43 RNA library. A , Western blot ( IB ) of fractions from HeLa nuclear extracts (± RNase A) applied to a size exclusion column, blotted for TDP-43, hnRNPA1, and lamin A/C. Fraction 6 = blue dextran (2000 kDa), fraction 30 = apoferritin (443 kDa), fraction 40 = alcohol dehydrogenase (150 kDa), and fraction 54 = bovine serum albumin (54 kDa) (data not shown).  B , Western blot of fractions from rat brain nuclear extracts (± micrococcal nuclease (±  MNase )) blotted for TDP-43. Fraction 5 = blue dextran (2,000 kDa), fraction 45 = bovine serum albumin (54 kDa), and fraction 54 = carbonic anhydrase (29 kDa) (data not shown). Note that different size exclusion columns were used in  A  and  B. C ,  panel i , diagram of TDP-43 RIP method.  C ,  panel ii , representative Western blot of TDP-43 RIP.  IP:CTL , control immunoprecipitation.  D , distribution of raw reads from the TDP-43 library mapped to exonic and intronic genes regions.  CDS , coding sequence.  E , read density, number of reads per 1,000 mappable nucleotides per million reads ( mRPKM ) of gene regions from the TDP-43 library.
Figure Legend Snippet: Genomic distribution of reads from TDP-43 RNA library. A , Western blot ( IB ) of fractions from HeLa nuclear extracts (± RNase A) applied to a size exclusion column, blotted for TDP-43, hnRNPA1, and lamin A/C. Fraction 6 = blue dextran (2000 kDa), fraction 30 = apoferritin (443 kDa), fraction 40 = alcohol dehydrogenase (150 kDa), and fraction 54 = bovine serum albumin (54 kDa) (data not shown). B , Western blot of fractions from rat brain nuclear extracts (± micrococcal nuclease (± MNase )) blotted for TDP-43. Fraction 5 = blue dextran (2,000 kDa), fraction 45 = bovine serum albumin (54 kDa), and fraction 54 = carbonic anhydrase (29 kDa) (data not shown). Note that different size exclusion columns were used in A and B. C , panel i , diagram of TDP-43 RIP method. C , panel ii , representative Western blot of TDP-43 RIP. IP:CTL , control immunoprecipitation. D , distribution of raw reads from the TDP-43 library mapped to exonic and intronic genes regions. CDS , coding sequence. E , read density, number of reads per 1,000 mappable nucleotides per million reads ( mRPKM ) of gene regions from the TDP-43 library.

Techniques Used: Western Blot, CTL Assay, Immunoprecipitation, Sequencing

4) Product Images from "Identification of Neuronal RNA Targets of TDP-43-containing Ribonucleoprotein Complexes *"

Article Title: Identification of Neuronal RNA Targets of TDP-43-containing Ribonucleoprotein Complexes *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.190884

Genomic distribution of reads from TDP-43 RNA library. A , Western blot ( IB ) of fractions from HeLa nuclear extracts (± RNase A) applied to a size exclusion column, blotted for TDP-43, hnRNPA1, and lamin A/C. Fraction 6 = blue dextran (2000 kDa), fraction 30 = apoferritin (443 kDa), fraction 40 = alcohol dehydrogenase (150 kDa), and fraction 54 = bovine serum albumin (54 kDa) (data not shown).  B , Western blot of fractions from rat brain nuclear extracts (± micrococcal nuclease (±  MNase )) blotted for TDP-43. Fraction 5 = blue dextran (2,000 kDa), fraction 45 = bovine serum albumin (54 kDa), and fraction 54 = carbonic anhydrase (29 kDa) (data not shown). Note that different size exclusion columns were used in  A  and  B. C ,  panel i , diagram of TDP-43 RIP method.  C ,  panel ii , representative Western blot of TDP-43 RIP.  IP:CTL , control immunoprecipitation.  D , distribution of raw reads from the TDP-43 library mapped to exonic and intronic genes regions.  CDS , coding sequence.  E , read density, number of reads per 1,000 mappable nucleotides per million reads ( mRPKM ) of gene regions from the TDP-43 library.
Figure Legend Snippet: Genomic distribution of reads from TDP-43 RNA library. A , Western blot ( IB ) of fractions from HeLa nuclear extracts (± RNase A) applied to a size exclusion column, blotted for TDP-43, hnRNPA1, and lamin A/C. Fraction 6 = blue dextran (2000 kDa), fraction 30 = apoferritin (443 kDa), fraction 40 = alcohol dehydrogenase (150 kDa), and fraction 54 = bovine serum albumin (54 kDa) (data not shown). B , Western blot of fractions from rat brain nuclear extracts (± micrococcal nuclease (± MNase )) blotted for TDP-43. Fraction 5 = blue dextran (2,000 kDa), fraction 45 = bovine serum albumin (54 kDa), and fraction 54 = carbonic anhydrase (29 kDa) (data not shown). Note that different size exclusion columns were used in A and B. C , panel i , diagram of TDP-43 RIP method. C , panel ii , representative Western blot of TDP-43 RIP. IP:CTL , control immunoprecipitation. D , distribution of raw reads from the TDP-43 library mapped to exonic and intronic genes regions. CDS , coding sequence. E , read density, number of reads per 1,000 mappable nucleotides per million reads ( mRPKM ) of gene regions from the TDP-43 library.

Techniques Used: Western Blot, CTL Assay, Immunoprecipitation, Sequencing

5) Product Images from "Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats"

Article Title: Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq935

RNA–DNA hybrid formation at a (CGG)39 · (CCG)39 FRAXA template. When the template DNA is transcribed with T7 RNA polymerase, heterogeneous RNA is produced generating a smear (Transcription lane). Treatment with RNase H alone which is specific to RNA base paired to template DNA digests only RNA that is base paired to its template DNA. Treatment with RNase A, which is specific to single-stranded RNA, digests all free, single-stranded RNA leaving template DNA and RNA:DNA hybrid structures. Note that RNA–DNA hybrids migrate more slowly than supercoiled DNA (as indicated schematically, RNA is in light blue). Hybrid structures generate a smear due to their heterogeneous sizes. With a larger RNA component, the DNA is open to a greater degree (more relaxed), hence migration is closer to open circular DNA. Treatment of the hybrids with RNase H along with RNase A removes any hybrids formed as well as transcript generated in the transcription reaction leaving only input template DNA. When transcription followed by RNase H or RNase A treatment alone or in combination is performed on an empty Bluescript vector [pBlueKS(+)], there is negligible hybrid formation.
Figure Legend Snippet: RNA–DNA hybrid formation at a (CGG)39 · (CCG)39 FRAXA template. When the template DNA is transcribed with T7 RNA polymerase, heterogeneous RNA is produced generating a smear (Transcription lane). Treatment with RNase H alone which is specific to RNA base paired to template DNA digests only RNA that is base paired to its template DNA. Treatment with RNase A, which is specific to single-stranded RNA, digests all free, single-stranded RNA leaving template DNA and RNA:DNA hybrid structures. Note that RNA–DNA hybrids migrate more slowly than supercoiled DNA (as indicated schematically, RNA is in light blue). Hybrid structures generate a smear due to their heterogeneous sizes. With a larger RNA component, the DNA is open to a greater degree (more relaxed), hence migration is closer to open circular DNA. Treatment of the hybrids with RNase H along with RNase A removes any hybrids formed as well as transcript generated in the transcription reaction leaving only input template DNA. When transcription followed by RNase H or RNase A treatment alone or in combination is performed on an empty Bluescript vector [pBlueKS(+)], there is negligible hybrid formation.

Techniques Used: Produced, Migration, Generated, Plasmid Preparation

Quantification of relative RNA–DNA hybrid formation at increasing repeat lengths. ( A ) In vitro transcription reactions were performed on FRAXA plasmids bearing repeat tracts of (CGG)17 · (CCG)17, (CGG)39 · (CCG)39 and (CGG)53 · (CCG)53 following which samples were treated with RNase A or A+H. Hybrid formation was quantified by densitometry analysis using image quant by measuring the proportion of products that migrate between open circular and supercoiled position (indicated by ‘R’) divided by the total products below open circular including supercoiled. RNase A+H treated samples were used to determine the position of the supercoiled DNA for each repeat length. To the left of the graph is a representative gel used to quantify relative hybrid formation. The RNA indicated in the graph represents the RNA component bound in the RNA:DNA hybrid. Error bars are derived from three separate experiments ( N =3). Using the t -test to compare R-loop formation revealed a significant difference between the 17 and 53 rCGG R-loop ( P =0.0413) as well as the 17 and 53 rCCG R-loop ( P =0.0092). ( B ) The same analysis was performed as in (A) but for SCA1 plasmids bearing repeat tracts of (CAG)30 · (CTG)30, (CAG) 49 · (CTG)49 and (CAG)74 · (CTG)74. Using the t -test to compare R-loop formation between each of the (CAG) · (CTG) repeat lengths did not reveal any statistically significant differences.
Figure Legend Snippet: Quantification of relative RNA–DNA hybrid formation at increasing repeat lengths. ( A ) In vitro transcription reactions were performed on FRAXA plasmids bearing repeat tracts of (CGG)17 · (CCG)17, (CGG)39 · (CCG)39 and (CGG)53 · (CCG)53 following which samples were treated with RNase A or A+H. Hybrid formation was quantified by densitometry analysis using image quant by measuring the proportion of products that migrate between open circular and supercoiled position (indicated by ‘R’) divided by the total products below open circular including supercoiled. RNase A+H treated samples were used to determine the position of the supercoiled DNA for each repeat length. To the left of the graph is a representative gel used to quantify relative hybrid formation. The RNA indicated in the graph represents the RNA component bound in the RNA:DNA hybrid. Error bars are derived from three separate experiments ( N =3). Using the t -test to compare R-loop formation revealed a significant difference between the 17 and 53 rCGG R-loop ( P =0.0413) as well as the 17 and 53 rCCG R-loop ( P =0.0092). ( B ) The same analysis was performed as in (A) but for SCA1 plasmids bearing repeat tracts of (CAG)30 · (CTG)30, (CAG) 49 · (CTG)49 and (CAG)74 · (CTG)74. Using the t -test to compare R-loop formation between each of the (CAG) · (CTG) repeat lengths did not reveal any statistically significant differences.

Techniques Used: In Vitro, Derivative Assay, CTG Assay

Effect of trinucleotide repeat interruptions on RNA–DNA hybrid formation. ( A ) In vitro transcription followed by RNase A or A + H treatment was performed with either pure (39p) FRAXA plasmids (CGG)39 · (CCG)39 or interrupted plasmids (39i) [(CGG)9(AGG) (CGG)9(AGG)(CGG)9(AGG)(CGG)9] · [(CCG)9(CCT) (CCG)9(CCT)(CCG)9(CCT)(CCG)9] as indicated. Repeat tract configurations are schematically presented where hollow dots are the CGG repeat units and the filled dots are the AGG interruptions. R-loops are indicated as ‘R’. ( B ) In vitro transcription followed by RNase A or A+H treatment was performed with either pure (49p) SCA1 plasmids (CAG)49 · (CTG)49 or interrupted plasmids (44i) [(CAG)12(CAT)(CAG)(CAT)(CAG)12(CAT)(CAG)(CAT)(CAG)14] · [(CTG)14(ATG)(CTG)(ATG)(CTG)12(ATG)(CAG)(ATG)(CTG)12] as indicated schematically where hollow dots are the CAG repeat units and the filled dots are the CAT interruptions.
Figure Legend Snippet: Effect of trinucleotide repeat interruptions on RNA–DNA hybrid formation. ( A ) In vitro transcription followed by RNase A or A + H treatment was performed with either pure (39p) FRAXA plasmids (CGG)39 · (CCG)39 or interrupted plasmids (39i) [(CGG)9(AGG) (CGG)9(AGG)(CGG)9(AGG)(CGG)9] · [(CCG)9(CCT) (CCG)9(CCT)(CCG)9(CCT)(CCG)9] as indicated. Repeat tract configurations are schematically presented where hollow dots are the CGG repeat units and the filled dots are the AGG interruptions. R-loops are indicated as ‘R’. ( B ) In vitro transcription followed by RNase A or A+H treatment was performed with either pure (49p) SCA1 plasmids (CAG)49 · (CTG)49 or interrupted plasmids (44i) [(CAG)12(CAT)(CAG)(CAT)(CAG)12(CAT)(CAG)(CAT)(CAG)14] · [(CTG)14(ATG)(CTG)(ATG)(CTG)12(ATG)(CAG)(ATG)(CTG)12] as indicated schematically where hollow dots are the CAG repeat units and the filled dots are the CAT interruptions.

Techniques Used: In Vitro, CTG Assay

Identification of R-loop structures formed in expanded DM1 (CTG)130 · (CAG)130 plasmids using EM following in vitro transcription and treatment with RNase A and SSB protein. SSB proteins bind to the looped-out non-template DNA in an R-loop structure. Thus, each R-loop structure is visualized as a loop within the DNA template as indicated by black arrowheads. ( A ) R-loops formed by using SP6 RNA polymerase, producing an rCAG:dCTG hybrid. ( B ) R-loops formed by using T7 RNA polymerase, producing an rCUG:dCAG hybrid.
Figure Legend Snippet: Identification of R-loop structures formed in expanded DM1 (CTG)130 · (CAG)130 plasmids using EM following in vitro transcription and treatment with RNase A and SSB protein. SSB proteins bind to the looped-out non-template DNA in an R-loop structure. Thus, each R-loop structure is visualized as a loop within the DNA template as indicated by black arrowheads. ( A ) R-loops formed by using SP6 RNA polymerase, producing an rCAG:dCTG hybrid. ( B ) R-loops formed by using T7 RNA polymerase, producing an rCUG:dCAG hybrid.

Techniques Used: CTG Assay, In Vitro

RNA–DNA hybrid formation during in vitro transcription of trinucleotide repeat-containing plasmids. ( A ) In vitro transcription of SCA1 plasmid containing (CAG)74 · (CTG)74 and FRAXA plasmid containing (CGG)39 · (CCG)39 repeats in either direction using T3 or T7 RNA polymerase. The repeat sequence contained within the RNA produced and bound in the hybrid is indicated below the transcribed template. Samples following transcription were subsequently treated with either RNase A or A+H as indicated to observe hybrid formation. R-loops are indicated on the gel as ‘R’. ( B ) Exact same reactions and gel conditions as in (A) but transcription was performed in the presence of 3.5 µCi [α- 32 P]-rCTP. Gel was dried and exposed to X-ray film as outlined in ‘Materials and Methods’ section.
Figure Legend Snippet: RNA–DNA hybrid formation during in vitro transcription of trinucleotide repeat-containing plasmids. ( A ) In vitro transcription of SCA1 plasmid containing (CAG)74 · (CTG)74 and FRAXA plasmid containing (CGG)39 · (CCG)39 repeats in either direction using T3 or T7 RNA polymerase. The repeat sequence contained within the RNA produced and bound in the hybrid is indicated below the transcribed template. Samples following transcription were subsequently treated with either RNase A or A+H as indicated to observe hybrid formation. R-loops are indicated on the gel as ‘R’. ( B ) Exact same reactions and gel conditions as in (A) but transcription was performed in the presence of 3.5 µCi [α- 32 P]-rCTP. Gel was dried and exposed to X-ray film as outlined in ‘Materials and Methods’ section.

Techniques Used: In Vitro, Plasmid Preparation, CTG Assay, Sequencing, Produced

Effect of convergent simultaneous bidirectional or serial transcription on R-loop formation. ( A ) In vitro transcription of FRAXA template (CGG)39 · (CCG)39 with either T3 or T7 RNA polymerase alone (rCCG or rCGG, respectively), or simultaneously (rCCG with rCGG) or serially: rCCG transcription then phenol chloroform extraction followed by rCGG transcription (rCCG then rCGG), and vice versa (rCGG then rCCG). R-loops are indicated as ‘R’. Note that in the case of bidirectional or serial transcription, complementary RNA is produced forming dsRNA as indicated on the gel by ‘*’. These products are not present in transcription reactions occurring in one direction. ( B ) Same as in (A) except in vitro transcription reactions were performed on a DM1 (CAG)79 · (CTG)79 template. ( C ) EM analysis of convergent transcription reaction products from DM1 (CAG)79 · (CTG)79 templates. Samples were transcribed convergently using T3 and T7 RNA polymerase promoters simultaneously then the products were treated with RNAse A and prepared for EM as described in the ‘Materials and Methods’ section (rCUGand rCAG RNase A). Samples were also subjected to RNase H treatment along with RNase A for comparison (rCUG and rCAG RNase A, H). Transcription was also performed on the same template in a single direction for further comparison (rCUG RNase A). The products observed for each type of transcription reaction is shown as a percentage of the total number of molecules analyzed. At least 100 molecules were analyzed for each type of transcription reaction.
Figure Legend Snippet: Effect of convergent simultaneous bidirectional or serial transcription on R-loop formation. ( A ) In vitro transcription of FRAXA template (CGG)39 · (CCG)39 with either T3 or T7 RNA polymerase alone (rCCG or rCGG, respectively), or simultaneously (rCCG with rCGG) or serially: rCCG transcription then phenol chloroform extraction followed by rCGG transcription (rCCG then rCGG), and vice versa (rCGG then rCCG). R-loops are indicated as ‘R’. Note that in the case of bidirectional or serial transcription, complementary RNA is produced forming dsRNA as indicated on the gel by ‘*’. These products are not present in transcription reactions occurring in one direction. ( B ) Same as in (A) except in vitro transcription reactions were performed on a DM1 (CAG)79 · (CTG)79 template. ( C ) EM analysis of convergent transcription reaction products from DM1 (CAG)79 · (CTG)79 templates. Samples were transcribed convergently using T3 and T7 RNA polymerase promoters simultaneously then the products were treated with RNAse A and prepared for EM as described in the ‘Materials and Methods’ section (rCUGand rCAG RNase A). Samples were also subjected to RNase H treatment along with RNase A for comparison (rCUG and rCAG RNase A, H). Transcription was also performed on the same template in a single direction for further comparison (rCUG RNase A). The products observed for each type of transcription reaction is shown as a percentage of the total number of molecules analyzed. At least 100 molecules were analyzed for each type of transcription reaction.

Techniques Used: In Vitro, Produced, CTG Assay

Mechanism of RNA:DNA hybrid formation during in vitro transcription. ( A ) Schematic of the two possible mechanisms for R-loop formation. By the thread-back model, the nascent transcript (depicted in light blue) that has been ejected from the RNA polymerase re-anneals with the complementary, free DNA template strand (depicted in red) following the progression of the RNA polymerase (light blue, oval). When transcription occurs in the presence of RNAse A (dark blue) the nascent transcript is degraded when it is ejected from the RNA polymerase hence cannot form the hybrid. By the extended-hybrid model, the nascent transcript remains bound to the template DNA and resists becoming ejected from the RNA polymerase. When transcribed in the presence of RNase A, as the nascent transcript is protected by being bound to the template DNA it is not degraded hence hybrid formation is not ablated. ( B ) FRAXA plasmid (CGG)39 · (CCG)39 transcribed in either direction in the absence (−) or presence (+) of RNase A during the transcription reaction. All transcription reactions were subjected to further RNase A or A + H treatment to analyze hybrid formation. ( C ) Same experiment as in (B) performed with SCA1 plasmid containing a (CAG)74 · (CTG)74 repeat tract.
Figure Legend Snippet: Mechanism of RNA:DNA hybrid formation during in vitro transcription. ( A ) Schematic of the two possible mechanisms for R-loop formation. By the thread-back model, the nascent transcript (depicted in light blue) that has been ejected from the RNA polymerase re-anneals with the complementary, free DNA template strand (depicted in red) following the progression of the RNA polymerase (light blue, oval). When transcription occurs in the presence of RNAse A (dark blue) the nascent transcript is degraded when it is ejected from the RNA polymerase hence cannot form the hybrid. By the extended-hybrid model, the nascent transcript remains bound to the template DNA and resists becoming ejected from the RNA polymerase. When transcribed in the presence of RNase A, as the nascent transcript is protected by being bound to the template DNA it is not degraded hence hybrid formation is not ablated. ( B ) FRAXA plasmid (CGG)39 · (CCG)39 transcribed in either direction in the absence (−) or presence (+) of RNase A during the transcription reaction. All transcription reactions were subjected to further RNase A or A + H treatment to analyze hybrid formation. ( C ) Same experiment as in (B) performed with SCA1 plasmid containing a (CAG)74 · (CTG)74 repeat tract.

Techniques Used: In Vitro, Plasmid Preparation, CTG Assay

6) Product Images from "Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats"

Article Title: Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq935

RNA–DNA hybrid formation at a (CGG)39 · (CCG)39 FRAXA template. When the template DNA is transcribed with T7 RNA polymerase, heterogeneous RNA is produced generating a smear (Transcription lane). Treatment with RNase H alone which is specific to RNA base paired to template DNA digests only RNA that is base paired to its template DNA. Treatment with RNase A, which is specific to single-stranded RNA, digests all free, single-stranded RNA leaving template DNA and RNA:DNA hybrid structures. Note that RNA–DNA hybrids migrate more slowly than supercoiled DNA (as indicated schematically, RNA is in light blue). Hybrid structures generate a smear due to their heterogeneous sizes. With a larger RNA component, the DNA is open to a greater degree (more relaxed), hence migration is closer to open circular DNA. Treatment of the hybrids with RNase H along with RNase A removes any hybrids formed as well as transcript generated in the transcription reaction leaving only input template DNA. When transcription followed by RNase H or RNase A treatment alone or in combination is performed on an empty Bluescript vector [pBlueKS(+)], there is negligible hybrid formation.
Figure Legend Snippet: RNA–DNA hybrid formation at a (CGG)39 · (CCG)39 FRAXA template. When the template DNA is transcribed with T7 RNA polymerase, heterogeneous RNA is produced generating a smear (Transcription lane). Treatment with RNase H alone which is specific to RNA base paired to template DNA digests only RNA that is base paired to its template DNA. Treatment with RNase A, which is specific to single-stranded RNA, digests all free, single-stranded RNA leaving template DNA and RNA:DNA hybrid structures. Note that RNA–DNA hybrids migrate more slowly than supercoiled DNA (as indicated schematically, RNA is in light blue). Hybrid structures generate a smear due to their heterogeneous sizes. With a larger RNA component, the DNA is open to a greater degree (more relaxed), hence migration is closer to open circular DNA. Treatment of the hybrids with RNase H along with RNase A removes any hybrids formed as well as transcript generated in the transcription reaction leaving only input template DNA. When transcription followed by RNase H or RNase A treatment alone or in combination is performed on an empty Bluescript vector [pBlueKS(+)], there is negligible hybrid formation.

Techniques Used: Produced, Migration, Generated, Plasmid Preparation

Quantification of relative RNA–DNA hybrid formation at increasing repeat lengths. ( A ) In vitro transcription reactions were performed on FRAXA plasmids bearing repeat tracts of (CGG)17 · (CCG)17, (CGG)39 · (CCG)39 and (CGG)53 · (CCG)53 following which samples were treated with RNase A or A+H. Hybrid formation was quantified by densitometry analysis using image quant by measuring the proportion of products that migrate between open circular and supercoiled position (indicated by ‘R’) divided by the total products below open circular including supercoiled. RNase A+H treated samples were used to determine the position of the supercoiled DNA for each repeat length. To the left of the graph is a representative gel used to quantify relative hybrid formation. The RNA indicated in the graph represents the RNA component bound in the RNA:DNA hybrid. Error bars are derived from three separate experiments ( N =3). Using the t -test to compare R-loop formation revealed a significant difference between the 17 and 53 rCGG R-loop ( P =0.0413) as well as the 17 and 53 rCCG R-loop ( P =0.0092). ( B ) The same analysis was performed as in (A) but for SCA1 plasmids bearing repeat tracts of (CAG)30 · (CTG)30, (CAG) 49 · (CTG)49 and (CAG)74 · (CTG)74. Using the t -test to compare R-loop formation between each of the (CAG) · (CTG) repeat lengths did not reveal any statistically significant differences.
Figure Legend Snippet: Quantification of relative RNA–DNA hybrid formation at increasing repeat lengths. ( A ) In vitro transcription reactions were performed on FRAXA plasmids bearing repeat tracts of (CGG)17 · (CCG)17, (CGG)39 · (CCG)39 and (CGG)53 · (CCG)53 following which samples were treated with RNase A or A+H. Hybrid formation was quantified by densitometry analysis using image quant by measuring the proportion of products that migrate between open circular and supercoiled position (indicated by ‘R’) divided by the total products below open circular including supercoiled. RNase A+H treated samples were used to determine the position of the supercoiled DNA for each repeat length. To the left of the graph is a representative gel used to quantify relative hybrid formation. The RNA indicated in the graph represents the RNA component bound in the RNA:DNA hybrid. Error bars are derived from three separate experiments ( N =3). Using the t -test to compare R-loop formation revealed a significant difference between the 17 and 53 rCGG R-loop ( P =0.0413) as well as the 17 and 53 rCCG R-loop ( P =0.0092). ( B ) The same analysis was performed as in (A) but for SCA1 plasmids bearing repeat tracts of (CAG)30 · (CTG)30, (CAG) 49 · (CTG)49 and (CAG)74 · (CTG)74. Using the t -test to compare R-loop formation between each of the (CAG) · (CTG) repeat lengths did not reveal any statistically significant differences.

Techniques Used: In Vitro, Derivative Assay, CTG Assay

Effect of trinucleotide repeat interruptions on RNA–DNA hybrid formation. ( A ) In vitro transcription followed by RNase A or A + H treatment was performed with either pure (39p) FRAXA plasmids (CGG)39 · (CCG)39 or interrupted plasmids (39i) [(CGG)9(AGG) (CGG)9(AGG)(CGG)9(AGG)(CGG)9] · [(CCG)9(CCT) (CCG)9(CCT)(CCG)9(CCT)(CCG)9] as indicated. Repeat tract configurations are schematically presented where hollow dots are the CGG repeat units and the filled dots are the AGG interruptions. R-loops are indicated as ‘R’. ( B ) In vitro transcription followed by RNase A or A+H treatment was performed with either pure (49p) SCA1 plasmids (CAG)49 · (CTG)49 or interrupted plasmids (44i) [(CAG)12(CAT)(CAG)(CAT)(CAG)12(CAT)(CAG)(CAT)(CAG)14] · [(CTG)14(ATG)(CTG)(ATG)(CTG)12(ATG)(CAG)(ATG)(CTG)12] as indicated schematically where hollow dots are the CAG repeat units and the filled dots are the CAT interruptions.
Figure Legend Snippet: Effect of trinucleotide repeat interruptions on RNA–DNA hybrid formation. ( A ) In vitro transcription followed by RNase A or A + H treatment was performed with either pure (39p) FRAXA plasmids (CGG)39 · (CCG)39 or interrupted plasmids (39i) [(CGG)9(AGG) (CGG)9(AGG)(CGG)9(AGG)(CGG)9] · [(CCG)9(CCT) (CCG)9(CCT)(CCG)9(CCT)(CCG)9] as indicated. Repeat tract configurations are schematically presented where hollow dots are the CGG repeat units and the filled dots are the AGG interruptions. R-loops are indicated as ‘R’. ( B ) In vitro transcription followed by RNase A or A+H treatment was performed with either pure (49p) SCA1 plasmids (CAG)49 · (CTG)49 or interrupted plasmids (44i) [(CAG)12(CAT)(CAG)(CAT)(CAG)12(CAT)(CAG)(CAT)(CAG)14] · [(CTG)14(ATG)(CTG)(ATG)(CTG)12(ATG)(CAG)(ATG)(CTG)12] as indicated schematically where hollow dots are the CAG repeat units and the filled dots are the CAT interruptions.

Techniques Used: In Vitro, CTG Assay

Identification of R-loop structures formed in expanded DM1 (CTG)130 · (CAG)130 plasmids using EM following in vitro transcription and treatment with RNase A and SSB protein. SSB proteins bind to the looped-out non-template DNA in an R-loop structure. Thus, each R-loop structure is visualized as a loop within the DNA template as indicated by black arrowheads. ( A ) R-loops formed by using SP6 RNA polymerase, producing an rCAG:dCTG hybrid. ( B ) R-loops formed by using T7 RNA polymerase, producing an rCUG:dCAG hybrid.
Figure Legend Snippet: Identification of R-loop structures formed in expanded DM1 (CTG)130 · (CAG)130 plasmids using EM following in vitro transcription and treatment with RNase A and SSB protein. SSB proteins bind to the looped-out non-template DNA in an R-loop structure. Thus, each R-loop structure is visualized as a loop within the DNA template as indicated by black arrowheads. ( A ) R-loops formed by using SP6 RNA polymerase, producing an rCAG:dCTG hybrid. ( B ) R-loops formed by using T7 RNA polymerase, producing an rCUG:dCAG hybrid.

Techniques Used: CTG Assay, In Vitro

RNA–DNA hybrid formation during in vitro transcription of trinucleotide repeat-containing plasmids. ( A ) In vitro transcription of SCA1 plasmid containing (CAG)74 · (CTG)74 and FRAXA plasmid containing (CGG)39 · (CCG)39 repeats in either direction using T3 or T7 RNA polymerase. The repeat sequence contained within the RNA produced and bound in the hybrid is indicated below the transcribed template. Samples following transcription were subsequently treated with either RNase A or A+H as indicated to observe hybrid formation. R-loops are indicated on the gel as ‘R’. ( B ) Exact same reactions and gel conditions as in (A) but transcription was performed in the presence of 3.5 µCi [α- 32 P]-rCTP. Gel was dried and exposed to X-ray film as outlined in ‘Materials and Methods’ section.
Figure Legend Snippet: RNA–DNA hybrid formation during in vitro transcription of trinucleotide repeat-containing plasmids. ( A ) In vitro transcription of SCA1 plasmid containing (CAG)74 · (CTG)74 and FRAXA plasmid containing (CGG)39 · (CCG)39 repeats in either direction using T3 or T7 RNA polymerase. The repeat sequence contained within the RNA produced and bound in the hybrid is indicated below the transcribed template. Samples following transcription were subsequently treated with either RNase A or A+H as indicated to observe hybrid formation. R-loops are indicated on the gel as ‘R’. ( B ) Exact same reactions and gel conditions as in (A) but transcription was performed in the presence of 3.5 µCi [α- 32 P]-rCTP. Gel was dried and exposed to X-ray film as outlined in ‘Materials and Methods’ section.

Techniques Used: In Vitro, Plasmid Preparation, CTG Assay, Sequencing, Produced

Effect of convergent simultaneous bidirectional or serial transcription on R-loop formation. ( A ) In vitro transcription of FRAXA template (CGG)39 · (CCG)39 with either T3 or T7 RNA polymerase alone (rCCG or rCGG, respectively), or simultaneously (rCCG with rCGG) or serially: rCCG transcription then phenol chloroform extraction followed by rCGG transcription (rCCG then rCGG), and vice versa (rCGG then rCCG). R-loops are indicated as ‘R’. Note that in the case of bidirectional or serial transcription, complementary RNA is produced forming dsRNA as indicated on the gel by ‘*’. These products are not present in transcription reactions occurring in one direction. ( B ) Same as in (A) except in vitro transcription reactions were performed on a DM1 (CAG)79 · (CTG)79 template. ( C ) EM analysis of convergent transcription reaction products from DM1 (CAG)79 · (CTG)79 templates. Samples were transcribed convergently using T3 and T7 RNA polymerase promoters simultaneously then the products were treated with RNAse A and prepared for EM as described in the ‘Materials and Methods’ section (rCUGand rCAG RNase A). Samples were also subjected to RNase H treatment along with RNase A for comparison (rCUG and rCAG RNase A, H). Transcription was also performed on the same template in a single direction for further comparison (rCUG RNase A). The products observed for each type of transcription reaction is shown as a percentage of the total number of molecules analyzed. At least 100 molecules were analyzed for each type of transcription reaction.
Figure Legend Snippet: Effect of convergent simultaneous bidirectional or serial transcription on R-loop formation. ( A ) In vitro transcription of FRAXA template (CGG)39 · (CCG)39 with either T3 or T7 RNA polymerase alone (rCCG or rCGG, respectively), or simultaneously (rCCG with rCGG) or serially: rCCG transcription then phenol chloroform extraction followed by rCGG transcription (rCCG then rCGG), and vice versa (rCGG then rCCG). R-loops are indicated as ‘R’. Note that in the case of bidirectional or serial transcription, complementary RNA is produced forming dsRNA as indicated on the gel by ‘*’. These products are not present in transcription reactions occurring in one direction. ( B ) Same as in (A) except in vitro transcription reactions were performed on a DM1 (CAG)79 · (CTG)79 template. ( C ) EM analysis of convergent transcription reaction products from DM1 (CAG)79 · (CTG)79 templates. Samples were transcribed convergently using T3 and T7 RNA polymerase promoters simultaneously then the products were treated with RNAse A and prepared for EM as described in the ‘Materials and Methods’ section (rCUGand rCAG RNase A). Samples were also subjected to RNase H treatment along with RNase A for comparison (rCUG and rCAG RNase A, H). Transcription was also performed on the same template in a single direction for further comparison (rCUG RNase A). The products observed for each type of transcription reaction is shown as a percentage of the total number of molecules analyzed. At least 100 molecules were analyzed for each type of transcription reaction.

Techniques Used: In Vitro, Produced, CTG Assay

Mechanism of RNA:DNA hybrid formation during in vitro transcription. ( A ) Schematic of the two possible mechanisms for R-loop formation. By the thread-back model, the nascent transcript (depicted in light blue) that has been ejected from the RNA polymerase re-anneals with the complementary, free DNA template strand (depicted in red) following the progression of the RNA polymerase (light blue, oval). When transcription occurs in the presence of RNAse A (dark blue) the nascent transcript is degraded when it is ejected from the RNA polymerase hence cannot form the hybrid. By the extended-hybrid model, the nascent transcript remains bound to the template DNA and resists becoming ejected from the RNA polymerase. When transcribed in the presence of RNase A, as the nascent transcript is protected by being bound to the template DNA it is not degraded hence hybrid formation is not ablated. ( B ) FRAXA plasmid (CGG)39 · (CCG)39 transcribed in either direction in the absence (−) or presence (+) of RNase A during the transcription reaction. All transcription reactions were subjected to further RNase A or A + H treatment to analyze hybrid formation. ( C ) Same experiment as in (B) performed with SCA1 plasmid containing a (CAG)74 · (CTG)74 repeat tract.
Figure Legend Snippet: Mechanism of RNA:DNA hybrid formation during in vitro transcription. ( A ) Schematic of the two possible mechanisms for R-loop formation. By the thread-back model, the nascent transcript (depicted in light blue) that has been ejected from the RNA polymerase re-anneals with the complementary, free DNA template strand (depicted in red) following the progression of the RNA polymerase (light blue, oval). When transcription occurs in the presence of RNAse A (dark blue) the nascent transcript is degraded when it is ejected from the RNA polymerase hence cannot form the hybrid. By the extended-hybrid model, the nascent transcript remains bound to the template DNA and resists becoming ejected from the RNA polymerase. When transcribed in the presence of RNase A, as the nascent transcript is protected by being bound to the template DNA it is not degraded hence hybrid formation is not ablated. ( B ) FRAXA plasmid (CGG)39 · (CCG)39 transcribed in either direction in the absence (−) or presence (+) of RNase A during the transcription reaction. All transcription reactions were subjected to further RNase A or A + H treatment to analyze hybrid formation. ( C ) Same experiment as in (B) performed with SCA1 plasmid containing a (CAG)74 · (CTG)74 repeat tract.

Techniques Used: In Vitro, Plasmid Preparation, CTG Assay

7) Product Images from "RNA-mediated dimerization of the human deoxycytidine deaminase APOBEC3H influences enzyme activity and interaction with nucleic acids"

Article Title: RNA-mediated dimerization of the human deoxycytidine deaminase APOBEC3H influences enzyme activity and interaction with nucleic acids

Journal: Journal of molecular biology

doi: 10.1016/j.jmb.2018.11.006

Deamination of ssDNA by GST-A3H. Deamination activity was tested on an 85 nt ssDNA with two 5′CTC deamination motifs separated by 30 nt. (A-B) A3H WT can deaminate ssDNA in the presence and absence of bound cellular RNA. (C-D) Monomeric A3H mutants have disrupted ssDNA deaminase activity. Using different MgCl 2  concentrations that were found to enhance GST-A3H WT activity, the deamination activity of A3H mutants were tested in the presence of RNase A. (C-D) The A3H GST-Y112A/, GST-W115A and GST-R175E/R176E were not active on ssDNA. A representative image is shown from three independent experiments. The S.D. was calculated from three independent experiments and is shown below the gel or for panel (B) is represented by error bars. Some error bars in (B) are obscured by the symbol.
Figure Legend Snippet: Deamination of ssDNA by GST-A3H. Deamination activity was tested on an 85 nt ssDNA with two 5′CTC deamination motifs separated by 30 nt. (A-B) A3H WT can deaminate ssDNA in the presence and absence of bound cellular RNA. (C-D) Monomeric A3H mutants have disrupted ssDNA deaminase activity. Using different MgCl 2 concentrations that were found to enhance GST-A3H WT activity, the deamination activity of A3H mutants were tested in the presence of RNase A. (C-D) The A3H GST-Y112A/, GST-W115A and GST-R175E/R176E were not active on ssDNA. A representative image is shown from three independent experiments. The S.D. was calculated from three independent experiments and is shown below the gel or for panel (B) is represented by error bars. Some error bars in (B) are obscured by the symbol.

Techniques Used: Activity Assay

8) Product Images from "Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats"

Article Title: Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq935

RNA–DNA hybrid formation at a (CGG)39 · (CCG)39 FRAXA template. When the template DNA is transcribed with T7 RNA polymerase, heterogeneous RNA is produced generating a smear (Transcription lane). Treatment with RNase H alone which is specific to RNA base paired to template DNA digests only RNA that is base paired to its template DNA. Treatment with RNase A, which is specific to single-stranded RNA, digests all free, single-stranded RNA leaving template DNA and RNA:DNA hybrid structures. Note that RNA–DNA hybrids migrate more slowly than supercoiled DNA (as indicated schematically, RNA is in light blue). Hybrid structures generate a smear due to their heterogeneous sizes. With a larger RNA component, the DNA is open to a greater degree (more relaxed), hence migration is closer to open circular DNA. Treatment of the hybrids with RNase H along with RNase A removes any hybrids formed as well as transcript generated in the transcription reaction leaving only input template DNA. When transcription followed by RNase H or RNase A treatment alone or in combination is performed on an empty Bluescript vector [pBlueKS(+)], there is negligible hybrid formation.
Figure Legend Snippet: RNA–DNA hybrid formation at a (CGG)39 · (CCG)39 FRAXA template. When the template DNA is transcribed with T7 RNA polymerase, heterogeneous RNA is produced generating a smear (Transcription lane). Treatment with RNase H alone which is specific to RNA base paired to template DNA digests only RNA that is base paired to its template DNA. Treatment with RNase A, which is specific to single-stranded RNA, digests all free, single-stranded RNA leaving template DNA and RNA:DNA hybrid structures. Note that RNA–DNA hybrids migrate more slowly than supercoiled DNA (as indicated schematically, RNA is in light blue). Hybrid structures generate a smear due to their heterogeneous sizes. With a larger RNA component, the DNA is open to a greater degree (more relaxed), hence migration is closer to open circular DNA. Treatment of the hybrids with RNase H along with RNase A removes any hybrids formed as well as transcript generated in the transcription reaction leaving only input template DNA. When transcription followed by RNase H or RNase A treatment alone or in combination is performed on an empty Bluescript vector [pBlueKS(+)], there is negligible hybrid formation.

Techniques Used: Produced, Migration, Generated, Plasmid Preparation

Quantification of relative RNA–DNA hybrid formation at increasing repeat lengths. ( A ) In vitro transcription reactions were performed on FRAXA plasmids bearing repeat tracts of (CGG)17 · (CCG)17, (CGG)39 · (CCG)39 and (CGG)53 · (CCG)53 following which samples were treated with RNase A or A+H. Hybrid formation was quantified by densitometry analysis using image quant by measuring the proportion of products that migrate between open circular and supercoiled position (indicated by ‘R’) divided by the total products below open circular including supercoiled. RNase A+H treated samples were used to determine the position of the supercoiled DNA for each repeat length. To the left of the graph is a representative gel used to quantify relative hybrid formation. The RNA indicated in the graph represents the RNA component bound in the RNA:DNA hybrid. Error bars are derived from three separate experiments ( N =3). Using the t -test to compare R-loop formation revealed a significant difference between the 17 and 53 rCGG R-loop ( P =0.0413) as well as the 17 and 53 rCCG R-loop ( P =0.0092). ( B ) The same analysis was performed as in (A) but for SCA1 plasmids bearing repeat tracts of (CAG)30 · (CTG)30, (CAG) 49 · (CTG)49 and (CAG)74 · (CTG)74. Using the t -test to compare R-loop formation between each of the (CAG) · (CTG) repeat lengths did not reveal any statistically significant differences.
Figure Legend Snippet: Quantification of relative RNA–DNA hybrid formation at increasing repeat lengths. ( A ) In vitro transcription reactions were performed on FRAXA plasmids bearing repeat tracts of (CGG)17 · (CCG)17, (CGG)39 · (CCG)39 and (CGG)53 · (CCG)53 following which samples were treated with RNase A or A+H. Hybrid formation was quantified by densitometry analysis using image quant by measuring the proportion of products that migrate between open circular and supercoiled position (indicated by ‘R’) divided by the total products below open circular including supercoiled. RNase A+H treated samples were used to determine the position of the supercoiled DNA for each repeat length. To the left of the graph is a representative gel used to quantify relative hybrid formation. The RNA indicated in the graph represents the RNA component bound in the RNA:DNA hybrid. Error bars are derived from three separate experiments ( N =3). Using the t -test to compare R-loop formation revealed a significant difference between the 17 and 53 rCGG R-loop ( P =0.0413) as well as the 17 and 53 rCCG R-loop ( P =0.0092). ( B ) The same analysis was performed as in (A) but for SCA1 plasmids bearing repeat tracts of (CAG)30 · (CTG)30, (CAG) 49 · (CTG)49 and (CAG)74 · (CTG)74. Using the t -test to compare R-loop formation between each of the (CAG) · (CTG) repeat lengths did not reveal any statistically significant differences.

Techniques Used: In Vitro, Derivative Assay, CTG Assay

Effect of trinucleotide repeat interruptions on RNA–DNA hybrid formation. ( A ) In vitro transcription followed by RNase A or A + H treatment was performed with either pure (39p) FRAXA plasmids (CGG)39 · (CCG)39 or interrupted plasmids (39i) [(CGG)9(AGG) (CGG)9(AGG)(CGG)9(AGG)(CGG)9] · [(CCG)9(CCT) (CCG)9(CCT)(CCG)9(CCT)(CCG)9] as indicated. Repeat tract configurations are schematically presented where hollow dots are the CGG repeat units and the filled dots are the AGG interruptions. R-loops are indicated as ‘R’. ( B ) In vitro transcription followed by RNase A or A+H treatment was performed with either pure (49p) SCA1 plasmids (CAG)49 · (CTG)49 or interrupted plasmids (44i) [(CAG)12(CAT)(CAG)(CAT)(CAG)12(CAT)(CAG)(CAT)(CAG)14] · [(CTG)14(ATG)(CTG)(ATG)(CTG)12(ATG)(CAG)(ATG)(CTG)12] as indicated schematically where hollow dots are the CAG repeat units and the filled dots are the CAT interruptions.
Figure Legend Snippet: Effect of trinucleotide repeat interruptions on RNA–DNA hybrid formation. ( A ) In vitro transcription followed by RNase A or A + H treatment was performed with either pure (39p) FRAXA plasmids (CGG)39 · (CCG)39 or interrupted plasmids (39i) [(CGG)9(AGG) (CGG)9(AGG)(CGG)9(AGG)(CGG)9] · [(CCG)9(CCT) (CCG)9(CCT)(CCG)9(CCT)(CCG)9] as indicated. Repeat tract configurations are schematically presented where hollow dots are the CGG repeat units and the filled dots are the AGG interruptions. R-loops are indicated as ‘R’. ( B ) In vitro transcription followed by RNase A or A+H treatment was performed with either pure (49p) SCA1 plasmids (CAG)49 · (CTG)49 or interrupted plasmids (44i) [(CAG)12(CAT)(CAG)(CAT)(CAG)12(CAT)(CAG)(CAT)(CAG)14] · [(CTG)14(ATG)(CTG)(ATG)(CTG)12(ATG)(CAG)(ATG)(CTG)12] as indicated schematically where hollow dots are the CAG repeat units and the filled dots are the CAT interruptions.

Techniques Used: In Vitro, CTG Assay

Identification of R-loop structures formed in expanded DM1 (CTG)130 · (CAG)130 plasmids using EM following in vitro transcription and treatment with RNase A and SSB protein. SSB proteins bind to the looped-out non-template DNA in an R-loop structure. Thus, each R-loop structure is visualized as a loop within the DNA template as indicated by black arrowheads. ( A ) R-loops formed by using SP6 RNA polymerase, producing an rCAG:dCTG hybrid. ( B ) R-loops formed by using T7 RNA polymerase, producing an rCUG:dCAG hybrid.
Figure Legend Snippet: Identification of R-loop structures formed in expanded DM1 (CTG)130 · (CAG)130 plasmids using EM following in vitro transcription and treatment with RNase A and SSB protein. SSB proteins bind to the looped-out non-template DNA in an R-loop structure. Thus, each R-loop structure is visualized as a loop within the DNA template as indicated by black arrowheads. ( A ) R-loops formed by using SP6 RNA polymerase, producing an rCAG:dCTG hybrid. ( B ) R-loops formed by using T7 RNA polymerase, producing an rCUG:dCAG hybrid.

Techniques Used: CTG Assay, In Vitro

RNA–DNA hybrid formation during in vitro transcription of trinucleotide repeat-containing plasmids. ( A ) In vitro transcription of SCA1 plasmid containing (CAG)74 · (CTG)74 and FRAXA plasmid containing (CGG)39 · (CCG)39 repeats in either direction using T3 or T7 RNA polymerase. The repeat sequence contained within the RNA produced and bound in the hybrid is indicated below the transcribed template. Samples following transcription were subsequently treated with either RNase A or A+H as indicated to observe hybrid formation. R-loops are indicated on the gel as ‘R’. ( B ) Exact same reactions and gel conditions as in (A) but transcription was performed in the presence of 3.5 µCi [α- 32 P]-rCTP. Gel was dried and exposed to X-ray film as outlined in ‘Materials and Methods’ section.
Figure Legend Snippet: RNA–DNA hybrid formation during in vitro transcription of trinucleotide repeat-containing plasmids. ( A ) In vitro transcription of SCA1 plasmid containing (CAG)74 · (CTG)74 and FRAXA plasmid containing (CGG)39 · (CCG)39 repeats in either direction using T3 or T7 RNA polymerase. The repeat sequence contained within the RNA produced and bound in the hybrid is indicated below the transcribed template. Samples following transcription were subsequently treated with either RNase A or A+H as indicated to observe hybrid formation. R-loops are indicated on the gel as ‘R’. ( B ) Exact same reactions and gel conditions as in (A) but transcription was performed in the presence of 3.5 µCi [α- 32 P]-rCTP. Gel was dried and exposed to X-ray film as outlined in ‘Materials and Methods’ section.

Techniques Used: In Vitro, Plasmid Preparation, CTG Assay, Sequencing, Produced

Effect of convergent simultaneous bidirectional or serial transcription on R-loop formation. ( A ) In vitro transcription of FRAXA template (CGG)39 · (CCG)39 with either T3 or T7 RNA polymerase alone (rCCG or rCGG, respectively), or simultaneously (rCCG with rCGG) or serially: rCCG transcription then phenol chloroform extraction followed by rCGG transcription (rCCG then rCGG), and vice versa (rCGG then rCCG). R-loops are indicated as ‘R’. Note that in the case of bidirectional or serial transcription, complementary RNA is produced forming dsRNA as indicated on the gel by ‘*’. These products are not present in transcription reactions occurring in one direction. ( B ) Same as in (A) except in vitro transcription reactions were performed on a DM1 (CAG)79 · (CTG)79 template. ( C ) EM analysis of convergent transcription reaction products from DM1 (CAG)79 · (CTG)79 templates. Samples were transcribed convergently using T3 and T7 RNA polymerase promoters simultaneously then the products were treated with RNAse A and prepared for EM as described in the ‘Materials and Methods’ section (rCUGand rCAG RNase A). Samples were also subjected to RNase H treatment along with RNase A for comparison (rCUG and rCAG RNase A, H). Transcription was also performed on the same template in a single direction for further comparison (rCUG RNase A). The products observed for each type of transcription reaction is shown as a percentage of the total number of molecules analyzed. At least 100 molecules were analyzed for each type of transcription reaction.
Figure Legend Snippet: Effect of convergent simultaneous bidirectional or serial transcription on R-loop formation. ( A ) In vitro transcription of FRAXA template (CGG)39 · (CCG)39 with either T3 or T7 RNA polymerase alone (rCCG or rCGG, respectively), or simultaneously (rCCG with rCGG) or serially: rCCG transcription then phenol chloroform extraction followed by rCGG transcription (rCCG then rCGG), and vice versa (rCGG then rCCG). R-loops are indicated as ‘R’. Note that in the case of bidirectional or serial transcription, complementary RNA is produced forming dsRNA as indicated on the gel by ‘*’. These products are not present in transcription reactions occurring in one direction. ( B ) Same as in (A) except in vitro transcription reactions were performed on a DM1 (CAG)79 · (CTG)79 template. ( C ) EM analysis of convergent transcription reaction products from DM1 (CAG)79 · (CTG)79 templates. Samples were transcribed convergently using T3 and T7 RNA polymerase promoters simultaneously then the products were treated with RNAse A and prepared for EM as described in the ‘Materials and Methods’ section (rCUGand rCAG RNase A). Samples were also subjected to RNase H treatment along with RNase A for comparison (rCUG and rCAG RNase A, H). Transcription was also performed on the same template in a single direction for further comparison (rCUG RNase A). The products observed for each type of transcription reaction is shown as a percentage of the total number of molecules analyzed. At least 100 molecules were analyzed for each type of transcription reaction.

Techniques Used: In Vitro, Produced, CTG Assay

Mechanism of RNA:DNA hybrid formation during in vitro transcription. ( A ) Schematic of the two possible mechanisms for R-loop formation. By the thread-back model, the nascent transcript (depicted in light blue) that has been ejected from the RNA polymerase re-anneals with the complementary, free DNA template strand (depicted in red) following the progression of the RNA polymerase (light blue, oval). When transcription occurs in the presence of RNAse A (dark blue) the nascent transcript is degraded when it is ejected from the RNA polymerase hence cannot form the hybrid. By the extended-hybrid model, the nascent transcript remains bound to the template DNA and resists becoming ejected from the RNA polymerase. When transcribed in the presence of RNase A, as the nascent transcript is protected by being bound to the template DNA it is not degraded hence hybrid formation is not ablated. ( B ) FRAXA plasmid (CGG)39 · (CCG)39 transcribed in either direction in the absence (−) or presence (+) of RNase A during the transcription reaction. All transcription reactions were subjected to further RNase A or A + H treatment to analyze hybrid formation. ( C ) Same experiment as in (B) performed with SCA1 plasmid containing a (CAG)74 · (CTG)74 repeat tract.
Figure Legend Snippet: Mechanism of RNA:DNA hybrid formation during in vitro transcription. ( A ) Schematic of the two possible mechanisms for R-loop formation. By the thread-back model, the nascent transcript (depicted in light blue) that has been ejected from the RNA polymerase re-anneals with the complementary, free DNA template strand (depicted in red) following the progression of the RNA polymerase (light blue, oval). When transcription occurs in the presence of RNAse A (dark blue) the nascent transcript is degraded when it is ejected from the RNA polymerase hence cannot form the hybrid. By the extended-hybrid model, the nascent transcript remains bound to the template DNA and resists becoming ejected from the RNA polymerase. When transcribed in the presence of RNase A, as the nascent transcript is protected by being bound to the template DNA it is not degraded hence hybrid formation is not ablated. ( B ) FRAXA plasmid (CGG)39 · (CCG)39 transcribed in either direction in the absence (−) or presence (+) of RNase A during the transcription reaction. All transcription reactions were subjected to further RNase A or A + H treatment to analyze hybrid formation. ( C ) Same experiment as in (B) performed with SCA1 plasmid containing a (CAG)74 · (CTG)74 repeat tract.

Techniques Used: In Vitro, Plasmid Preparation, CTG Assay

9) Product Images from "ATX-2, the C. elegans Ortholog of Human Ataxin-2, Regulates Centrosome Size and Microtubule Dynamics"

Article Title: ATX-2, the C. elegans Ortholog of Human Ataxin-2, Regulates Centrosome Size and Microtubule Dynamics

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1006370

ATX-2 physically associates with SZY-20 and functions in early cell division. (A) ATX-2 co-precipitates with SZY-20 in the presence of RNAse inhibitor or RNAse A. For input, 10% of the total embryonic lysates was loaded for comparison. (B) At 24°C,  atx-2(RNAi)  and  szy-20(bs52)  animals produce 50–60% of embryonic lethality but  pab-1(RNAi)  produces
Figure Legend Snippet: ATX-2 physically associates with SZY-20 and functions in early cell division. (A) ATX-2 co-precipitates with SZY-20 in the presence of RNAse inhibitor or RNAse A. For input, 10% of the total embryonic lysates was loaded for comparison. (B) At 24°C, atx-2(RNAi) and szy-20(bs52) animals produce 50–60% of embryonic lethality but pab-1(RNAi) produces

Techniques Used:

10) Product Images from "Specific Recognition and Cleavage of the Plus-Strand Primer by Reverse Transcriptase †"

Article Title: Specific Recognition and Cleavage of the Plus-Strand Primer by Reverse Transcriptase †

Journal: Journal of Virology

doi: 10.1128/JVI.79.23.14863-14875.2005

Analysis of the 5′ end of plus-strand cDNA by primer extension. The cDNA produced by wild-type Tf1 and the mutants R786H and N782S was isolated from virus-like particles and analyzed by primer extension. The cDNAs were either not treated (−), or were pretreated with RNase A (+). The extension products were mapped using sequence ladders generated with the same primers used in the extension reactions. The triangles indicate the products that corresponded to the first nucleotide of the LTR. The asterisks mark the positions where products would map if PPT sequence were present at the 5′ end of the cDNA.
Figure Legend Snippet: Analysis of the 5′ end of plus-strand cDNA by primer extension. The cDNA produced by wild-type Tf1 and the mutants R786H and N782S was isolated from virus-like particles and analyzed by primer extension. The cDNAs were either not treated (−), or were pretreated with RNase A (+). The extension products were mapped using sequence ladders generated with the same primers used in the extension reactions. The triangles indicate the products that corresponded to the first nucleotide of the LTR. The asterisks mark the positions where products would map if PPT sequence were present at the 5′ end of the cDNA.

Techniques Used: Produced, Isolation, Sequencing, Generated

11) Product Images from "Sequence Variability of Borna Disease Virus: Resistance to Superinfection May Contribute to High Genome Stability in Persistently Infected Cells"

Article Title: Sequence Variability of Borna Disease Virus: Resistance to Superinfection May Contribute to High Genome Stability in Persistently Infected Cells

Journal: Journal of Virology

doi:

RPA for the detection of sequence variations between different strains of BDV. Nucleotide differences between the BDV strains He/80 and V were visualized by RPA using a 535-nt RNA probe (lane 1) complementary to the M and G open reading frames of strain He/80. Analysis was carried out using 10 μg of tRNA (lane 2), RNA samples (10 μg) prepared from total cell lysates of uninfected (uninf.) C6 cells (lane 3), C6 cells infected with BDV strain He/80 (lanes 4 and 5), or C6 cells infected with BDV strain V (lanes 6 and 7). Low (0.02-μg) or high (4-μg) concentrations of RNase A were used (+) as indicated. The repertoire of RNA species and the expected gel positions of the corresponding RPA signals are indicated on the right. The arrows mark signals resulting from RNA species with slightly different sequences. The numbers to the left indicate the mobility of molecular size markers (in nucleotides).
Figure Legend Snippet: RPA for the detection of sequence variations between different strains of BDV. Nucleotide differences between the BDV strains He/80 and V were visualized by RPA using a 535-nt RNA probe (lane 1) complementary to the M and G open reading frames of strain He/80. Analysis was carried out using 10 μg of tRNA (lane 2), RNA samples (10 μg) prepared from total cell lysates of uninfected (uninf.) C6 cells (lane 3), C6 cells infected with BDV strain He/80 (lanes 4 and 5), or C6 cells infected with BDV strain V (lanes 6 and 7). Low (0.02-μg) or high (4-μg) concentrations of RNase A were used (+) as indicated. The repertoire of RNA species and the expected gel positions of the corresponding RPA signals are indicated on the right. The arrows mark signals resulting from RNA species with slightly different sequences. The numbers to the left indicate the mobility of molecular size markers (in nucleotides).

Techniques Used: Recombinase Polymerase Amplification, Sequencing, Infection

12) Product Images from "Evidence that Lin28 stimulates translation by recruiting RNA helicase A to polysomes"

Article Title: Evidence that Lin28 stimulates translation by recruiting RNA helicase A to polysomes

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq1350

The N- and C-terminal regions of RHA interact with Lin28. ( A ) Domain organization of human RHA protein. Double-stranded RNA binding domain I and II (dsRBD I and II), C-terminal domain rich in arginine-glycine-glycine (RGG) repeats and the Walker helicase motifs of the conserved DEAD-box RNA helicases are depicted. Numbers indicate corresponding amino acid residue. ( B ) GST pulldown results. HEK293 cell lysate containing Flag-Lin28 was incubated with bacterial lysate containing the indicated recombinant RHA domains or GST alone in the presence of RNase A, followed by GST pulldown assays. Left panel, anti-Flag and anti-PABP antibodies were used in the upper and lower blots, respectively. Input was 0.5% of the total amount of proteins used for each GST pulldown. Right panel, Coomassie staining determined comparable amounts of the recombinant proteins used in the GST pulldown assays. 1% of the input was loaded in each lane. Molecular size markers are on the right. ( C ) Flag-Lin28 and Flag-N300 were co-transfected into HEK293 cells. Co-IP was carried out in the presence of RNase A 24 h later using anti-Lin28 antibody to bring down Flag-Lin28 together with its associated proteins, followed by western blot analysis. Antibodies used in the western blot were anti-RHA (top two blots, note, this antibody recognizes both full-length RHA and Flag-N300), anti-NXF1 (third blot from top), and anti-Flag M2 (bottom blot). Total proteins (2%) used for each immunoprecipitation was loaded as input.
Figure Legend Snippet: The N- and C-terminal regions of RHA interact with Lin28. ( A ) Domain organization of human RHA protein. Double-stranded RNA binding domain I and II (dsRBD I and II), C-terminal domain rich in arginine-glycine-glycine (RGG) repeats and the Walker helicase motifs of the conserved DEAD-box RNA helicases are depicted. Numbers indicate corresponding amino acid residue. ( B ) GST pulldown results. HEK293 cell lysate containing Flag-Lin28 was incubated with bacterial lysate containing the indicated recombinant RHA domains or GST alone in the presence of RNase A, followed by GST pulldown assays. Left panel, anti-Flag and anti-PABP antibodies were used in the upper and lower blots, respectively. Input was 0.5% of the total amount of proteins used for each GST pulldown. Right panel, Coomassie staining determined comparable amounts of the recombinant proteins used in the GST pulldown assays. 1% of the input was loaded in each lane. Molecular size markers are on the right. ( C ) Flag-Lin28 and Flag-N300 were co-transfected into HEK293 cells. Co-IP was carried out in the presence of RNase A 24 h later using anti-Lin28 antibody to bring down Flag-Lin28 together with its associated proteins, followed by western blot analysis. Antibodies used in the western blot were anti-RHA (top two blots, note, this antibody recognizes both full-length RHA and Flag-N300), anti-NXF1 (third blot from top), and anti-Flag M2 (bottom blot). Total proteins (2%) used for each immunoprecipitation was loaded as input.

Techniques Used: RNA Binding Assay, Incubation, Recombinant, Staining, Transfection, Co-Immunoprecipitation Assay, Western Blot, Immunoprecipitation

C-terminus deletion reduces Lin28′s ability to interact with RHA. ( A ) Schematic of wild-type and mutant Lin28 protein. Numbers are in amino acids. ( B ) Flag-Lin28, Flag-Lin28ΔN, Flag-Lin28ΔC or empty vector were each transfected into HEK293 cells. Co-IP was carried out in the presence of RNase A using anti-Flag antibody. Resulting protein complexes were resolved by SDS–PAGE, followed by western blot analysis. Anti-RHA and polyclonal anti-Flag antibody were used in the top and bottom blots, respectively. Three percent of input was loaded. ( C ) Flag-Lin28ΔC was transfected into HEK293 cells. Cell lysate containing Flag-Lin28ΔC was incubated with bacterial lysate containing the indicated recombinant RHA domains or GST, followed by GST pulldown assays. Top panel, anti-Flag M2 antibody was used to detect Flag-Lin28ΔC. About 0.5% of the input was loaded in lane 1. Bottom panel, Coomassie staining of the recombinant proteins used in the GST pulldown assays. About 1% of the input was loaded in each lane. Molecular size markers are on the right.
Figure Legend Snippet: C-terminus deletion reduces Lin28′s ability to interact with RHA. ( A ) Schematic of wild-type and mutant Lin28 protein. Numbers are in amino acids. ( B ) Flag-Lin28, Flag-Lin28ΔN, Flag-Lin28ΔC or empty vector were each transfected into HEK293 cells. Co-IP was carried out in the presence of RNase A using anti-Flag antibody. Resulting protein complexes were resolved by SDS–PAGE, followed by western blot analysis. Anti-RHA and polyclonal anti-Flag antibody were used in the top and bottom blots, respectively. Three percent of input was loaded. ( C ) Flag-Lin28ΔC was transfected into HEK293 cells. Cell lysate containing Flag-Lin28ΔC was incubated with bacterial lysate containing the indicated recombinant RHA domains or GST, followed by GST pulldown assays. Top panel, anti-Flag M2 antibody was used to detect Flag-Lin28ΔC. About 0.5% of the input was loaded in lane 1. Bottom panel, Coomassie staining of the recombinant proteins used in the GST pulldown assays. About 1% of the input was loaded in each lane. Molecular size markers are on the right.

Techniques Used: Mutagenesis, Plasmid Preparation, Transfection, Co-Immunoprecipitation Assay, SDS Page, Western Blot, Incubation, Recombinant, Staining

Lin28 interacts with RHA in PA-1 cells. Lin28-containing protein complexes were immunoprecipitated in the presence of excess amounts of RNase A from PA-1 cells using anti-Lin28 or pre-immune IgG (as a negative control for non-specific binding). Co-IP complexes (lanes 1 and 2) and 3% input (lane 3) were resolved by SDS–PAGE, followed by western blot analysis using anti-RHA (top blot) and anti-NXF1 (bottom blot) antiboddies, respectively.
Figure Legend Snippet: Lin28 interacts with RHA in PA-1 cells. Lin28-containing protein complexes were immunoprecipitated in the presence of excess amounts of RNase A from PA-1 cells using anti-Lin28 or pre-immune IgG (as a negative control for non-specific binding). Co-IP complexes (lanes 1 and 2) and 3% input (lane 3) were resolved by SDS–PAGE, followed by western blot analysis using anti-RHA (top blot) and anti-NXF1 (bottom blot) antiboddies, respectively.

Techniques Used: Immunoprecipitation, Negative Control, Binding Assay, Co-Immunoprecipitation Assay, SDS Page, Western Blot

13) Product Images from "RNA Polymerase V Functions in Arabidopsis Interphase Heterochromatin Organization Independently of the 24-nt siRNA-Directed DNA Methylation Pathway"

Article Title: RNA Polymerase V Functions in Arabidopsis Interphase Heterochromatin Organization Independently of the 24-nt siRNA-Directed DNA Methylation Pathway

Journal: Molecular Plant

doi: 10.1093/mp/ssp006

Pericentromere-Repeat RNAs Localized by RNA-FISH during Interphase. The probe specifically detects forward transcripts (CEN-F, red). RNase A treatment prior to in-situ hybridization eliminates the FISH signals, indicating that the method predominantly detects RNA (bottom nuclei). DNA was DAPI-counterstained (gray). The size bar corresponds to 5 μm.
Figure Legend Snippet: Pericentromere-Repeat RNAs Localized by RNA-FISH during Interphase. The probe specifically detects forward transcripts (CEN-F, red). RNase A treatment prior to in-situ hybridization eliminates the FISH signals, indicating that the method predominantly detects RNA (bottom nuclei). DNA was DAPI-counterstained (gray). The size bar corresponds to 5 μm.

Techniques Used: Fluorescence In Situ Hybridization, In Situ Hybridization

Effects of RNase A Treatment on the Interphase Organization of Centromere Repeats (Green), H3K9me2 (Red), and 5-mC (Red) Detected by DNA-FISH and Immunostaining. Representative images of untreated and nuclease treated (+RNase) nuclei are shown. The right-most nuclei depict immunostaining of HTR12 (red) and 180-bp pericentromeric repeats (green) in non-treated and RNase A-treated nuclei. DNA was visualized by DAPI (gray). Size bars indicate 5 μm.
Figure Legend Snippet: Effects of RNase A Treatment on the Interphase Organization of Centromere Repeats (Green), H3K9me2 (Red), and 5-mC (Red) Detected by DNA-FISH and Immunostaining. Representative images of untreated and nuclease treated (+RNase) nuclei are shown. The right-most nuclei depict immunostaining of HTR12 (red) and 180-bp pericentromeric repeats (green) in non-treated and RNase A-treated nuclei. DNA was visualized by DAPI (gray). Size bars indicate 5 μm.

Techniques Used: Fluorescence In Situ Hybridization, Immunostaining

14) Product Images from "Structural Protein Requirements in Equine Arteritis Virus Assembly"

Article Title: Structural Protein Requirements in Equine Arteritis Virus Assembly

Journal: Journal of Virology

doi: 10.1128/JVI.78.23.13019-13027.2004

Packaging of genomic EAV RNA in viral particles. KO-E particles were treated with DNase I and RNase A in the presence or absence of SDS and Triton X-100. After inactivation of these enzymes by proteinase K, RNA was isolated from each sample and used for RT-PCR with primers located at either side of the StuI site that was introduced as a marker mutation in the KO-E RNA. When indicated, the PCR fragments were digested with StuI. The reaction products were analyzed by agarose gel electrophoresis. The numbers on the right refer to the anticipated sizes of the PCR fragments before and after StuI digestion. On the left, the positions and sizes of marker DNA fragments that were analyzed in the same gel are indicated (in nucleotides).
Figure Legend Snippet: Packaging of genomic EAV RNA in viral particles. KO-E particles were treated with DNase I and RNase A in the presence or absence of SDS and Triton X-100. After inactivation of these enzymes by proteinase K, RNA was isolated from each sample and used for RT-PCR with primers located at either side of the StuI site that was introduced as a marker mutation in the KO-E RNA. When indicated, the PCR fragments were digested with StuI. The reaction products were analyzed by agarose gel electrophoresis. The numbers on the right refer to the anticipated sizes of the PCR fragments before and after StuI digestion. On the left, the positions and sizes of marker DNA fragments that were analyzed in the same gel are indicated (in nucleotides).

Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction, Marker, Mutagenesis, Polymerase Chain Reaction, Agarose Gel Electrophoresis

15) Product Images from "Structural Protein Requirements in Equine Arteritis Virus Assembly"

Article Title: Structural Protein Requirements in Equine Arteritis Virus Assembly

Journal: Journal of Virology

doi: 10.1128/JVI.78.23.13019-13027.2004

Packaging of genomic EAV RNA in viral particles. KO-E particles were treated with DNase I and RNase A in the presence or absence of SDS and Triton X-100. After inactivation of these enzymes by proteinase K, RNA was isolated from each sample and used for RT-PCR with primers located at either side of the StuI site that was introduced as a marker mutation in the KO-E RNA. When indicated, the PCR fragments were digested with StuI. The reaction products were analyzed by agarose gel electrophoresis. The numbers on the right refer to the anticipated sizes of the PCR fragments before and after StuI digestion. On the left, the positions and sizes of marker DNA fragments that were analyzed in the same gel are indicated (in nucleotides).
Figure Legend Snippet: Packaging of genomic EAV RNA in viral particles. KO-E particles were treated with DNase I and RNase A in the presence or absence of SDS and Triton X-100. After inactivation of these enzymes by proteinase K, RNA was isolated from each sample and used for RT-PCR with primers located at either side of the StuI site that was introduced as a marker mutation in the KO-E RNA. When indicated, the PCR fragments were digested with StuI. The reaction products were analyzed by agarose gel electrophoresis. The numbers on the right refer to the anticipated sizes of the PCR fragments before and after StuI digestion. On the left, the positions and sizes of marker DNA fragments that were analyzed in the same gel are indicated (in nucleotides).

Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction, Marker, Mutagenesis, Polymerase Chain Reaction, Agarose Gel Electrophoresis

16) Product Images from "Capturing RNA-dependent pathways for cryo-EM analysis"

Article Title: Capturing RNA-dependent pathways for cryo-EM analysis

Journal: Computational and Structural Biotechnology Journal

doi: 10.5936/csbj.201204003

Digestion of mRNA . Bacterial lysates were incubated for 30 minutes in the absence (a) and presence (b) of RNase A. Scale bar is 50 nm. Samples incubated with RNase A show a more homogeneous population of translational complexes.
Figure Legend Snippet: Digestion of mRNA . Bacterial lysates were incubated for 30 minutes in the absence (a) and presence (b) of RNase A. Scale bar is 50 nm. Samples incubated with RNase A show a more homogeneous population of translational complexes.

Techniques Used: Incubation

17) Product Images from "Processing of double-R-loops in (CAG)·(CTG) and C9orf72 (GGGGCC)·(GGCCCC) repeats causes instability"

Article Title: Processing of double-R-loops in (CAG)·(CTG) and C9orf72 (GGGGCC)·(GGCCCC) repeats causes instability

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku658

C9orf72 double-R-loops when processed by neuron cell extracts lead to instability. ( A ) Products of double-R-loop processing by gel analysis. DNA control template with (GGGGCC) 40 ·(GGCCCC) 40 was not transcribed, while the other templates were transcribed and treated with RNase A to remove single-stranded RNA, but retain the R-loop configuration as indicated schematically above each gel. These templates were treated with SH-SY5Y cell extract. Products of SH-SY5Y extract processing were extracted, treated with RNase A+H to remove residual R-loops and transformed into E. coli. Following minimal culturing, products of each R-loop configuration were resolved on polyacrylamide gels alongside known size markers. For each gel, the first four lanes show a titration of DNA concentration of the starting DNA, to reveal repeat length heterogeneity. A sample of products with lengths larger, smaller or similar to the starting lengths are shown. Specific colony numbers are as follows: DNA-168; rGGGGCC+rGGCCCC+RNase A-167; rGGGGGCC+rGGCCCC+RNase A+H-157. The arrowhead indicates the size of the major starting length of 40 repeats. ( B ) Distribution of unstable products of R-loop processing. Sizes were estimated for each unstable product of processing from electrophoretic migration position relative to known size markers as previously described ( 26 ) and plotted. Only unstable products are shown; the starting major repeat size of 40 is indicated by the dashed vertical line.
Figure Legend Snippet: C9orf72 double-R-loops when processed by neuron cell extracts lead to instability. ( A ) Products of double-R-loop processing by gel analysis. DNA control template with (GGGGCC) 40 ·(GGCCCC) 40 was not transcribed, while the other templates were transcribed and treated with RNase A to remove single-stranded RNA, but retain the R-loop configuration as indicated schematically above each gel. These templates were treated with SH-SY5Y cell extract. Products of SH-SY5Y extract processing were extracted, treated with RNase A+H to remove residual R-loops and transformed into E. coli. Following minimal culturing, products of each R-loop configuration were resolved on polyacrylamide gels alongside known size markers. For each gel, the first four lanes show a titration of DNA concentration of the starting DNA, to reveal repeat length heterogeneity. A sample of products with lengths larger, smaller or similar to the starting lengths are shown. Specific colony numbers are as follows: DNA-168; rGGGGCC+rGGCCCC+RNase A-167; rGGGGGCC+rGGCCCC+RNase A+H-157. The arrowhead indicates the size of the major starting length of 40 repeats. ( B ) Distribution of unstable products of R-loop processing. Sizes were estimated for each unstable product of processing from electrophoretic migration position relative to known size markers as previously described ( 26 ) and plotted. Only unstable products are shown; the starting major repeat size of 40 is indicated by the dashed vertical line.

Techniques Used: Transformation Assay, Titration, Concentration Assay, Migration

R-loop processing by human cell extract. In vitro transcription of a (CAG) 79 ·(CTG) 79 repeat-containing plasmid with [α- 32 P]rCTP was performed followed by RNase A treatment (to cleave single-stranded RNA); labeled ‘A’ or RNase H treatment (to also cleave RNA:DNA hybrids of the R-loop); labeled ‘H’ or human cell extract treatment; labeled ‘Ext.’ as indicated. The configuration of the R-loop generated is schematically represented above the gel. Autoradiographic signal in the gel represents R-loop formation. The position of supercoiled plasmid in dimer and monomer form is indicated by ‘sc’ where the top ‘sc’ represents linked dimers and bottom ‘sc’ represents monomers.
Figure Legend Snippet: R-loop processing by human cell extract. In vitro transcription of a (CAG) 79 ·(CTG) 79 repeat-containing plasmid with [α- 32 P]rCTP was performed followed by RNase A treatment (to cleave single-stranded RNA); labeled ‘A’ or RNase H treatment (to also cleave RNA:DNA hybrids of the R-loop); labeled ‘H’ or human cell extract treatment; labeled ‘Ext.’ as indicated. The configuration of the R-loop generated is schematically represented above the gel. Autoradiographic signal in the gel represents R-loop formation. The position of supercoiled plasmid in dimer and monomer form is indicated by ‘sc’ where the top ‘sc’ represents linked dimers and bottom ‘sc’ represents monomers.

Techniques Used: In Vitro, CTG Assay, Plasmid Preparation, Labeling, Generated

Products of R-loop processing by gel analysis. R-loop templates were formed by in vitro transcription; the DNA control template was not transcribed, while the other templates were transcribed and treated with RNase A to remove single-stranded RNA but retain the R-loop configuration as indicated schematically above each gel. These templates were treated with HeLa cell extract. Products of HeLa extract processing were extracted, treated with RNase A+H to remove residual R-loops and transformed into E. coli. Following minimal culturing, products of each R-loop configuration were resolved on polyacrylamide gels alongside known size markers. The product immediately adjacent to the ladder lane in each gel (indicated by arrow) is the untranscribed, unprocessed parental DNA template that serves as a size marker containing 79 (CAG)·(CTG) repeats.
Figure Legend Snippet: Products of R-loop processing by gel analysis. R-loop templates were formed by in vitro transcription; the DNA control template was not transcribed, while the other templates were transcribed and treated with RNase A to remove single-stranded RNA but retain the R-loop configuration as indicated schematically above each gel. These templates were treated with HeLa cell extract. Products of HeLa extract processing were extracted, treated with RNase A+H to remove residual R-loops and transformed into E. coli. Following minimal culturing, products of each R-loop configuration were resolved on polyacrylamide gels alongside known size markers. The product immediately adjacent to the ladder lane in each gel (indicated by arrow) is the untranscribed, unprocessed parental DNA template that serves as a size marker containing 79 (CAG)·(CTG) repeats.

Techniques Used: In Vitro, Transformation Assay, Marker, CTG Assay

R-loop formation in (CAG) 79 ·(CTG) 79 templates. Templates were in vitro transcribed with T3 and/or T7 RNA polymerases and treated with RNase A (which digests single-stranded RNA) to form each R-loop configuration indicated schematically above the gel. The presence of R-loops forces the plasmid into a more open configuration, thus reducing electrophoretic migration within the gel. Treatment of the R-loop with RNase H cleaves RNA that is base-paired to DNA (the RNA:DNA hybrid) and thus collapses the R-loop, returning DNA to supercoiled form. The position of supercoiled plasmid is indicated as ‘sc’ and open circular plasmid as ‘oc’. Products above these are catenated multimers, which also form R-loops.
Figure Legend Snippet: R-loop formation in (CAG) 79 ·(CTG) 79 templates. Templates were in vitro transcribed with T3 and/or T7 RNA polymerases and treated with RNase A (which digests single-stranded RNA) to form each R-loop configuration indicated schematically above the gel. The presence of R-loops forces the plasmid into a more open configuration, thus reducing electrophoretic migration within the gel. Treatment of the R-loop with RNase H cleaves RNA that is base-paired to DNA (the RNA:DNA hybrid) and thus collapses the R-loop, returning DNA to supercoiled form. The position of supercoiled plasmid is indicated as ‘sc’ and open circular plasmid as ‘oc’. Products above these are catenated multimers, which also form R-loops.

Techniques Used: CTG Assay, In Vitro, Plasmid Preparation, Migration

R-loop instability assay. Plasmids bearing an expanded (CAG) 79 ·(CTG) 79  repeat tract were in vitro transcribed and treated with the appropriate RNase to produce R-loop templates of each configuration as schematically depicted. R-loop templates were subsequently treated with human (HeLa) cell extract to allow processing to occur. Nucleic acid products were extracted and subjected to a final RNase A+H treatment to remove any residual R-loop products and transformed into E. coli bacteria and plated overnight. Individual colonies were picked (representing individual products of R-loop processing) and minimally cultured (see Materials and Methods). DNA was then extracted and restriction digested to release the repeat tract. Products were electrophoresed alongside known size markers to determine repeat length.
Figure Legend Snippet: R-loop instability assay. Plasmids bearing an expanded (CAG) 79 ·(CTG) 79 repeat tract were in vitro transcribed and treated with the appropriate RNase to produce R-loop templates of each configuration as schematically depicted. R-loop templates were subsequently treated with human (HeLa) cell extract to allow processing to occur. Nucleic acid products were extracted and subjected to a final RNase A+H treatment to remove any residual R-loop products and transformed into E. coli bacteria and plated overnight. Individual colonies were picked (representing individual products of R-loop processing) and minimally cultured (see Materials and Methods). DNA was then extracted and restriction digested to release the repeat tract. Products were electrophoresed alongside known size markers to determine repeat length.

Techniques Used: CTG Assay, In Vitro, Transformation Assay, Cell Culture

Instability analysis following R-loop removal. ( A ) Detection of S-DNA structures following R-loop removal with RNase H. DNA templates were transcribed to generate r(CAG), r(CUG) or r(CAG)+r(CUG) R-loops and then treated with RNase A and RNase H to remove all single-stranded RNA and RNA:DNA hybrids. EM was performed on individual molecules in the presence of bacterial SSB to detect unpaired DNA strands that exist in S-DNA structures (see ‘Materials and Methods’). A total of 20 molecules were analyzed for each sample type. DNA controls that were not transcribed contained 2/20 (10%) molecules bound by SSB. The number of samples bound by SSB at a single position following R-loop removal is indicated above each image and expressed as a percentage. ( B ) Percentage of unstable products following processing of RNase H-treated R-loops. R-loop products of each configuration were treated with RNase H prior to cell extract processing and assessed for instability through STRIP analysis as in (A). Products from (A) (RNase A only) were compared to RNase H-treated R-loops (RNase A+H) using the χ 2 test. Data for RNase H-treated R-loop processing are derived from three independent in vitro transcription and human cell extract processing reactions with ∼150 colonies representing 150 individual products of cell extract treatment for each RNase H-treated R-loop configuration. Specific colony numbers are as follows: rCAG-148, rCUG-152, rCAG+rCUG-153. Dashed line indicates DNA control level of instability (21%) for comparison. ( C ) Distribution of unstable products of HeLa extract processing following RNase-H-mediated R-loop removal. Sizes were determined for each unstable product of processing from electrophoretic migration position relative to known size markers as previously described ( 26 ) and plotted. Only unstable products are shown; the stable repeat size of 79 is indicated by the dashed vertical line.
Figure Legend Snippet: Instability analysis following R-loop removal. ( A ) Detection of S-DNA structures following R-loop removal with RNase H. DNA templates were transcribed to generate r(CAG), r(CUG) or r(CAG)+r(CUG) R-loops and then treated with RNase A and RNase H to remove all single-stranded RNA and RNA:DNA hybrids. EM was performed on individual molecules in the presence of bacterial SSB to detect unpaired DNA strands that exist in S-DNA structures (see ‘Materials and Methods’). A total of 20 molecules were analyzed for each sample type. DNA controls that were not transcribed contained 2/20 (10%) molecules bound by SSB. The number of samples bound by SSB at a single position following R-loop removal is indicated above each image and expressed as a percentage. ( B ) Percentage of unstable products following processing of RNase H-treated R-loops. R-loop products of each configuration were treated with RNase H prior to cell extract processing and assessed for instability through STRIP analysis as in (A). Products from (A) (RNase A only) were compared to RNase H-treated R-loops (RNase A+H) using the χ 2 test. Data for RNase H-treated R-loop processing are derived from three independent in vitro transcription and human cell extract processing reactions with ∼150 colonies representing 150 individual products of cell extract treatment for each RNase H-treated R-loop configuration. Specific colony numbers are as follows: rCAG-148, rCUG-152, rCAG+rCUG-153. Dashed line indicates DNA control level of instability (21%) for comparison. ( C ) Distribution of unstable products of HeLa extract processing following RNase-H-mediated R-loop removal. Sizes were determined for each unstable product of processing from electrophoretic migration position relative to known size markers as previously described ( 26 ) and plotted. Only unstable products are shown; the stable repeat size of 79 is indicated by the dashed vertical line.

Techniques Used: Stripping Membranes, Derivative Assay, In Vitro, Migration

C9orf72 repeat forms R-loops and double-R-loops. ( A ) Templates with (GGGGCC) 60 ·(GGCCCC) 60 were in vitro transcribed with T3 and/or T7 RNA polymerases and treated with RNase A (which digests single-stranded RNA) to form each R-loop configuration indicated schematically above the gel. The presence of R-loops forces the plasmid into a more open configuration, thus reducing electrophoretic migration within the gel. Treatment of the R-loop with RNase H cleaves RNA that is base-paired to DNA (the RNA:DNA hybrid) and thus collapses the R-loop, returning DNA to supercoiled form. The slower migrating products above these are catenated multimers, which also form R-loops. ( B ) Templates with 13, 21 or 60 C9orf72 repeats were transcribed as in panel (A) to reveal single and double-R-loop formation.
Figure Legend Snippet: C9orf72 repeat forms R-loops and double-R-loops. ( A ) Templates with (GGGGCC) 60 ·(GGCCCC) 60 were in vitro transcribed with T3 and/or T7 RNA polymerases and treated with RNase A (which digests single-stranded RNA) to form each R-loop configuration indicated schematically above the gel. The presence of R-loops forces the plasmid into a more open configuration, thus reducing electrophoretic migration within the gel. Treatment of the R-loop with RNase H cleaves RNA that is base-paired to DNA (the RNA:DNA hybrid) and thus collapses the R-loop, returning DNA to supercoiled form. The slower migrating products above these are catenated multimers, which also form R-loops. ( B ) Templates with 13, 21 or 60 C9orf72 repeats were transcribed as in panel (A) to reveal single and double-R-loop formation.

Techniques Used: In Vitro, Plasmid Preparation, Migration

Instability analysis of products from double-R-loop processing by neuron cell extract. ( A ) Retinoic acid-differentiated SH-SY5Y cells. ( B ) Percentage of total unstable products following processing. Products were characterized as either stable (having 79 repeats) or unstable (having fewer than or greater than 79 repeats), based on electrophoretic migration and plotted. Data are derived from three independent  in vitro  transcription and retinoic acid human SH-SY5Y cell extract processing reactions with ∼150 colonies (∼50 colonies per replicate) representing 150 individual products of cell extract treatment analyzed for each R-loop configuration. Individual experiments were compared with each other within a triplicate using the  χ 2  test to ensure there were no significant differences between experiments and then data were pooled for each experimental condition. Specific colony numbers are as follows: DNA-194; rCAG+rCUG+RNase A-121; rCAG+rCUG+RNase A+H-149. Products of R-loop processing were compared to the DNA control processing products using the  χ 2  test. ( C ) Percentage of contractions and expansions from processing. Unstable products were further separated into contractions (fewer than 79 repeats) and expansions (greater than 79 repeats) and plotted. The distribution of contractions and expansions were compared between R-loop products and DNA control products using the  χ 2  test. ( D ) Distribution of unstable products of R-loop processing. Sizes were estimated for each unstable product of processing from electrophoretic migration position relative to known size markers as previously described (  26 ) and plotted. Only unstable products are shown; the stable repeat size of 79 is indicated by the dashed vertical line.
Figure Legend Snippet: Instability analysis of products from double-R-loop processing by neuron cell extract. ( A ) Retinoic acid-differentiated SH-SY5Y cells. ( B ) Percentage of total unstable products following processing. Products were characterized as either stable (having 79 repeats) or unstable (having fewer than or greater than 79 repeats), based on electrophoretic migration and plotted. Data are derived from three independent in vitro transcription and retinoic acid human SH-SY5Y cell extract processing reactions with ∼150 colonies (∼50 colonies per replicate) representing 150 individual products of cell extract treatment analyzed for each R-loop configuration. Individual experiments were compared with each other within a triplicate using the χ 2 test to ensure there were no significant differences between experiments and then data were pooled for each experimental condition. Specific colony numbers are as follows: DNA-194; rCAG+rCUG+RNase A-121; rCAG+rCUG+RNase A+H-149. Products of R-loop processing were compared to the DNA control processing products using the χ 2 test. ( C ) Percentage of contractions and expansions from processing. Unstable products were further separated into contractions (fewer than 79 repeats) and expansions (greater than 79 repeats) and plotted. The distribution of contractions and expansions were compared between R-loop products and DNA control products using the χ 2 test. ( D ) Distribution of unstable products of R-loop processing. Sizes were estimated for each unstable product of processing from electrophoretic migration position relative to known size markers as previously described ( 26 ) and plotted. Only unstable products are shown; the stable repeat size of 79 is indicated by the dashed vertical line.

Techniques Used: Migration, Derivative Assay, In Vitro

18) Product Images from "Murine Leukemia Virus Nucleocapsid Mutant Particles Lacking Viral RNA Encapsidate Ribosomes"

Article Title: Murine Leukemia Virus Nucleocapsid Mutant Particles Lacking Viral RNA Encapsidate Ribosomes

Journal: Journal of Virology

doi: 10.1128/JVI.76.22.11405-11413.2002

Effects of detergent and RNase A on NC mutant core stability. Gag VLPs were incubated with or without detergent (1% NP-40 + 1% Triton X-100) and were then incubated in the presence or the absence of RNase A before fractionation by centrifugation. The Gag proteins present in the supernatant (S) and pellet (P) were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were analyzed by immunoblotting with rabbit anti-MuLV CA antiserum (A). Gels in panel A: a, wild-type Gag VLPs; b, C39S Gag VLPs; c, NC(Δ16-23)-Gag VLPs; d, C17− Gag VLPs; and e, C37− Gag VLPs. Lanes 1 to 4, virion cores in detergent; lanes 5 to 6, no detergent; and lanes 3 and 4, + RNase A. Panel B represents the quantification of gels from multiple experiments like that shown in panel A. The bands representative of Gag in supernatants (S) and pellets (P) were scanned by densitometry and were quantified by the Bio-Rad Quantity One software. The percentage of Gag released in supernatant can be evaluated for each mutant in the presence (+) or in the absence (−) of detergent (D) and RNase (R). n is the number of experiments.
Figure Legend Snippet: Effects of detergent and RNase A on NC mutant core stability. Gag VLPs were incubated with or without detergent (1% NP-40 + 1% Triton X-100) and were then incubated in the presence or the absence of RNase A before fractionation by centrifugation. The Gag proteins present in the supernatant (S) and pellet (P) were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were analyzed by immunoblotting with rabbit anti-MuLV CA antiserum (A). Gels in panel A: a, wild-type Gag VLPs; b, C39S Gag VLPs; c, NC(Δ16-23)-Gag VLPs; d, C17− Gag VLPs; and e, C37− Gag VLPs. Lanes 1 to 4, virion cores in detergent; lanes 5 to 6, no detergent; and lanes 3 and 4, + RNase A. Panel B represents the quantification of gels from multiple experiments like that shown in panel A. The bands representative of Gag in supernatants (S) and pellets (P) were scanned by densitometry and were quantified by the Bio-Rad Quantity One software. The percentage of Gag released in supernatant can be evaluated for each mutant in the presence (+) or in the absence (−) of detergent (D) and RNase (R). n is the number of experiments.

Techniques Used: Mutagenesis, Incubation, Fractionation, Centrifugation, Polyacrylamide Gel Electrophoresis, Software

19) Product Images from "Knockdown of BC200 RNA expression reduces cell migration and invasion by destabilizing mRNA for calcium-binding protein S100A11"

Article Title: Knockdown of BC200 RNA expression reduces cell migration and invasion by destabilizing mRNA for calcium-binding protein S100A11

Journal: RNA Biology

doi: 10.1080/15476286.2017.1297913

BC200 RNA-knockdown-associated changes in the ribosome footprint profile. (A) A schematic of the workflow used to profile ribosome footprints. First, ribosome bounded mRNAs were purified from HeLa cells transfected with siRNAs. RNase I degraded unbounded fractions of mRNAs. Next, ribosomes were purified by sucrose cushioning and mRNAs bounded by ribosomes were selectively extracted. Finally, the remained mRNA fragments were converted to sequencing libraries and sequencing was performed. (B) A volcano plot representing the fold change (x-axis, log 2  [Fold change]) and statistical significance (y-axis, −log 10   (C) GO of genes upregulated by knockdown of BC200 RNA. (D) GO of genes downregulated by knockdown of BC200 RNA.
Figure Legend Snippet: BC200 RNA-knockdown-associated changes in the ribosome footprint profile. (A) A schematic of the workflow used to profile ribosome footprints. First, ribosome bounded mRNAs were purified from HeLa cells transfected with siRNAs. RNase I degraded unbounded fractions of mRNAs. Next, ribosomes were purified by sucrose cushioning and mRNAs bounded by ribosomes were selectively extracted. Finally, the remained mRNA fragments were converted to sequencing libraries and sequencing was performed. (B) A volcano plot representing the fold change (x-axis, log 2 [Fold change]) and statistical significance (y-axis, −log 10 (C) GO of genes upregulated by knockdown of BC200 RNA. (D) GO of genes downregulated by knockdown of BC200 RNA.

Techniques Used: Purification, Transfection, Sequencing

20) Product Images from "Oxidative protein folding by an endoplasmic reticulum localized peroxiredoxin"

Article Title: Oxidative protein folding by an endoplasmic reticulum localized peroxiredoxin

Journal: Molecular cell

doi: 10.1016/j.molcel.2010.11.010

PRDX4 catalyzes H 2 O 2 and PDI-dependent oxidative refolding of RNase A in vitro (a) Coomassie stained reducing SDS-PAGE of purified PDI, wildtype and mutant PRDX4-GST tagged proteins used in the experiments shown below. (b) Time dependent change in absorbance of Ellman’s reagent reacted with reduced PDI (150 μM) after introduction of glucose (2.5 mM) and glucose oxidase (GO, 10 mU/mL) as a source of H 2 O 2 and the indicated wildtype or mutant PRDX4 proteins shown in panel “a” (5 μM). The absorbance of each reaction mixture at t=0 is set at 100%. Shown are means ± SEM of a typical assay conducted in triplicate and reproduced 4 times (n=3, * P
Figure Legend Snippet: PRDX4 catalyzes H 2 O 2 and PDI-dependent oxidative refolding of RNase A in vitro (a) Coomassie stained reducing SDS-PAGE of purified PDI, wildtype and mutant PRDX4-GST tagged proteins used in the experiments shown below. (b) Time dependent change in absorbance of Ellman’s reagent reacted with reduced PDI (150 μM) after introduction of glucose (2.5 mM) and glucose oxidase (GO, 10 mU/mL) as a source of H 2 O 2 and the indicated wildtype or mutant PRDX4 proteins shown in panel “a” (5 μM). The absorbance of each reaction mixture at t=0 is set at 100%. Shown are means ± SEM of a typical assay conducted in triplicate and reproduced 4 times (n=3, * P

Techniques Used: In Vitro, Staining, SDS Page, Purification, Mutagenesis

21) Product Images from "Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats"

Article Title: Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq935

RNA–DNA hybrid formation at a (CGG)39 · (CCG)39 FRAXA template. When the template DNA is transcribed with T7 RNA polymerase, heterogeneous RNA is produced generating a smear (Transcription lane). Treatment with RNase H alone which is specific to RNA base paired to template DNA digests only RNA that is base paired to its template DNA. Treatment with RNase A, which is specific to single-stranded RNA, digests all free, single-stranded RNA leaving template DNA and RNA:DNA hybrid structures. Note that RNA–DNA hybrids migrate more slowly than supercoiled DNA (as indicated schematically, RNA is in light blue). Hybrid structures generate a smear due to their heterogeneous sizes. With a larger RNA component, the DNA is open to a greater degree (more relaxed), hence migration is closer to open circular DNA. Treatment of the hybrids with RNase H along with RNase A removes any hybrids formed as well as transcript generated in the transcription reaction leaving only input template DNA. When transcription followed by RNase H or RNase A treatment alone or in combination is performed on an empty Bluescript vector [pBlueKS(+)], there is negligible hybrid formation.
Figure Legend Snippet: RNA–DNA hybrid formation at a (CGG)39 · (CCG)39 FRAXA template. When the template DNA is transcribed with T7 RNA polymerase, heterogeneous RNA is produced generating a smear (Transcription lane). Treatment with RNase H alone which is specific to RNA base paired to template DNA digests only RNA that is base paired to its template DNA. Treatment with RNase A, which is specific to single-stranded RNA, digests all free, single-stranded RNA leaving template DNA and RNA:DNA hybrid structures. Note that RNA–DNA hybrids migrate more slowly than supercoiled DNA (as indicated schematically, RNA is in light blue). Hybrid structures generate a smear due to their heterogeneous sizes. With a larger RNA component, the DNA is open to a greater degree (more relaxed), hence migration is closer to open circular DNA. Treatment of the hybrids with RNase H along with RNase A removes any hybrids formed as well as transcript generated in the transcription reaction leaving only input template DNA. When transcription followed by RNase H or RNase A treatment alone or in combination is performed on an empty Bluescript vector [pBlueKS(+)], there is negligible hybrid formation.

Techniques Used: Produced, Migration, Generated, Plasmid Preparation

Quantification of relative RNA–DNA hybrid formation at increasing repeat lengths. ( A ) In vitro transcription reactions were performed on FRAXA plasmids bearing repeat tracts of (CGG)17 · (CCG)17, (CGG)39 · (CCG)39 and (CGG)53 · (CCG)53 following which samples were treated with RNase A or A+H. Hybrid formation was quantified by densitometry analysis using image quant by measuring the proportion of products that migrate between open circular and supercoiled position (indicated by ‘R’) divided by the total products below open circular including supercoiled. RNase A+H treated samples were used to determine the position of the supercoiled DNA for each repeat length. To the left of the graph is a representative gel used to quantify relative hybrid formation. The RNA indicated in the graph represents the RNA component bound in the RNA:DNA hybrid. Error bars are derived from three separate experiments ( N =3). Using the t -test to compare R-loop formation revealed a significant difference between the 17 and 53 rCGG R-loop ( P =0.0413) as well as the 17 and 53 rCCG R-loop ( P =0.0092). ( B ) The same analysis was performed as in (A) but for SCA1 plasmids bearing repeat tracts of (CAG)30 · (CTG)30, (CAG) 49 · (CTG)49 and (CAG)74 · (CTG)74. Using the t -test to compare R-loop formation between each of the (CAG) · (CTG) repeat lengths did not reveal any statistically significant differences.
Figure Legend Snippet: Quantification of relative RNA–DNA hybrid formation at increasing repeat lengths. ( A ) In vitro transcription reactions were performed on FRAXA plasmids bearing repeat tracts of (CGG)17 · (CCG)17, (CGG)39 · (CCG)39 and (CGG)53 · (CCG)53 following which samples were treated with RNase A or A+H. Hybrid formation was quantified by densitometry analysis using image quant by measuring the proportion of products that migrate between open circular and supercoiled position (indicated by ‘R’) divided by the total products below open circular including supercoiled. RNase A+H treated samples were used to determine the position of the supercoiled DNA for each repeat length. To the left of the graph is a representative gel used to quantify relative hybrid formation. The RNA indicated in the graph represents the RNA component bound in the RNA:DNA hybrid. Error bars are derived from three separate experiments ( N =3). Using the t -test to compare R-loop formation revealed a significant difference between the 17 and 53 rCGG R-loop ( P =0.0413) as well as the 17 and 53 rCCG R-loop ( P =0.0092). ( B ) The same analysis was performed as in (A) but for SCA1 plasmids bearing repeat tracts of (CAG)30 · (CTG)30, (CAG) 49 · (CTG)49 and (CAG)74 · (CTG)74. Using the t -test to compare R-loop formation between each of the (CAG) · (CTG) repeat lengths did not reveal any statistically significant differences.

Techniques Used: In Vitro, Derivative Assay, CTG Assay

Effect of trinucleotide repeat interruptions on RNA–DNA hybrid formation. ( A ) In vitro transcription followed by RNase A or A + H treatment was performed with either pure (39p) FRAXA plasmids (CGG)39 · (CCG)39 or interrupted plasmids (39i) [(CGG)9(AGG) (CGG)9(AGG)(CGG)9(AGG)(CGG)9] · [(CCG)9(CCT) (CCG)9(CCT)(CCG)9(CCT)(CCG)9] as indicated. Repeat tract configurations are schematically presented where hollow dots are the CGG repeat units and the filled dots are the AGG interruptions. R-loops are indicated as ‘R’. ( B ) In vitro transcription followed by RNase A or A+H treatment was performed with either pure (49p) SCA1 plasmids (CAG)49 · (CTG)49 or interrupted plasmids (44i) [(CAG)12(CAT)(CAG)(CAT)(CAG)12(CAT)(CAG)(CAT)(CAG)14] · [(CTG)14(ATG)(CTG)(ATG)(CTG)12(ATG)(CAG)(ATG)(CTG)12] as indicated schematically where hollow dots are the CAG repeat units and the filled dots are the CAT interruptions.
Figure Legend Snippet: Effect of trinucleotide repeat interruptions on RNA–DNA hybrid formation. ( A ) In vitro transcription followed by RNase A or A + H treatment was performed with either pure (39p) FRAXA plasmids (CGG)39 · (CCG)39 or interrupted plasmids (39i) [(CGG)9(AGG) (CGG)9(AGG)(CGG)9(AGG)(CGG)9] · [(CCG)9(CCT) (CCG)9(CCT)(CCG)9(CCT)(CCG)9] as indicated. Repeat tract configurations are schematically presented where hollow dots are the CGG repeat units and the filled dots are the AGG interruptions. R-loops are indicated as ‘R’. ( B ) In vitro transcription followed by RNase A or A+H treatment was performed with either pure (49p) SCA1 plasmids (CAG)49 · (CTG)49 or interrupted plasmids (44i) [(CAG)12(CAT)(CAG)(CAT)(CAG)12(CAT)(CAG)(CAT)(CAG)14] · [(CTG)14(ATG)(CTG)(ATG)(CTG)12(ATG)(CAG)(ATG)(CTG)12] as indicated schematically where hollow dots are the CAG repeat units and the filled dots are the CAT interruptions.

Techniques Used: In Vitro, CTG Assay

Identification of R-loop structures formed in expanded DM1 (CTG)130 · (CAG)130 plasmids using EM following in vitro transcription and treatment with RNase A and SSB protein. SSB proteins bind to the looped-out non-template DNA in an R-loop structure. Thus, each R-loop structure is visualized as a loop within the DNA template as indicated by black arrowheads. ( A ) R-loops formed by using SP6 RNA polymerase, producing an rCAG:dCTG hybrid. ( B ) R-loops formed by using T7 RNA polymerase, producing an rCUG:dCAG hybrid.
Figure Legend Snippet: Identification of R-loop structures formed in expanded DM1 (CTG)130 · (CAG)130 plasmids using EM following in vitro transcription and treatment with RNase A and SSB protein. SSB proteins bind to the looped-out non-template DNA in an R-loop structure. Thus, each R-loop structure is visualized as a loop within the DNA template as indicated by black arrowheads. ( A ) R-loops formed by using SP6 RNA polymerase, producing an rCAG:dCTG hybrid. ( B ) R-loops formed by using T7 RNA polymerase, producing an rCUG:dCAG hybrid.

Techniques Used: CTG Assay, In Vitro

RNA–DNA hybrid formation during in vitro transcription of trinucleotide repeat-containing plasmids. ( A ) In vitro transcription of SCA1 plasmid containing (CAG)74 · (CTG)74 and FRAXA plasmid containing (CGG)39 · (CCG)39 repeats in either direction using T3 or T7 RNA polymerase. The repeat sequence contained within the RNA produced and bound in the hybrid is indicated below the transcribed template. Samples following transcription were subsequently treated with either RNase A or A+H as indicated to observe hybrid formation. R-loops are indicated on the gel as ‘R’. ( B ) Exact same reactions and gel conditions as in (A) but transcription was performed in the presence of 3.5 µCi [α- 32 P]-rCTP. Gel was dried and exposed to X-ray film as outlined in ‘Materials and Methods’ section.
Figure Legend Snippet: RNA–DNA hybrid formation during in vitro transcription of trinucleotide repeat-containing plasmids. ( A ) In vitro transcription of SCA1 plasmid containing (CAG)74 · (CTG)74 and FRAXA plasmid containing (CGG)39 · (CCG)39 repeats in either direction using T3 or T7 RNA polymerase. The repeat sequence contained within the RNA produced and bound in the hybrid is indicated below the transcribed template. Samples following transcription were subsequently treated with either RNase A or A+H as indicated to observe hybrid formation. R-loops are indicated on the gel as ‘R’. ( B ) Exact same reactions and gel conditions as in (A) but transcription was performed in the presence of 3.5 µCi [α- 32 P]-rCTP. Gel was dried and exposed to X-ray film as outlined in ‘Materials and Methods’ section.

Techniques Used: In Vitro, Plasmid Preparation, CTG Assay, Sequencing, Produced

Effect of convergent simultaneous bidirectional or serial transcription on R-loop formation. ( A ) In vitro transcription of FRAXA template (CGG)39 · (CCG)39 with either T3 or T7 RNA polymerase alone (rCCG or rCGG, respectively), or simultaneously (rCCG with rCGG) or serially: rCCG transcription then phenol chloroform extraction followed by rCGG transcription (rCCG then rCGG), and vice versa (rCGG then rCCG). R-loops are indicated as ‘R’. Note that in the case of bidirectional or serial transcription, complementary RNA is produced forming dsRNA as indicated on the gel by ‘*’. These products are not present in transcription reactions occurring in one direction. ( B ) Same as in (A) except in vitro transcription reactions were performed on a DM1 (CAG)79 · (CTG)79 template. ( C ) EM analysis of convergent transcription reaction products from DM1 (CAG)79 · (CTG)79 templates. Samples were transcribed convergently using T3 and T7 RNA polymerase promoters simultaneously then the products were treated with RNAse A and prepared for EM as described in the ‘Materials and Methods’ section (rCUGand rCAG RNase A). Samples were also subjected to RNase H treatment along with RNase A for comparison (rCUG and rCAG RNase A, H). Transcription was also performed on the same template in a single direction for further comparison (rCUG RNase A). The products observed for each type of transcription reaction is shown as a percentage of the total number of molecules analyzed. At least 100 molecules were analyzed for each type of transcription reaction.
Figure Legend Snippet: Effect of convergent simultaneous bidirectional or serial transcription on R-loop formation. ( A ) In vitro transcription of FRAXA template (CGG)39 · (CCG)39 with either T3 or T7 RNA polymerase alone (rCCG or rCGG, respectively), or simultaneously (rCCG with rCGG) or serially: rCCG transcription then phenol chloroform extraction followed by rCGG transcription (rCCG then rCGG), and vice versa (rCGG then rCCG). R-loops are indicated as ‘R’. Note that in the case of bidirectional or serial transcription, complementary RNA is produced forming dsRNA as indicated on the gel by ‘*’. These products are not present in transcription reactions occurring in one direction. ( B ) Same as in (A) except in vitro transcription reactions were performed on a DM1 (CAG)79 · (CTG)79 template. ( C ) EM analysis of convergent transcription reaction products from DM1 (CAG)79 · (CTG)79 templates. Samples were transcribed convergently using T3 and T7 RNA polymerase promoters simultaneously then the products were treated with RNAse A and prepared for EM as described in the ‘Materials and Methods’ section (rCUGand rCAG RNase A). Samples were also subjected to RNase H treatment along with RNase A for comparison (rCUG and rCAG RNase A, H). Transcription was also performed on the same template in a single direction for further comparison (rCUG RNase A). The products observed for each type of transcription reaction is shown as a percentage of the total number of molecules analyzed. At least 100 molecules were analyzed for each type of transcription reaction.

Techniques Used: In Vitro, Produced, CTG Assay

Mechanism of RNA:DNA hybrid formation during in vitro transcription. ( A ) Schematic of the two possible mechanisms for R-loop formation. By the thread-back model, the nascent transcript (depicted in light blue) that has been ejected from the RNA polymerase re-anneals with the complementary, free DNA template strand (depicted in red) following the progression of the RNA polymerase (light blue, oval). When transcription occurs in the presence of RNAse A (dark blue) the nascent transcript is degraded when it is ejected from the RNA polymerase hence cannot form the hybrid. By the extended-hybrid model, the nascent transcript remains bound to the template DNA and resists becoming ejected from the RNA polymerase. When transcribed in the presence of RNase A, as the nascent transcript is protected by being bound to the template DNA it is not degraded hence hybrid formation is not ablated. ( B ) FRAXA plasmid (CGG)39 · (CCG)39 transcribed in either direction in the absence (−) or presence (+) of RNase A during the transcription reaction. All transcription reactions were subjected to further RNase A or A + H treatment to analyze hybrid formation. ( C ) Same experiment as in (B) performed with SCA1 plasmid containing a (CAG)74 · (CTG)74 repeat tract.
Figure Legend Snippet: Mechanism of RNA:DNA hybrid formation during in vitro transcription. ( A ) Schematic of the two possible mechanisms for R-loop formation. By the thread-back model, the nascent transcript (depicted in light blue) that has been ejected from the RNA polymerase re-anneals with the complementary, free DNA template strand (depicted in red) following the progression of the RNA polymerase (light blue, oval). When transcription occurs in the presence of RNAse A (dark blue) the nascent transcript is degraded when it is ejected from the RNA polymerase hence cannot form the hybrid. By the extended-hybrid model, the nascent transcript remains bound to the template DNA and resists becoming ejected from the RNA polymerase. When transcribed in the presence of RNase A, as the nascent transcript is protected by being bound to the template DNA it is not degraded hence hybrid formation is not ablated. ( B ) FRAXA plasmid (CGG)39 · (CCG)39 transcribed in either direction in the absence (−) or presence (+) of RNase A during the transcription reaction. All transcription reactions were subjected to further RNase A or A + H treatment to analyze hybrid formation. ( C ) Same experiment as in (B) performed with SCA1 plasmid containing a (CAG)74 · (CTG)74 repeat tract.

Techniques Used: In Vitro, Plasmid Preparation, CTG Assay

22) Product Images from "SAMMSON fosters cancer cell fitness by enhancing concertedly mitochondrial and cytosolic translation"

Article Title: SAMMSON fosters cancer cell fitness by enhancing concertedly mitochondrial and cytosolic translation

Journal: Nature structural & molecular biology

doi: 10.1038/s41594-018-0143-4

SAMMSON promotes CARF localization to the cytoplasm and its binding to p32. ( a ) Representative CARF (red) and p32 (yellow) IF in SK-MEL-28 cells 30 hours after transfection with a non-targeting GapmeR(Ctrl), GapmeR3 or GapmeR11 or untransfected (Mock). Cell nuclei are stained with DAPI (blue). Scale bar low magnification, 10 µm; high magnification, 2 µm. Representative images of three independent experiments. ( b ) CARF IP in SK-MEL-28 cells in the presence (+) or absence (-) of RNase A and western blotting. Representative image of three independent experiments. ( c ) CARF IP in LCL cells described in Figure 1a and western blotting, where (-) represents cells infected with an empty control plasmid and (+) the SAMMSON -expressing cells. Representative image of three independent experiments. ( d ) SAMMSON relative expression measured by RT-qPCR in SK-MEL-28 cells 30 hours after transfection with a non-targeting GapmeR (Ctrl) or GapmeR3 (G3). Error bars represent mean ± s.e.m.; n=3. ( e ) CARF IP in SK-MEL-28 cells treated as described in d and western blotting. Representative images of three independent experiments. (f) Representative Proximity Ligation Assay (PLA, cyan) assay using antibodies against CARF and p32 in SKMEL-28 cells 30 hours after transfection with a non-targeting (Ctrl), GapmeR3 or GapmeR11. Cell nuclei are stained with DAPI (blue). Scale bar low magnification, 10 µm; high magnification, 2 µm. Representative images of three independent experiments. ( g ) SAMMSON relative expression measured by RT-qPCR in SK-MEL-28 cells 30 hours after transfection with a non-targeting GapmeR(Ctrl), GapmeR3 (G3) or GapmeR11 (G11). Error bars represent mean ± s.e.m.; n=3. ( h ) Quantification of PLA assay described in f and g . Error bars represent mean ± s.e.m.; n=3. P values were calculated by paired two-tailed Student’s t-test. * P
Figure Legend Snippet: SAMMSON promotes CARF localization to the cytoplasm and its binding to p32. ( a ) Representative CARF (red) and p32 (yellow) IF in SK-MEL-28 cells 30 hours after transfection with a non-targeting GapmeR(Ctrl), GapmeR3 or GapmeR11 or untransfected (Mock). Cell nuclei are stained with DAPI (blue). Scale bar low magnification, 10 µm; high magnification, 2 µm. Representative images of three independent experiments. ( b ) CARF IP in SK-MEL-28 cells in the presence (+) or absence (-) of RNase A and western blotting. Representative image of three independent experiments. ( c ) CARF IP in LCL cells described in Figure 1a and western blotting, where (-) represents cells infected with an empty control plasmid and (+) the SAMMSON -expressing cells. Representative image of three independent experiments. ( d ) SAMMSON relative expression measured by RT-qPCR in SK-MEL-28 cells 30 hours after transfection with a non-targeting GapmeR (Ctrl) or GapmeR3 (G3). Error bars represent mean ± s.e.m.; n=3. ( e ) CARF IP in SK-MEL-28 cells treated as described in d and western blotting. Representative images of three independent experiments. (f) Representative Proximity Ligation Assay (PLA, cyan) assay using antibodies against CARF and p32 in SKMEL-28 cells 30 hours after transfection with a non-targeting (Ctrl), GapmeR3 or GapmeR11. Cell nuclei are stained with DAPI (blue). Scale bar low magnification, 10 µm; high magnification, 2 µm. Representative images of three independent experiments. ( g ) SAMMSON relative expression measured by RT-qPCR in SK-MEL-28 cells 30 hours after transfection with a non-targeting GapmeR(Ctrl), GapmeR3 (G3) or GapmeR11 (G11). Error bars represent mean ± s.e.m.; n=3. ( h ) Quantification of PLA assay described in f and g . Error bars represent mean ± s.e.m.; n=3. P values were calculated by paired two-tailed Student’s t-test. * P

Techniques Used: Binding Assay, Transfection, Staining, Western Blot, Infection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Proximity Ligation Assay, Two Tailed Test

23) Product Images from "Formation of telomeric repeat-containing RNA (TERRA) foci in highly proliferating mouse cerebellar neuronal progenitors and medulloblastoma"

Article Title: Formation of telomeric repeat-containing RNA (TERRA) foci in highly proliferating mouse cerebellar neuronal progenitors and medulloblastoma

Journal: Journal of Cell Science

doi: 10.1242/jcs.108118

TERRA expression can be induced in purified progenitor cells stimulated with high SHH signaling activation in vitro. ( A ) GNPs were purified from wild-type mouse P5 cerebella and cultured for 12 hours with or without SHH (600 ng/ml). TERRA RNA was analyzed by northern blotting using a 32 P-labeled (TAACCC) 4 oligonucleotide probe. 18S RNA expression is shown as an internal control for RNA loading. RNase A treatment is shown in the right panel. ( B ) Quantification of TERRA levels from at least three independent northern blot analyses using RNA isolated from GNPs treated with SHH for 12 hours or left untreated, one of which is shown in A. Bar graph represents TERRA signal intensity relative to 18S signal, and relative intensity for SHH (−) was set at 100. The P -value was calculated using a two-tailed Student's t -test. ( C ) qRT-PCR analysis for expression of Gli1 and Math1 is shown as a control for SHH activity. ΔΔCT methods relative to untreated GNPs and Gapdh were used to calculate relative RT-PCR by SHH treatment. Bar graph represents mean ± standard deviations from three independent experiments. ( D ) qRT-PCR analysis of individual TERRA expression using subtelomere-specific primers for chromosomes 2q, 11q, TeloCen, 18q or 5q as described in C.
Figure Legend Snippet: TERRA expression can be induced in purified progenitor cells stimulated with high SHH signaling activation in vitro. ( A ) GNPs were purified from wild-type mouse P5 cerebella and cultured for 12 hours with or without SHH (600 ng/ml). TERRA RNA was analyzed by northern blotting using a 32 P-labeled (TAACCC) 4 oligonucleotide probe. 18S RNA expression is shown as an internal control for RNA loading. RNase A treatment is shown in the right panel. ( B ) Quantification of TERRA levels from at least three independent northern blot analyses using RNA isolated from GNPs treated with SHH for 12 hours or left untreated, one of which is shown in A. Bar graph represents TERRA signal intensity relative to 18S signal, and relative intensity for SHH (−) was set at 100. The P -value was calculated using a two-tailed Student's t -test. ( C ) qRT-PCR analysis for expression of Gli1 and Math1 is shown as a control for SHH activity. ΔΔCT methods relative to untreated GNPs and Gapdh were used to calculate relative RT-PCR by SHH treatment. Bar graph represents mean ± standard deviations from three independent experiments. ( D ) qRT-PCR analysis of individual TERRA expression using subtelomere-specific primers for chromosomes 2q, 11q, TeloCen, 18q or 5q as described in C.

Techniques Used: Expressing, Purification, Activation Assay, In Vitro, Cell Culture, Northern Blot, Labeling, RNA Expression, Isolation, Two Tailed Test, Quantitative RT-PCR, Activity Assay, Reverse Transcription Polymerase Chain Reaction

TERRA foci formation in mouse medulloblastoma. ( A ) (Top panel) Hematoxylin and Eosin staining of a region of a Ptch1 +/− mouse cerebellum with non-tumor (left panel; indicated by an arrow) and tumor tissue (right panel; no. 6850). The normal morphology of cerebellar folia with the IGL (containing the mature granule neurons) and the Purkinje layer containing the Purkinje neurons is seen in left panel. Scale bar: 200 µm. (Lower panel) Sections of cerebellum containing both normal and tumor tissue showing the results of in situ hybridizations with specific digoxigenin-labeled RNA probes for Gli1 and Math1 . Note that Gli1 and Math1 are highly expressed in the tumor part of the cerebellum. Asterisk in left panel denotes the normal expression of Gli1 in the normal Bergmann glial cells that form a layer in this, still organized, non-tumor part of the cerebellum. Scale bars: 100 µm. ( B ) Confocal images of FISH analyses of TERRA expression in sections of the cerebellum of the same mouse as above containing both normal and tumor tissue. Note the presence of TERRA expression (strong red labeling) in the tumor as compared to the low or undetectable expression in the non-tumor cerebellar tissue. Asterisk denote non-specific TERRA signal in the Purkinje layer. RNase A treatment leads to an elimination of the tumor-specific TERRA signal (middle panels). Images were taken with 40× lens (lower panels) to reveal details of TERRA localization in the nuclei. Scale bar: 20 µm. Other images were taken with 20× lens. ( C ) Histogram comparing relative TERRA fluorescence signal intensity (FU) in non-tumor (black) vs tumor (red) tissue, analyzed by RNA FISH and ImageProPlus software. ( D ) TERRA expression measured as total mean fluorescence intensity (FU). Values are means ± standard error from three independent experiments. ( E ) Quantification of cells with ≧1 TERRA signals of mean intensity > 40 FU. More than 800 nuclei from at least three independent experiments were counted for quantification. The P -value was calculated using a two-tailed Student's t -test in all cases. ( F ) Confocal images of FISH analyses of TERRA expression in tumor (top panel) or non-tumor (middle panel) using an Alexa-Fluor-488-conjugated DNA oligonucleotide probe (TAACCC) 7 (top two panels) or a control mutant probe (TAACAC) 7 containing both non-tumor and tumor tissue were used. Scale bar: 20 µm.
Figure Legend Snippet: TERRA foci formation in mouse medulloblastoma. ( A ) (Top panel) Hematoxylin and Eosin staining of a region of a Ptch1 +/− mouse cerebellum with non-tumor (left panel; indicated by an arrow) and tumor tissue (right panel; no. 6850). The normal morphology of cerebellar folia with the IGL (containing the mature granule neurons) and the Purkinje layer containing the Purkinje neurons is seen in left panel. Scale bar: 200 µm. (Lower panel) Sections of cerebellum containing both normal and tumor tissue showing the results of in situ hybridizations with specific digoxigenin-labeled RNA probes for Gli1 and Math1 . Note that Gli1 and Math1 are highly expressed in the tumor part of the cerebellum. Asterisk in left panel denotes the normal expression of Gli1 in the normal Bergmann glial cells that form a layer in this, still organized, non-tumor part of the cerebellum. Scale bars: 100 µm. ( B ) Confocal images of FISH analyses of TERRA expression in sections of the cerebellum of the same mouse as above containing both normal and tumor tissue. Note the presence of TERRA expression (strong red labeling) in the tumor as compared to the low or undetectable expression in the non-tumor cerebellar tissue. Asterisk denote non-specific TERRA signal in the Purkinje layer. RNase A treatment leads to an elimination of the tumor-specific TERRA signal (middle panels). Images were taken with 40× lens (lower panels) to reveal details of TERRA localization in the nuclei. Scale bar: 20 µm. Other images were taken with 20× lens. ( C ) Histogram comparing relative TERRA fluorescence signal intensity (FU) in non-tumor (black) vs tumor (red) tissue, analyzed by RNA FISH and ImageProPlus software. ( D ) TERRA expression measured as total mean fluorescence intensity (FU). Values are means ± standard error from three independent experiments. ( E ) Quantification of cells with ≧1 TERRA signals of mean intensity > 40 FU. More than 800 nuclei from at least three independent experiments were counted for quantification. The P -value was calculated using a two-tailed Student's t -test in all cases. ( F ) Confocal images of FISH analyses of TERRA expression in tumor (top panel) or non-tumor (middle panel) using an Alexa-Fluor-488-conjugated DNA oligonucleotide probe (TAACCC) 7 (top two panels) or a control mutant probe (TAACAC) 7 containing both non-tumor and tumor tissue were used. Scale bar: 20 µm.

Techniques Used: Staining, In Situ, Labeling, Expressing, Fluorescence In Situ Hybridization, Fluorescence, Software, Two Tailed Test, Mutagenesis

24) Product Images from "Two Forms of Activation-Induced Cytidine Deaminase Differing in Their Ability to Bind Agarose"

Article Title: Two Forms of Activation-Induced Cytidine Deaminase Differing in Their Ability to Bind Agarose

Journal: PLoS ONE

doi: 10.1371/journal.pone.0008883

AID's binding to agarose is not RNase sensitive. (A) Agarose binding to AID in the absence (\) and presence of RNase A at various concentrations: low (L) (1 µg/ml), medium (M) (100 µg/ml), or high (H) (1 mg/ml). GFP control (–AID) or AID-transfected (+ AID) human embryonic kidney cell lysates were treated with RNase and incubated with two successive rounds of agarose beads (1st and 2nd Round). The washed 1st and 2nd round beads and the unbound lysate after the second bead incubation (Supernatant) were western blotted and probed with a monoclonal anti-AID antibody. Input, 1st Round, 2nd Round, and Supernatant, all equivalent to 5×10 5 cells. The positions of the molecular mass standards are indicated next to the blots. (B) Incubation of 1 µg RNA in the absence (\) and presence of RNase A at various concentrations: low (L) (1 µg/ml), medium (M) (100 µg/ml), or high (H) (1 mg/ml). The positions of the size standards and the 18S and 28S rRNA are indicated next to the agarose gel.
Figure Legend Snippet: AID's binding to agarose is not RNase sensitive. (A) Agarose binding to AID in the absence (\) and presence of RNase A at various concentrations: low (L) (1 µg/ml), medium (M) (100 µg/ml), or high (H) (1 mg/ml). GFP control (–AID) or AID-transfected (+ AID) human embryonic kidney cell lysates were treated with RNase and incubated with two successive rounds of agarose beads (1st and 2nd Round). The washed 1st and 2nd round beads and the unbound lysate after the second bead incubation (Supernatant) were western blotted and probed with a monoclonal anti-AID antibody. Input, 1st Round, 2nd Round, and Supernatant, all equivalent to 5×10 5 cells. The positions of the molecular mass standards are indicated next to the blots. (B) Incubation of 1 µg RNA in the absence (\) and presence of RNase A at various concentrations: low (L) (1 µg/ml), medium (M) (100 µg/ml), or high (H) (1 mg/ml). The positions of the size standards and the 18S and 28S rRNA are indicated next to the agarose gel.

Techniques Used: Binding Assay, Transfection, Incubation, Western Blot, Agarose Gel Electrophoresis

25) Product Images from "Type I Interferon Production Induced by Streptococcus pyogenes-Derived Nucleic Acids Is Required for Host Protection"

Article Title: Type I Interferon Production Induced by Streptococcus pyogenes-Derived Nucleic Acids Is Required for Host Protection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1001345

S. pyogenes -derived RNA and DNA delivered to endosomes induce IFN-β in cDCs and BMDMs, respectively. S. pyogenes  cells were sonicated and the extracts were treated with either DNase I, RNase A, Proteinase K, or left untreated (control extract). The extracts and a reagent control were delivered into cDCs derived from TLR7 -/-  ( A ), MyD88 -/-  ( B ) and MAVS -/-  ( C ) or control (WT) mice using DOTAP. After stimulation for 6 h, supernatants were collected and IFN-β release was measured using ELISA. Values represent mean ± SD; n = 3. ( D ) BMDMs from TLR9 -/-  and control (WT) mice were transfected with streptococcal extracts as described in (A). After 8 h supernatants were collected for IFN-β measurements by ELISA. ( E)  Purified RNA (5 µg) from  S. pyogenes  (SP), Group B streptococcus (GBS),  Staphylococcus aureus  (SA),  Listeria monocytogenes  (LM) and RAW 264.7 cells (RAW) were delivered into cDCs using DOTAP. After stimulation for 6 h supernatants were collected and IFN-β release was measured. Values represent mean ± SD; n = 3. ( F ) DNA (5 µg) from same organisms as in (E) as well as poly(dA:dT) were delivered into BMDMs and 8 h later IFN-β release was determined as in (E).
Figure Legend Snippet: S. pyogenes -derived RNA and DNA delivered to endosomes induce IFN-β in cDCs and BMDMs, respectively. S. pyogenes cells were sonicated and the extracts were treated with either DNase I, RNase A, Proteinase K, or left untreated (control extract). The extracts and a reagent control were delivered into cDCs derived from TLR7 -/- ( A ), MyD88 -/- ( B ) and MAVS -/- ( C ) or control (WT) mice using DOTAP. After stimulation for 6 h, supernatants were collected and IFN-β release was measured using ELISA. Values represent mean ± SD; n = 3. ( D ) BMDMs from TLR9 -/- and control (WT) mice were transfected with streptococcal extracts as described in (A). After 8 h supernatants were collected for IFN-β measurements by ELISA. ( E) Purified RNA (5 µg) from S. pyogenes (SP), Group B streptococcus (GBS), Staphylococcus aureus (SA), Listeria monocytogenes (LM) and RAW 264.7 cells (RAW) were delivered into cDCs using DOTAP. After stimulation for 6 h supernatants were collected and IFN-β release was measured. Values represent mean ± SD; n = 3. ( F ) DNA (5 µg) from same organisms as in (E) as well as poly(dA:dT) were delivered into BMDMs and 8 h later IFN-β release was determined as in (E).

Techniques Used: Derivative Assay, Sonication, Mouse Assay, Enzyme-linked Immunosorbent Assay, Transfection, Purification

26) Product Images from "Structural Protein Requirements in Equine Arteritis Virus Assembly"

Article Title: Structural Protein Requirements in Equine Arteritis Virus Assembly

Journal: Journal of Virology

doi: 10.1128/JVI.78.23.13019-13027.2004

Packaging of genomic EAV RNA in viral particles. KO-E particles were treated with DNase I and RNase A in the presence or absence of SDS and Triton X-100. After inactivation of these enzymes by proteinase K, RNA was isolated from each sample and used for RT-PCR with primers located at either side of the StuI site that was introduced as a marker mutation in the KO-E RNA. When indicated, the PCR fragments were digested with StuI. The reaction products were analyzed by agarose gel electrophoresis. The numbers on the right refer to the anticipated sizes of the PCR fragments before and after StuI digestion. On the left, the positions and sizes of marker DNA fragments that were analyzed in the same gel are indicated (in nucleotides).
Figure Legend Snippet: Packaging of genomic EAV RNA in viral particles. KO-E particles were treated with DNase I and RNase A in the presence or absence of SDS and Triton X-100. After inactivation of these enzymes by proteinase K, RNA was isolated from each sample and used for RT-PCR with primers located at either side of the StuI site that was introduced as a marker mutation in the KO-E RNA. When indicated, the PCR fragments were digested with StuI. The reaction products were analyzed by agarose gel electrophoresis. The numbers on the right refer to the anticipated sizes of the PCR fragments before and after StuI digestion. On the left, the positions and sizes of marker DNA fragments that were analyzed in the same gel are indicated (in nucleotides).

Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction, Marker, Mutagenesis, Polymerase Chain Reaction, Agarose Gel Electrophoresis

27) Product Images from "The spliceosome-associated protein Nrl1 suppresses homologous recombination-dependent R-loop formation in fission yeast"

Article Title: The spliceosome-associated protein Nrl1 suppresses homologous recombination-dependent R-loop formation in fission yeast

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv1473

Nrl1 associates with spliceosome proteins. ( A ) Comparison of S. pombe (Spo) Nrl1, C. elegans (Cele) and Homo sapiens (Hs) NRDE-2 like proteins. HAT = halfa-tetratricopeptide domain. ( B ) Nrl1-associated proteins were isolated from exponentially growing WT cells harboring a TAP-tagged nrl1 allele (17106) in the presence or absence of RNase A by tandem affinity purification and identified by mass spectrometry (MS) analysis. A core complex consisting of Nrl1, Mtl1, Ctr1, Ntr2 and Syf3 associates through RNA-dependent interactions with the spliceosome. Blue: Nrl1; pink: splicing factors; orange: mRNA processing factors. Only the top 30 proteins based on spectral counting are shown. ( C ) Yeast-two-hybrid interaction map of Nrl1 interactome. All constructs were made using vectors supplied in the Matchmaker GAL4 2-hybrid system (Clontech). Two-hybrid DNA-binding domain (BD) constructs were made in the pAS2–1 vector containing the TRP1 gene for selection on tryptophan-deficient media and activation domain (AD) constructs were made in the pGADT7 vector containing the LEU2 gene for selection on leucine-deficient media.
Figure Legend Snippet: Nrl1 associates with spliceosome proteins. ( A ) Comparison of S. pombe (Spo) Nrl1, C. elegans (Cele) and Homo sapiens (Hs) NRDE-2 like proteins. HAT = halfa-tetratricopeptide domain. ( B ) Nrl1-associated proteins were isolated from exponentially growing WT cells harboring a TAP-tagged nrl1 allele (17106) in the presence or absence of RNase A by tandem affinity purification and identified by mass spectrometry (MS) analysis. A core complex consisting of Nrl1, Mtl1, Ctr1, Ntr2 and Syf3 associates through RNA-dependent interactions with the spliceosome. Blue: Nrl1; pink: splicing factors; orange: mRNA processing factors. Only the top 30 proteins based on spectral counting are shown. ( C ) Yeast-two-hybrid interaction map of Nrl1 interactome. All constructs were made using vectors supplied in the Matchmaker GAL4 2-hybrid system (Clontech). Two-hybrid DNA-binding domain (BD) constructs were made in the pAS2–1 vector containing the TRP1 gene for selection on tryptophan-deficient media and activation domain (AD) constructs were made in the pGADT7 vector containing the LEU2 gene for selection on leucine-deficient media.

Techniques Used: HAT Assay, Isolation, Affinity Purification, Mass Spectrometry, Construct, Binding Assay, Plasmid Preparation, Selection, Activation Assay

28) Product Images from "Nuclear ARVCF Protein Binds Splicing Factors and Contributes to the Regulation of Alternative Splicing *"

Article Title: Nuclear ARVCF Protein Binds Splicing Factors and Contributes to the Regulation of Alternative Splicing *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.530717

ARVCF is part of an RNP containing classical mRNAs and other RNA-binding proteins. Sucrose gradient centrifugation with lysates from HEK 293 cells was done with the addition of RNasin ( A ) or RNase A ( B ). Western blot was performed with antibodies against
Figure Legend Snippet: ARVCF is part of an RNP containing classical mRNAs and other RNA-binding proteins. Sucrose gradient centrifugation with lysates from HEK 293 cells was done with the addition of RNasin ( A ) or RNase A ( B ). Western blot was performed with antibodies against

Techniques Used: RNA Binding Assay, Gradient Centrifugation, Western Blot

29) Product Images from "Biochemical Analysis of Yeast Suppressor of Ty 4/5 (Spt4/5) Reveals the Importance of Nucleic Acid Interactions in the Prevention of RNA Polymerase II Arrest *Biochemical Analysis of Yeast Suppressor of Ty 4/5 (Spt4/5) Reveals the Importance of Nucleic Acid Interactions in the Prevention of RNA Polymerase II Arrest * ♦"

Article Title: Biochemical Analysis of Yeast Suppressor of Ty 4/5 (Spt4/5) Reveals the Importance of Nucleic Acid Interactions in the Prevention of RNA Polymerase II Arrest *Biochemical Analysis of Yeast Suppressor of Ty 4/5 (Spt4/5) Reveals the Importance of Nucleic Acid Interactions in the Prevention of RNA Polymerase II Arrest * ♦

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.716001

Spt4/5 contacts the emerging transcript. A,  EMSA analysis of EC42 treated with ( lanes 7–12 ) and without RNase I ( lanes 1–6 ). Spt4/5 ΔCTR was titrated in and complexes were resolved on native polyacrylamide gels.  B,  RNase I footprint
Figure Legend Snippet: Spt4/5 contacts the emerging transcript. A, EMSA analysis of EC42 treated with ( lanes 7–12 ) and without RNase I ( lanes 1–6 ). Spt4/5 ΔCTR was titrated in and complexes were resolved on native polyacrylamide gels. B, RNase I footprint

Techniques Used:

30) Product Images from "A Cross-chiral RNA Polymerase Ribozyme"

Article Title: A Cross-chiral RNA Polymerase Ribozyme

Journal: Nature

doi: 10.1038/nature13900

Analysis of the regiospecificity of ligation. a, D-RNA substrates and template for ligation, catalyzed by the L-16.12tx enzyme. Dot indicates the ligation junction, which is also the site for RNase A cleavage that is closest to the 3′ end of the ligated product. The downstream substrate is labeled at the 3′ end with fluorescein (circled F). b, RNase A digestion of the ligated products (LP) in comparison to authentic all-3′,5′-linked RNA of the same sequence (S10). Reaction conditions: 0–100 μg/μL RNase A, 2 μM RNA, 50 mM Tris (pH 7.6), 23 °C, 1 min.
Figure Legend Snippet: Analysis of the regiospecificity of ligation. a, D-RNA substrates and template for ligation, catalyzed by the L-16.12tx enzyme. Dot indicates the ligation junction, which is also the site for RNase A cleavage that is closest to the 3′ end of the ligated product. The downstream substrate is labeled at the 3′ end with fluorescein (circled F). b, RNase A digestion of the ligated products (LP) in comparison to authentic all-3′,5′-linked RNA of the same sequence (S10). Reaction conditions: 0–100 μg/μL RNase A, 2 μM RNA, 50 mM Tris (pH 7.6), 23 °C, 1 min.

Techniques Used: Ligation, Labeling, Sequencing

31) Product Images from "eIF4GI Facilitates the MicroRNA-Mediated Gene Silencing"

Article Title: eIF4GI Facilitates the MicroRNA-Mediated Gene Silencing

Journal: PLoS ONE

doi: 10.1371/journal.pone.0055725

eIF4GI associates with Ago2. ( A ) Schematic diagram of human eIF4GI. ‘4E’ means ‘the eIF4E-binding motif’. Plasmids were constructed for expression of various Flag-tagged eIF4GI fragments in human cells. ( B ) The N-terminal and middle domains of eIF4GI participate in the eIF4GI-Ago2 association. Plasmids expressing Flag-tagged eIF4GI variants and myc-tagged full-length Ago2 were co-transfected in 293FT cells, and their associations were examined by Flag immunoprecipitation (Flag-IP) with the Flag-resin. The levels of Ago2 and the eIF4GI mutants (left panel) and the amount of co-precipitated Ago2 (right panel) were monitored by Western blotting using the indicated antibodies. ( C ) Schematic diagram of the N-terminal constructs of eIF4GI for fine mapping of the region required for the association with Ago2. ( D ) Determination of the Ago2-associated region in eIF4GI. WCEs from 293FT cells expressing myc-tagged full-length Ago2 and N-terminal variants of eIF4GI serially deleted from the C- or N-termini were subjected to Flag-IP. The expressions of Ago2 and the eIF4GI derivatives (lower panel) and the amount of precipitated Ago2 (upper panel) were examined using the indicated antibodies. ( E ) RNA-independent association of Ago2 with eIF4GI (aa 42–202). WCEs from 293FT cells expressing myc-tagged Ago2 and Flag-tagged eIF4GI-NtP were treated with (lanes 2 and 4) and without (lanes 1 and 3) RNase A and subjected to Flag-IP.
Figure Legend Snippet: eIF4GI associates with Ago2. ( A ) Schematic diagram of human eIF4GI. ‘4E’ means ‘the eIF4E-binding motif’. Plasmids were constructed for expression of various Flag-tagged eIF4GI fragments in human cells. ( B ) The N-terminal and middle domains of eIF4GI participate in the eIF4GI-Ago2 association. Plasmids expressing Flag-tagged eIF4GI variants and myc-tagged full-length Ago2 were co-transfected in 293FT cells, and their associations were examined by Flag immunoprecipitation (Flag-IP) with the Flag-resin. The levels of Ago2 and the eIF4GI mutants (left panel) and the amount of co-precipitated Ago2 (right panel) were monitored by Western blotting using the indicated antibodies. ( C ) Schematic diagram of the N-terminal constructs of eIF4GI for fine mapping of the region required for the association with Ago2. ( D ) Determination of the Ago2-associated region in eIF4GI. WCEs from 293FT cells expressing myc-tagged full-length Ago2 and N-terminal variants of eIF4GI serially deleted from the C- or N-termini were subjected to Flag-IP. The expressions of Ago2 and the eIF4GI derivatives (lower panel) and the amount of precipitated Ago2 (upper panel) were examined using the indicated antibodies. ( E ) RNA-independent association of Ago2 with eIF4GI (aa 42–202). WCEs from 293FT cells expressing myc-tagged Ago2 and Flag-tagged eIF4GI-NtP were treated with (lanes 2 and 4) and without (lanes 1 and 3) RNase A and subjected to Flag-IP.

Techniques Used: Binding Assay, Construct, Expressing, Transfection, Immunoprecipitation, Western Blot

Human Ago associates with the cap-binding complex. ( A ) The cap-association of endogenous Ago2 proteins from HeLa cells were examined by a cap-pulldown assay, using 2 mg of whole-cell extracts (WCEs) for incubation with either control-resin (lane 6) or cap-resin in the presence (lane 8) or absence (lane 7) of the cap analog. ( B ) The cap-association of miRNAs. WCEs from HeLa cells (2 mg) were subjected to a cap-pulldown assay, and the cap-associated RNAs were extracted and subjected to UREA-PAGE followed by Northern blotting using radiolabeled probes against the indicated miRNAs. For comparison, various amounts equal to 4.4–0.8% of the total RNAs contained in WCEs used for the cap-pulldown assays (∼10–2 µg each) were loaded in lanes 1–5. ( C ) The cap-associations of ectopically expressed proteins were monitored as in panel A , except for using 2 mg of WCEs from 293FT cells transfected with plasmids expressing Flag-tagged Ago1, Ago2 or Dicer. ( D ) Cap-pulldown assays were done using 2 mg of WCEs from 293FT cells expressing Flag-Ago2 with 200 µM of G(5′)ppp(3′)G (lane 3) or m 7 G(5′)ppp(3′)G (lane 4). ( E ) The RNA-independent cap-association of Ago2. 2 mg of WCEs from 293FT cells ectopically expressing myc-tagged Ago2 were treated with (lanes 2 and 5) or without (lanes 1, 3 and 4) RNase A and subjected to cap-pulldown assays. In panels A and C , various amounts corresponding to 2–0.4% of WCEs used in the pulldown assay were loaded in lanes 1–5 for comparison. In panels A , C , D and E , Western blot analyses were performed using the indicated antibodies.
Figure Legend Snippet: Human Ago associates with the cap-binding complex. ( A ) The cap-association of endogenous Ago2 proteins from HeLa cells were examined by a cap-pulldown assay, using 2 mg of whole-cell extracts (WCEs) for incubation with either control-resin (lane 6) or cap-resin in the presence (lane 8) or absence (lane 7) of the cap analog. ( B ) The cap-association of miRNAs. WCEs from HeLa cells (2 mg) were subjected to a cap-pulldown assay, and the cap-associated RNAs were extracted and subjected to UREA-PAGE followed by Northern blotting using radiolabeled probes against the indicated miRNAs. For comparison, various amounts equal to 4.4–0.8% of the total RNAs contained in WCEs used for the cap-pulldown assays (∼10–2 µg each) were loaded in lanes 1–5. ( C ) The cap-associations of ectopically expressed proteins were monitored as in panel A , except for using 2 mg of WCEs from 293FT cells transfected with plasmids expressing Flag-tagged Ago1, Ago2 or Dicer. ( D ) Cap-pulldown assays were done using 2 mg of WCEs from 293FT cells expressing Flag-Ago2 with 200 µM of G(5′)ppp(3′)G (lane 3) or m 7 G(5′)ppp(3′)G (lane 4). ( E ) The RNA-independent cap-association of Ago2. 2 mg of WCEs from 293FT cells ectopically expressing myc-tagged Ago2 were treated with (lanes 2 and 5) or without (lanes 1, 3 and 4) RNase A and subjected to cap-pulldown assays. In panels A and C , various amounts corresponding to 2–0.4% of WCEs used in the pulldown assay were loaded in lanes 1–5 for comparison. In panels A , C , D and E , Western blot analyses were performed using the indicated antibodies.

Techniques Used: Binding Assay, Incubation, Polyacrylamide Gel Electrophoresis, Northern Blot, Transfection, Expressing, Western Blot

32) Product Images from "LINE-1 Retroelements Complexed and Inhibited by Activation Induced Cytidine Deaminase"

Article Title: LINE-1 Retroelements Complexed and Inhibited by Activation Induced Cytidine Deaminase

Journal: PLoS ONE

doi: 10.1371/journal.pone.0049358

AID forms high-molecular-mass complexes in the cytoplasm. Fractionation according to size by gel filtration of A3G (A) and AID (B and C), followed by Western blot analysis. Numbers above panels, fractions of separation by FPLC; numbers to the left of panels, molecular mass standards (in kDa) of the SDS gel run; numbers below panels, molecular mass standards (in kDa) of the fractions of the FPLC run. ( A ) Western blots of fractions of FPLC eluates were developed with polyclonal antibody to A3G. Input, untreated lysate of A3G-positive cells; input + RNase, RNase A-treated lysate of A3G-positive cells; A3G minus, lysate of A3G-negative cells; H9, A3G-positive cell line; fractions 3–16, fractionated lysates of LPS- plus IL-4-activated B lymphocytes from human A3 transgenic mice. Upper panel, untreated cell lysate (devoid of nuclei); lower panel, treated with RNase A before fractionation on FPLC. ( B and C ) Western blots of fractions of FPLC eluates developed with monoclonal antibody to AID. AID minus, lysate of AID-negative HeLa cells; AID plus, lysate of AID-positive HeLa cells; fractions 3–15, fractionated lysates of LPS- plus IL-4-activated B lymphocytes from AID-sufficient mice (B) and of AID-positive HeLa cells (C). Top panel, untreated cell lysate (devoid of nuclei); middle panel, treated with RNase A before fractionation on FPLC; bottom panel, treated with RNase A and RNase inhibitors before fractionation. Concomitant with RNase A treatment, proteins were somewhat digested for unknown reasons.
Figure Legend Snippet: AID forms high-molecular-mass complexes in the cytoplasm. Fractionation according to size by gel filtration of A3G (A) and AID (B and C), followed by Western blot analysis. Numbers above panels, fractions of separation by FPLC; numbers to the left of panels, molecular mass standards (in kDa) of the SDS gel run; numbers below panels, molecular mass standards (in kDa) of the fractions of the FPLC run. ( A ) Western blots of fractions of FPLC eluates were developed with polyclonal antibody to A3G. Input, untreated lysate of A3G-positive cells; input + RNase, RNase A-treated lysate of A3G-positive cells; A3G minus, lysate of A3G-negative cells; H9, A3G-positive cell line; fractions 3–16, fractionated lysates of LPS- plus IL-4-activated B lymphocytes from human A3 transgenic mice. Upper panel, untreated cell lysate (devoid of nuclei); lower panel, treated with RNase A before fractionation on FPLC. ( B and C ) Western blots of fractions of FPLC eluates developed with monoclonal antibody to AID. AID minus, lysate of AID-negative HeLa cells; AID plus, lysate of AID-positive HeLa cells; fractions 3–15, fractionated lysates of LPS- plus IL-4-activated B lymphocytes from AID-sufficient mice (B) and of AID-positive HeLa cells (C). Top panel, untreated cell lysate (devoid of nuclei); middle panel, treated with RNase A before fractionation on FPLC; bottom panel, treated with RNase A and RNase inhibitors before fractionation. Concomitant with RNase A treatment, proteins were somewhat digested for unknown reasons.

Techniques Used: Fractionation, Filtration, Western Blot, Fast Protein Liquid Chromatography, SDS-Gel, Transgenic Assay, Mouse Assay

33) Product Images from "Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats"

Article Title: Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq935

RNA–DNA hybrid formation at a (CGG)39 · (CCG)39 FRAXA template. When the template DNA is transcribed with T7 RNA polymerase, heterogeneous RNA is produced generating a smear (Transcription lane). Treatment with RNase H alone which is specific to RNA base paired to template DNA digests only RNA that is base paired to its template DNA. Treatment with RNase A, which is specific to single-stranded RNA, digests all free, single-stranded RNA leaving template DNA and RNA:DNA hybrid structures. Note that RNA–DNA hybrids migrate more slowly than supercoiled DNA (as indicated schematically, RNA is in light blue). Hybrid structures generate a smear due to their heterogeneous sizes. With a larger RNA component, the DNA is open to a greater degree (more relaxed), hence migration is closer to open circular DNA. Treatment of the hybrids with RNase H along with RNase A removes any hybrids formed as well as transcript generated in the transcription reaction leaving only input template DNA. When transcription followed by RNase H or RNase A treatment alone or in combination is performed on an empty Bluescript vector [pBlueKS(+)], there is negligible hybrid formation.
Figure Legend Snippet: RNA–DNA hybrid formation at a (CGG)39 · (CCG)39 FRAXA template. When the template DNA is transcribed with T7 RNA polymerase, heterogeneous RNA is produced generating a smear (Transcription lane). Treatment with RNase H alone which is specific to RNA base paired to template DNA digests only RNA that is base paired to its template DNA. Treatment with RNase A, which is specific to single-stranded RNA, digests all free, single-stranded RNA leaving template DNA and RNA:DNA hybrid structures. Note that RNA–DNA hybrids migrate more slowly than supercoiled DNA (as indicated schematically, RNA is in light blue). Hybrid structures generate a smear due to their heterogeneous sizes. With a larger RNA component, the DNA is open to a greater degree (more relaxed), hence migration is closer to open circular DNA. Treatment of the hybrids with RNase H along with RNase A removes any hybrids formed as well as transcript generated in the transcription reaction leaving only input template DNA. When transcription followed by RNase H or RNase A treatment alone or in combination is performed on an empty Bluescript vector [pBlueKS(+)], there is negligible hybrid formation.

Techniques Used: Produced, Migration, Generated, Plasmid Preparation

Quantification of relative RNA–DNA hybrid formation at increasing repeat lengths. ( A ) In vitro transcription reactions were performed on FRAXA plasmids bearing repeat tracts of (CGG)17 · (CCG)17, (CGG)39 · (CCG)39 and (CGG)53 · (CCG)53 following which samples were treated with RNase A or A+H. Hybrid formation was quantified by densitometry analysis using image quant by measuring the proportion of products that migrate between open circular and supercoiled position (indicated by ‘R’) divided by the total products below open circular including supercoiled. RNase A+H treated samples were used to determine the position of the supercoiled DNA for each repeat length. To the left of the graph is a representative gel used to quantify relative hybrid formation. The RNA indicated in the graph represents the RNA component bound in the RNA:DNA hybrid. Error bars are derived from three separate experiments ( N =3). Using the t -test to compare R-loop formation revealed a significant difference between the 17 and 53 rCGG R-loop ( P =0.0413) as well as the 17 and 53 rCCG R-loop ( P =0.0092). ( B ) The same analysis was performed as in (A) but for SCA1 plasmids bearing repeat tracts of (CAG)30 · (CTG)30, (CAG) 49 · (CTG)49 and (CAG)74 · (CTG)74. Using the t -test to compare R-loop formation between each of the (CAG) · (CTG) repeat lengths did not reveal any statistically significant differences.
Figure Legend Snippet: Quantification of relative RNA–DNA hybrid formation at increasing repeat lengths. ( A ) In vitro transcription reactions were performed on FRAXA plasmids bearing repeat tracts of (CGG)17 · (CCG)17, (CGG)39 · (CCG)39 and (CGG)53 · (CCG)53 following which samples were treated with RNase A or A+H. Hybrid formation was quantified by densitometry analysis using image quant by measuring the proportion of products that migrate between open circular and supercoiled position (indicated by ‘R’) divided by the total products below open circular including supercoiled. RNase A+H treated samples were used to determine the position of the supercoiled DNA for each repeat length. To the left of the graph is a representative gel used to quantify relative hybrid formation. The RNA indicated in the graph represents the RNA component bound in the RNA:DNA hybrid. Error bars are derived from three separate experiments ( N =3). Using the t -test to compare R-loop formation revealed a significant difference between the 17 and 53 rCGG R-loop ( P =0.0413) as well as the 17 and 53 rCCG R-loop ( P =0.0092). ( B ) The same analysis was performed as in (A) but for SCA1 plasmids bearing repeat tracts of (CAG)30 · (CTG)30, (CAG) 49 · (CTG)49 and (CAG)74 · (CTG)74. Using the t -test to compare R-loop formation between each of the (CAG) · (CTG) repeat lengths did not reveal any statistically significant differences.

Techniques Used: In Vitro, Derivative Assay, CTG Assay

Effect of trinucleotide repeat interruptions on RNA–DNA hybrid formation. ( A ) In vitro transcription followed by RNase A or A + H treatment was performed with either pure (39p) FRAXA plasmids (CGG)39 · (CCG)39 or interrupted plasmids (39i) [(CGG)9(AGG) (CGG)9(AGG)(CGG)9(AGG)(CGG)9] · [(CCG)9(CCT) (CCG)9(CCT)(CCG)9(CCT)(CCG)9] as indicated. Repeat tract configurations are schematically presented where hollow dots are the CGG repeat units and the filled dots are the AGG interruptions. R-loops are indicated as ‘R’. ( B ) In vitro transcription followed by RNase A or A+H treatment was performed with either pure (49p) SCA1 plasmids (CAG)49 · (CTG)49 or interrupted plasmids (44i) [(CAG)12(CAT)(CAG)(CAT)(CAG)12(CAT)(CAG)(CAT)(CAG)14] · [(CTG)14(ATG)(CTG)(ATG)(CTG)12(ATG)(CAG)(ATG)(CTG)12] as indicated schematically where hollow dots are the CAG repeat units and the filled dots are the CAT interruptions.
Figure Legend Snippet: Effect of trinucleotide repeat interruptions on RNA–DNA hybrid formation. ( A ) In vitro transcription followed by RNase A or A + H treatment was performed with either pure (39p) FRAXA plasmids (CGG)39 · (CCG)39 or interrupted plasmids (39i) [(CGG)9(AGG) (CGG)9(AGG)(CGG)9(AGG)(CGG)9] · [(CCG)9(CCT) (CCG)9(CCT)(CCG)9(CCT)(CCG)9] as indicated. Repeat tract configurations are schematically presented where hollow dots are the CGG repeat units and the filled dots are the AGG interruptions. R-loops are indicated as ‘R’. ( B ) In vitro transcription followed by RNase A or A+H treatment was performed with either pure (49p) SCA1 plasmids (CAG)49 · (CTG)49 or interrupted plasmids (44i) [(CAG)12(CAT)(CAG)(CAT)(CAG)12(CAT)(CAG)(CAT)(CAG)14] · [(CTG)14(ATG)(CTG)(ATG)(CTG)12(ATG)(CAG)(ATG)(CTG)12] as indicated schematically where hollow dots are the CAG repeat units and the filled dots are the CAT interruptions.

Techniques Used: In Vitro, CTG Assay

Identification of R-loop structures formed in expanded DM1 (CTG)130 · (CAG)130 plasmids using EM following in vitro transcription and treatment with RNase A and SSB protein. SSB proteins bind to the looped-out non-template DNA in an R-loop structure. Thus, each R-loop structure is visualized as a loop within the DNA template as indicated by black arrowheads. ( A ) R-loops formed by using SP6 RNA polymerase, producing an rCAG:dCTG hybrid. ( B ) R-loops formed by using T7 RNA polymerase, producing an rCUG:dCAG hybrid.
Figure Legend Snippet: Identification of R-loop structures formed in expanded DM1 (CTG)130 · (CAG)130 plasmids using EM following in vitro transcription and treatment with RNase A and SSB protein. SSB proteins bind to the looped-out non-template DNA in an R-loop structure. Thus, each R-loop structure is visualized as a loop within the DNA template as indicated by black arrowheads. ( A ) R-loops formed by using SP6 RNA polymerase, producing an rCAG:dCTG hybrid. ( B ) R-loops formed by using T7 RNA polymerase, producing an rCUG:dCAG hybrid.

Techniques Used: CTG Assay, In Vitro

RNA–DNA hybrid formation during in vitro transcription of trinucleotide repeat-containing plasmids. ( A ) In vitro transcription of SCA1 plasmid containing (CAG)74 · (CTG)74 and FRAXA plasmid containing (CGG)39 · (CCG)39 repeats in either direction using T3 or T7 RNA polymerase. The repeat sequence contained within the RNA produced and bound in the hybrid is indicated below the transcribed template. Samples following transcription were subsequently treated with either RNase A or A+H as indicated to observe hybrid formation. R-loops are indicated on the gel as ‘R’. ( B ) Exact same reactions and gel conditions as in (A) but transcription was performed in the presence of 3.5 µCi [α- 32 P]-rCTP. Gel was dried and exposed to X-ray film as outlined in ‘Materials and Methods’ section.
Figure Legend Snippet: RNA–DNA hybrid formation during in vitro transcription of trinucleotide repeat-containing plasmids. ( A ) In vitro transcription of SCA1 plasmid containing (CAG)74 · (CTG)74 and FRAXA plasmid containing (CGG)39 · (CCG)39 repeats in either direction using T3 or T7 RNA polymerase. The repeat sequence contained within the RNA produced and bound in the hybrid is indicated below the transcribed template. Samples following transcription were subsequently treated with either RNase A or A+H as indicated to observe hybrid formation. R-loops are indicated on the gel as ‘R’. ( B ) Exact same reactions and gel conditions as in (A) but transcription was performed in the presence of 3.5 µCi [α- 32 P]-rCTP. Gel was dried and exposed to X-ray film as outlined in ‘Materials and Methods’ section.

Techniques Used: In Vitro, Plasmid Preparation, CTG Assay, Sequencing, Produced

Effect of convergent simultaneous bidirectional or serial transcription on R-loop formation. ( A ) In vitro transcription of FRAXA template (CGG)39 · (CCG)39 with either T3 or T7 RNA polymerase alone (rCCG or rCGG, respectively), or simultaneously (rCCG with rCGG) or serially: rCCG transcription then phenol chloroform extraction followed by rCGG transcription (rCCG then rCGG), and vice versa (rCGG then rCCG). R-loops are indicated as ‘R’. Note that in the case of bidirectional or serial transcription, complementary RNA is produced forming dsRNA as indicated on the gel by ‘*’. These products are not present in transcription reactions occurring in one direction. ( B ) Same as in (A) except in vitro transcription reactions were performed on a DM1 (CAG)79 · (CTG)79 template. ( C ) EM analysis of convergent transcription reaction products from DM1 (CAG)79 · (CTG)79 templates. Samples were transcribed convergently using T3 and T7 RNA polymerase promoters simultaneously then the products were treated with RNAse A and prepared for EM as described in the ‘Materials and Methods’ section (rCUGand rCAG RNase A). Samples were also subjected to RNase H treatment along with RNase A for comparison (rCUG and rCAG RNase A, H). Transcription was also performed on the same template in a single direction for further comparison (rCUG RNase A). The products observed for each type of transcription reaction is shown as a percentage of the total number of molecules analyzed. At least 100 molecules were analyzed for each type of transcription reaction.
Figure Legend Snippet: Effect of convergent simultaneous bidirectional or serial transcription on R-loop formation. ( A ) In vitro transcription of FRAXA template (CGG)39 · (CCG)39 with either T3 or T7 RNA polymerase alone (rCCG or rCGG, respectively), or simultaneously (rCCG with rCGG) or serially: rCCG transcription then phenol chloroform extraction followed by rCGG transcription (rCCG then rCGG), and vice versa (rCGG then rCCG). R-loops are indicated as ‘R’. Note that in the case of bidirectional or serial transcription, complementary RNA is produced forming dsRNA as indicated on the gel by ‘*’. These products are not present in transcription reactions occurring in one direction. ( B ) Same as in (A) except in vitro transcription reactions were performed on a DM1 (CAG)79 · (CTG)79 template. ( C ) EM analysis of convergent transcription reaction products from DM1 (CAG)79 · (CTG)79 templates. Samples were transcribed convergently using T3 and T7 RNA polymerase promoters simultaneously then the products were treated with RNAse A and prepared for EM as described in the ‘Materials and Methods’ section (rCUGand rCAG RNase A). Samples were also subjected to RNase H treatment along with RNase A for comparison (rCUG and rCAG RNase A, H). Transcription was also performed on the same template in a single direction for further comparison (rCUG RNase A). The products observed for each type of transcription reaction is shown as a percentage of the total number of molecules analyzed. At least 100 molecules were analyzed for each type of transcription reaction.

Techniques Used: In Vitro, Produced, CTG Assay

Mechanism of RNA:DNA hybrid formation during in vitro transcription. ( A ) Schematic of the two possible mechanisms for R-loop formation. By the thread-back model, the nascent transcript (depicted in light blue) that has been ejected from the RNA polymerase re-anneals with the complementary, free DNA template strand (depicted in red) following the progression of the RNA polymerase (light blue, oval). When transcription occurs in the presence of RNAse A (dark blue) the nascent transcript is degraded when it is ejected from the RNA polymerase hence cannot form the hybrid. By the extended-hybrid model, the nascent transcript remains bound to the template DNA and resists becoming ejected from the RNA polymerase. When transcribed in the presence of RNase A, as the nascent transcript is protected by being bound to the template DNA it is not degraded hence hybrid formation is not ablated. ( B ) FRAXA plasmid (CGG)39 · (CCG)39 transcribed in either direction in the absence (−) or presence (+) of RNase A during the transcription reaction. All transcription reactions were subjected to further RNase A or A + H treatment to analyze hybrid formation. ( C ) Same experiment as in (B) performed with SCA1 plasmid containing a (CAG)74 · (CTG)74 repeat tract.
Figure Legend Snippet: Mechanism of RNA:DNA hybrid formation during in vitro transcription. ( A ) Schematic of the two possible mechanisms for R-loop formation. By the thread-back model, the nascent transcript (depicted in light blue) that has been ejected from the RNA polymerase re-anneals with the complementary, free DNA template strand (depicted in red) following the progression of the RNA polymerase (light blue, oval). When transcription occurs in the presence of RNAse A (dark blue) the nascent transcript is degraded when it is ejected from the RNA polymerase hence cannot form the hybrid. By the extended-hybrid model, the nascent transcript remains bound to the template DNA and resists becoming ejected from the RNA polymerase. When transcribed in the presence of RNase A, as the nascent transcript is protected by being bound to the template DNA it is not degraded hence hybrid formation is not ablated. ( B ) FRAXA plasmid (CGG)39 · (CCG)39 transcribed in either direction in the absence (−) or presence (+) of RNase A during the transcription reaction. All transcription reactions were subjected to further RNase A or A + H treatment to analyze hybrid formation. ( C ) Same experiment as in (B) performed with SCA1 plasmid containing a (CAG)74 · (CTG)74 repeat tract.

Techniques Used: In Vitro, Plasmid Preparation, CTG Assay

34) Product Images from "Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats"

Article Title: Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq935

RNA–DNA hybrid formation at a (CGG)39 · (CCG)39 FRAXA template. When the template DNA is transcribed with T7 RNA polymerase, heterogeneous RNA is produced generating a smear (Transcription lane). Treatment with RNase H alone which is specific to RNA base paired to template DNA digests only RNA that is base paired to its template DNA. Treatment with RNase A, which is specific to single-stranded RNA, digests all free, single-stranded RNA leaving template DNA and RNA:DNA hybrid structures. Note that RNA–DNA hybrids migrate more slowly than supercoiled DNA (as indicated schematically, RNA is in light blue). Hybrid structures generate a smear due to their heterogeneous sizes. With a larger RNA component, the DNA is open to a greater degree (more relaxed), hence migration is closer to open circular DNA. Treatment of the hybrids with RNase H along with RNase A removes any hybrids formed as well as transcript generated in the transcription reaction leaving only input template DNA. When transcription followed by RNase H or RNase A treatment alone or in combination is performed on an empty Bluescript vector [pBlueKS(+)], there is negligible hybrid formation.
Figure Legend Snippet: RNA–DNA hybrid formation at a (CGG)39 · (CCG)39 FRAXA template. When the template DNA is transcribed with T7 RNA polymerase, heterogeneous RNA is produced generating a smear (Transcription lane). Treatment with RNase H alone which is specific to RNA base paired to template DNA digests only RNA that is base paired to its template DNA. Treatment with RNase A, which is specific to single-stranded RNA, digests all free, single-stranded RNA leaving template DNA and RNA:DNA hybrid structures. Note that RNA–DNA hybrids migrate more slowly than supercoiled DNA (as indicated schematically, RNA is in light blue). Hybrid structures generate a smear due to their heterogeneous sizes. With a larger RNA component, the DNA is open to a greater degree (more relaxed), hence migration is closer to open circular DNA. Treatment of the hybrids with RNase H along with RNase A removes any hybrids formed as well as transcript generated in the transcription reaction leaving only input template DNA. When transcription followed by RNase H or RNase A treatment alone or in combination is performed on an empty Bluescript vector [pBlueKS(+)], there is negligible hybrid formation.

Techniques Used: Produced, Migration, Generated, Plasmid Preparation

Quantification of relative RNA–DNA hybrid formation at increasing repeat lengths. ( A ) In vitro transcription reactions were performed on FRAXA plasmids bearing repeat tracts of (CGG)17 · (CCG)17, (CGG)39 · (CCG)39 and (CGG)53 · (CCG)53 following which samples were treated with RNase A or A+H. Hybrid formation was quantified by densitometry analysis using image quant by measuring the proportion of products that migrate between open circular and supercoiled position (indicated by ‘R’) divided by the total products below open circular including supercoiled. RNase A+H treated samples were used to determine the position of the supercoiled DNA for each repeat length. To the left of the graph is a representative gel used to quantify relative hybrid formation. The RNA indicated in the graph represents the RNA component bound in the RNA:DNA hybrid. Error bars are derived from three separate experiments ( N =3). Using the t -test to compare R-loop formation revealed a significant difference between the 17 and 53 rCGG R-loop ( P =0.0413) as well as the 17 and 53 rCCG R-loop ( P =0.0092). ( B ) The same analysis was performed as in (A) but for SCA1 plasmids bearing repeat tracts of (CAG)30 · (CTG)30, (CAG) 49 · (CTG)49 and (CAG)74 · (CTG)74. Using the t -test to compare R-loop formation between each of the (CAG) · (CTG) repeat lengths did not reveal any statistically significant differences.
Figure Legend Snippet: Quantification of relative RNA–DNA hybrid formation at increasing repeat lengths. ( A ) In vitro transcription reactions were performed on FRAXA plasmids bearing repeat tracts of (CGG)17 · (CCG)17, (CGG)39 · (CCG)39 and (CGG)53 · (CCG)53 following which samples were treated with RNase A or A+H. Hybrid formation was quantified by densitometry analysis using image quant by measuring the proportion of products that migrate between open circular and supercoiled position (indicated by ‘R’) divided by the total products below open circular including supercoiled. RNase A+H treated samples were used to determine the position of the supercoiled DNA for each repeat length. To the left of the graph is a representative gel used to quantify relative hybrid formation. The RNA indicated in the graph represents the RNA component bound in the RNA:DNA hybrid. Error bars are derived from three separate experiments ( N =3). Using the t -test to compare R-loop formation revealed a significant difference between the 17 and 53 rCGG R-loop ( P =0.0413) as well as the 17 and 53 rCCG R-loop ( P =0.0092). ( B ) The same analysis was performed as in (A) but for SCA1 plasmids bearing repeat tracts of (CAG)30 · (CTG)30, (CAG) 49 · (CTG)49 and (CAG)74 · (CTG)74. Using the t -test to compare R-loop formation between each of the (CAG) · (CTG) repeat lengths did not reveal any statistically significant differences.

Techniques Used: In Vitro, Derivative Assay, CTG Assay

Effect of trinucleotide repeat interruptions on RNA–DNA hybrid formation. ( A ) In vitro transcription followed by RNase A or A + H treatment was performed with either pure (39p) FRAXA plasmids (CGG)39 · (CCG)39 or interrupted plasmids (39i) [(CGG)9(AGG) (CGG)9(AGG)(CGG)9(AGG)(CGG)9] · [(CCG)9(CCT) (CCG)9(CCT)(CCG)9(CCT)(CCG)9] as indicated. Repeat tract configurations are schematically presented where hollow dots are the CGG repeat units and the filled dots are the AGG interruptions. R-loops are indicated as ‘R’. ( B ) In vitro transcription followed by RNase A or A+H treatment was performed with either pure (49p) SCA1 plasmids (CAG)49 · (CTG)49 or interrupted plasmids (44i) [(CAG)12(CAT)(CAG)(CAT)(CAG)12(CAT)(CAG)(CAT)(CAG)14] · [(CTG)14(ATG)(CTG)(ATG)(CTG)12(ATG)(CAG)(ATG)(CTG)12] as indicated schematically where hollow dots are the CAG repeat units and the filled dots are the CAT interruptions.
Figure Legend Snippet: Effect of trinucleotide repeat interruptions on RNA–DNA hybrid formation. ( A ) In vitro transcription followed by RNase A or A + H treatment was performed with either pure (39p) FRAXA plasmids (CGG)39 · (CCG)39 or interrupted plasmids (39i) [(CGG)9(AGG) (CGG)9(AGG)(CGG)9(AGG)(CGG)9] · [(CCG)9(CCT) (CCG)9(CCT)(CCG)9(CCT)(CCG)9] as indicated. Repeat tract configurations are schematically presented where hollow dots are the CGG repeat units and the filled dots are the AGG interruptions. R-loops are indicated as ‘R’. ( B ) In vitro transcription followed by RNase A or A+H treatment was performed with either pure (49p) SCA1 plasmids (CAG)49 · (CTG)49 or interrupted plasmids (44i) [(CAG)12(CAT)(CAG)(CAT)(CAG)12(CAT)(CAG)(CAT)(CAG)14] · [(CTG)14(ATG)(CTG)(ATG)(CTG)12(ATG)(CAG)(ATG)(CTG)12] as indicated schematically where hollow dots are the CAG repeat units and the filled dots are the CAT interruptions.

Techniques Used: In Vitro, CTG Assay

Identification of R-loop structures formed in expanded DM1 (CTG)130 · (CAG)130 plasmids using EM following in vitro transcription and treatment with RNase A and SSB protein. SSB proteins bind to the looped-out non-template DNA in an R-loop structure. Thus, each R-loop structure is visualized as a loop within the DNA template as indicated by black arrowheads. ( A ) R-loops formed by using SP6 RNA polymerase, producing an rCAG:dCTG hybrid. ( B ) R-loops formed by using T7 RNA polymerase, producing an rCUG:dCAG hybrid.
Figure Legend Snippet: Identification of R-loop structures formed in expanded DM1 (CTG)130 · (CAG)130 plasmids using EM following in vitro transcription and treatment with RNase A and SSB protein. SSB proteins bind to the looped-out non-template DNA in an R-loop structure. Thus, each R-loop structure is visualized as a loop within the DNA template as indicated by black arrowheads. ( A ) R-loops formed by using SP6 RNA polymerase, producing an rCAG:dCTG hybrid. ( B ) R-loops formed by using T7 RNA polymerase, producing an rCUG:dCAG hybrid.

Techniques Used: CTG Assay, In Vitro

RNA–DNA hybrid formation during in vitro transcription of trinucleotide repeat-containing plasmids. ( A ) In vitro transcription of SCA1 plasmid containing (CAG)74 · (CTG)74 and FRAXA plasmid containing (CGG)39 · (CCG)39 repeats in either direction using T3 or T7 RNA polymerase. The repeat sequence contained within the RNA produced and bound in the hybrid is indicated below the transcribed template. Samples following transcription were subsequently treated with either RNase A or A+H as indicated to observe hybrid formation. R-loops are indicated on the gel as ‘R’. ( B ) Exact same reactions and gel conditions as in (A) but transcription was performed in the presence of 3.5 µCi [α- 32 P]-rCTP. Gel was dried and exposed to X-ray film as outlined in ‘Materials and Methods’ section.
Figure Legend Snippet: RNA–DNA hybrid formation during in vitro transcription of trinucleotide repeat-containing plasmids. ( A ) In vitro transcription of SCA1 plasmid containing (CAG)74 · (CTG)74 and FRAXA plasmid containing (CGG)39 · (CCG)39 repeats in either direction using T3 or T7 RNA polymerase. The repeat sequence contained within the RNA produced and bound in the hybrid is indicated below the transcribed template. Samples following transcription were subsequently treated with either RNase A or A+H as indicated to observe hybrid formation. R-loops are indicated on the gel as ‘R’. ( B ) Exact same reactions and gel conditions as in (A) but transcription was performed in the presence of 3.5 µCi [α- 32 P]-rCTP. Gel was dried and exposed to X-ray film as outlined in ‘Materials and Methods’ section.

Techniques Used: In Vitro, Plasmid Preparation, CTG Assay, Sequencing, Produced

Effect of convergent simultaneous bidirectional or serial transcription on R-loop formation. ( A ) In vitro transcription of FRAXA template (CGG)39 · (CCG)39 with either T3 or T7 RNA polymerase alone (rCCG or rCGG, respectively), or simultaneously (rCCG with rCGG) or serially: rCCG transcription then phenol chloroform extraction followed by rCGG transcription (rCCG then rCGG), and vice versa (rCGG then rCCG). R-loops are indicated as ‘R’. Note that in the case of bidirectional or serial transcription, complementary RNA is produced forming dsRNA as indicated on the gel by ‘*’. These products are not present in transcription reactions occurring in one direction. ( B ) Same as in (A) except in vitro transcription reactions were performed on a DM1 (CAG)79 · (CTG)79 template. ( C ) EM analysis of convergent transcription reaction products from DM1 (CAG)79 · (CTG)79 templates. Samples were transcribed convergently using T3 and T7 RNA polymerase promoters simultaneously then the products were treated with RNAse A and prepared for EM as described in the ‘Materials and Methods’ section (rCUGand rCAG RNase A). Samples were also subjected to RNase H treatment along with RNase A for comparison (rCUG and rCAG RNase A, H). Transcription was also performed on the same template in a single direction for further comparison (rCUG RNase A). The products observed for each type of transcription reaction is shown as a percentage of the total number of molecules analyzed. At least 100 molecules were analyzed for each type of transcription reaction.
Figure Legend Snippet: Effect of convergent simultaneous bidirectional or serial transcription on R-loop formation. ( A ) In vitro transcription of FRAXA template (CGG)39 · (CCG)39 with either T3 or T7 RNA polymerase alone (rCCG or rCGG, respectively), or simultaneously (rCCG with rCGG) or serially: rCCG transcription then phenol chloroform extraction followed by rCGG transcription (rCCG then rCGG), and vice versa (rCGG then rCCG). R-loops are indicated as ‘R’. Note that in the case of bidirectional or serial transcription, complementary RNA is produced forming dsRNA as indicated on the gel by ‘*’. These products are not present in transcription reactions occurring in one direction. ( B ) Same as in (A) except in vitro transcription reactions were performed on a DM1 (CAG)79 · (CTG)79 template. ( C ) EM analysis of convergent transcription reaction products from DM1 (CAG)79 · (CTG)79 templates. Samples were transcribed convergently using T3 and T7 RNA polymerase promoters simultaneously then the products were treated with RNAse A and prepared for EM as described in the ‘Materials and Methods’ section (rCUGand rCAG RNase A). Samples were also subjected to RNase H treatment along with RNase A for comparison (rCUG and rCAG RNase A, H). Transcription was also performed on the same template in a single direction for further comparison (rCUG RNase A). The products observed for each type of transcription reaction is shown as a percentage of the total number of molecules analyzed. At least 100 molecules were analyzed for each type of transcription reaction.

Techniques Used: In Vitro, Produced, CTG Assay

Mechanism of RNA:DNA hybrid formation during in vitro transcription. ( A ) Schematic of the two possible mechanisms for R-loop formation. By the thread-back model, the nascent transcript (depicted in light blue) that has been ejected from the RNA polymerase re-anneals with the complementary, free DNA template strand (depicted in red) following the progression of the RNA polymerase (light blue, oval). When transcription occurs in the presence of RNAse A (dark blue) the nascent transcript is degraded when it is ejected from the RNA polymerase hence cannot form the hybrid. By the extended-hybrid model, the nascent transcript remains bound to the template DNA and resists becoming ejected from the RNA polymerase. When transcribed in the presence of RNase A, as the nascent transcript is protected by being bound to the template DNA it is not degraded hence hybrid formation is not ablated. ( B ) FRAXA plasmid (CGG)39 · (CCG)39 transcribed in either direction in the absence (−) or presence (+) of RNase A during the transcription reaction. All transcription reactions were subjected to further RNase A or A + H treatment to analyze hybrid formation. ( C ) Same experiment as in (B) performed with SCA1 plasmid containing a (CAG)74 · (CTG)74 repeat tract.
Figure Legend Snippet: Mechanism of RNA:DNA hybrid formation during in vitro transcription. ( A ) Schematic of the two possible mechanisms for R-loop formation. By the thread-back model, the nascent transcript (depicted in light blue) that has been ejected from the RNA polymerase re-anneals with the complementary, free DNA template strand (depicted in red) following the progression of the RNA polymerase (light blue, oval). When transcription occurs in the presence of RNAse A (dark blue) the nascent transcript is degraded when it is ejected from the RNA polymerase hence cannot form the hybrid. By the extended-hybrid model, the nascent transcript remains bound to the template DNA and resists becoming ejected from the RNA polymerase. When transcribed in the presence of RNase A, as the nascent transcript is protected by being bound to the template DNA it is not degraded hence hybrid formation is not ablated. ( B ) FRAXA plasmid (CGG)39 · (CCG)39 transcribed in either direction in the absence (−) or presence (+) of RNase A during the transcription reaction. All transcription reactions were subjected to further RNase A or A + H treatment to analyze hybrid formation. ( C ) Same experiment as in (B) performed with SCA1 plasmid containing a (CAG)74 · (CTG)74 repeat tract.

Techniques Used: In Vitro, Plasmid Preparation, CTG Assay

35) Product Images from "Evidence for a Posttranscriptional Role of a TFIIIC?-like Protein in Chironomus tentans"

Article Title: Evidence for a Posttranscriptional Role of a TFIIIC?-like Protein in Chironomus tentans

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.01-09-0436

Chromatographic analysis of TFIIIC-α–containing complexes. Nuclear proteins from tissue culture cells of C. tentans were fractionated on a gel filtration Superose HR6 column. (A) The chromatogram, showing the fractionation of some molecular mass standards, in kDa, used for calibration. V O : void volume. (B) Fractions were pooled two by two (for example, lane 12 contains fractions 12 and 13), separated by SDS-PAGE, and analyzed by immunoblotting using mAb 2D10. The mobilities of molecular mass standards in SDS-PAGE are shown on the left. (C) Nuclear extract was preincubated with 25 μg/ml RNase A for 20 min before chromatography and Western blot analysis as in B.
Figure Legend Snippet: Chromatographic analysis of TFIIIC-α–containing complexes. Nuclear proteins from tissue culture cells of C. tentans were fractionated on a gel filtration Superose HR6 column. (A) The chromatogram, showing the fractionation of some molecular mass standards, in kDa, used for calibration. V O : void volume. (B) Fractions were pooled two by two (for example, lane 12 contains fractions 12 and 13), separated by SDS-PAGE, and analyzed by immunoblotting using mAb 2D10. The mobilities of molecular mass standards in SDS-PAGE are shown on the left. (C) Nuclear extract was preincubated with 25 μg/ml RNase A for 20 min before chromatography and Western blot analysis as in B.

Techniques Used: Filtration, Fractionation, SDS Page, Chromatography, Western Blot

36) Product Images from "Role of the C-Terminal Domain of RNA Polymerase II in U2 snRNA Transcription and 3? Processing"

Article Title: Role of the C-Terminal Domain of RNA Polymerase II in U2 snRNA Transcription and 3? Processing

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.2.846-855.2004

); and no tagged LS is seen upon transfection with a control β-galactosidase expression construct. Assayed by Western blotting against the N terminus of Pol II (lower panel), the α-amanitin-resistant LS constructs are vastly overexpressed compared to endogenous Pol II (compare right two lanes with left two), and endogenous Pol II is degraded after exposure to 2 μg of α-amanitin/ml for 24 h (compare left two lanes). (B) Nascent U2 snRNA assayed by run-on transcription, using 5S rRNA as an internal control. Cells were harvested 24 h after transfection, and nuclear run-on transcription was conducted in the presence or absence of α-amanitin (2 μg/ml). (C) U2+10 precursor assayed by primer extension. α-Amanitin was added 24 h after transfection, and cells were incubated for an additional 24 h to degrade endogenous Pol II LS before harvesting RNA. (D) U2 snRNA primary transcript assayed by RNase protection. RNA was harvested as for panel C and assayed using the probe shown in panel A. The U2+152 signal reflects transcripts extending beyond position +152 downstream of the U2 coding region. The small amount of full-length probe seen reflects incomplete RNase digestion. RNase protection conducted with RNase A as well as T1 results in the absence of undigested probe and sharper U2+152 bands but also reveals a spurious band at U2+110 (data not shown), perhaps due to an AT-rich region in this area. Minor degradation of the high-specific-activity probe reflects autoradiolysis and does not affect our results because the probe cannot protect itself (see lane with probe only plus RNase).
Figure Legend Snippet: ); and no tagged LS is seen upon transfection with a control β-galactosidase expression construct. Assayed by Western blotting against the N terminus of Pol II (lower panel), the α-amanitin-resistant LS constructs are vastly overexpressed compared to endogenous Pol II (compare right two lanes with left two), and endogenous Pol II is degraded after exposure to 2 μg of α-amanitin/ml for 24 h (compare left two lanes). (B) Nascent U2 snRNA assayed by run-on transcription, using 5S rRNA as an internal control. Cells were harvested 24 h after transfection, and nuclear run-on transcription was conducted in the presence or absence of α-amanitin (2 μg/ml). (C) U2+10 precursor assayed by primer extension. α-Amanitin was added 24 h after transfection, and cells were incubated for an additional 24 h to degrade endogenous Pol II LS before harvesting RNA. (D) U2 snRNA primary transcript assayed by RNase protection. RNA was harvested as for panel C and assayed using the probe shown in panel A. The U2+152 signal reflects transcripts extending beyond position +152 downstream of the U2 coding region. The small amount of full-length probe seen reflects incomplete RNase digestion. RNase protection conducted with RNase A as well as T1 results in the absence of undigested probe and sharper U2+152 bands but also reveals a spurious band at U2+110 (data not shown), perhaps due to an AT-rich region in this area. Minor degradation of the high-specific-activity probe reflects autoradiolysis and does not affect our results because the probe cannot protect itself (see lane with probe only plus RNase).

Techniques Used: Transfection, Expressing, Construct, Western Blot, Incubation, Activity Assay

37) Product Images from "REST-Mediated Recruitment of Polycomb Repressor Complexes in Mammalian Cells"

Article Title: REST-Mediated Recruitment of Polycomb Repressor Complexes in Mammalian Cells

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1002494

REST and Polycomb Repressor Complex 1 (PRC1) and PRC2 interact in vivo . (A) Nuclear extracts from NT2-D1 cells were processed for size-exclusion chromatography followed by Western blotting to reveal the profiles of Polycomb proteins, the transcription factors REST and E2F6. Pooled fractions (1: Fractions F7–9; 2: F11–13; 3: F21–22) were used for immunoprecipitation (IP) with anti-REST or control IgG and processed for Western blotting with antibodies as indicated (total lysate: 12 µg of protein). (B) IPs using REST or control IgG on total nuclear extract (NT2-D1 cells; 500 µg per IP). After IPs the samples were either treated with a combination of RNase V1 and RNase A or left without RNase followed by repeated washes. Eluted proteins were processed for Western blotting using antibodies as indicated. Lower panel: Control experiment for the efficiency of RNase treatment using either 2 µg (left part) or 4 µg (right part) of RNA. Samples were incubated under the conditions used for REST IPs. (C–D) Nuclear protein extracts from mouse embryonic stem cells (mES) of different genetic background ( Wt , Eed−/− or Rnf2−/− ) were separated by size-exclusion chromatography (C–D: upper panels) and pooled fractions (1: F8–10; 2: F11–13; 3: F22) were processed for IPs (C–D: lower panels) using antibodies for Rest or control IgG. Western blots were processed with antibodies as indicated. Input corresponds to 3% of the material used for each IP. (C) Represents IPs comparing Wt and Eed−/− mES cells and (D) represents IPs in the Rnf2−/− mES cells. The samples were processed for Western blotting with antibodies as indicated. Lanes marked “M” represents loading of a pre-stained molecular weight marker.
Figure Legend Snippet: REST and Polycomb Repressor Complex 1 (PRC1) and PRC2 interact in vivo . (A) Nuclear extracts from NT2-D1 cells were processed for size-exclusion chromatography followed by Western blotting to reveal the profiles of Polycomb proteins, the transcription factors REST and E2F6. Pooled fractions (1: Fractions F7–9; 2: F11–13; 3: F21–22) were used for immunoprecipitation (IP) with anti-REST or control IgG and processed for Western blotting with antibodies as indicated (total lysate: 12 µg of protein). (B) IPs using REST or control IgG on total nuclear extract (NT2-D1 cells; 500 µg per IP). After IPs the samples were either treated with a combination of RNase V1 and RNase A or left without RNase followed by repeated washes. Eluted proteins were processed for Western blotting using antibodies as indicated. Lower panel: Control experiment for the efficiency of RNase treatment using either 2 µg (left part) or 4 µg (right part) of RNA. Samples were incubated under the conditions used for REST IPs. (C–D) Nuclear protein extracts from mouse embryonic stem cells (mES) of different genetic background ( Wt , Eed−/− or Rnf2−/− ) were separated by size-exclusion chromatography (C–D: upper panels) and pooled fractions (1: F8–10; 2: F11–13; 3: F22) were processed for IPs (C–D: lower panels) using antibodies for Rest or control IgG. Western blots were processed with antibodies as indicated. Input corresponds to 3% of the material used for each IP. (C) Represents IPs comparing Wt and Eed−/− mES cells and (D) represents IPs in the Rnf2−/− mES cells. The samples were processed for Western blotting with antibodies as indicated. Lanes marked “M” represents loading of a pre-stained molecular weight marker.

Techniques Used: In Vivo, Size-exclusion Chromatography, Western Blot, Immunoprecipitation, Incubation, Staining, Molecular Weight, Marker

38) Product Images from "Identification of Neuronal RNA Targets of TDP-43-containing Ribonucleoprotein Complexes *"

Article Title: Identification of Neuronal RNA Targets of TDP-43-containing Ribonucleoprotein Complexes *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.190884

Genomic distribution of reads from TDP-43 RNA library. A , Western blot ( IB ) of fractions from HeLa nuclear extracts (± RNase A) applied to a size exclusion column, blotted for TDP-43, hnRNPA1, and lamin A/C. Fraction 6 = blue dextran (2000 kDa), fraction 30 = apoferritin (443 kDa), fraction 40 = alcohol dehydrogenase (150 kDa), and fraction 54 = bovine serum albumin (54 kDa) (data not shown).  B , Western blot of fractions from rat brain nuclear extracts (± micrococcal nuclease (±  MNase )) blotted for TDP-43. Fraction 5 = blue dextran (2,000 kDa), fraction 45 = bovine serum albumin (54 kDa), and fraction 54 = carbonic anhydrase (29 kDa) (data not shown). Note that different size exclusion columns were used in  A  and  B. C ,  panel i , diagram of TDP-43 RIP method.  C ,  panel ii , representative Western blot of TDP-43 RIP.  IP:CTL , control immunoprecipitation.  D , distribution of raw reads from the TDP-43 library mapped to exonic and intronic genes regions.  CDS , coding sequence.  E , read density, number of reads per 1,000 mappable nucleotides per million reads ( mRPKM ) of gene regions from the TDP-43 library.
Figure Legend Snippet: Genomic distribution of reads from TDP-43 RNA library. A , Western blot ( IB ) of fractions from HeLa nuclear extracts (± RNase A) applied to a size exclusion column, blotted for TDP-43, hnRNPA1, and lamin A/C. Fraction 6 = blue dextran (2000 kDa), fraction 30 = apoferritin (443 kDa), fraction 40 = alcohol dehydrogenase (150 kDa), and fraction 54 = bovine serum albumin (54 kDa) (data not shown). B , Western blot of fractions from rat brain nuclear extracts (± micrococcal nuclease (± MNase )) blotted for TDP-43. Fraction 5 = blue dextran (2,000 kDa), fraction 45 = bovine serum albumin (54 kDa), and fraction 54 = carbonic anhydrase (29 kDa) (data not shown). Note that different size exclusion columns were used in A and B. C , panel i , diagram of TDP-43 RIP method. C , panel ii , representative Western blot of TDP-43 RIP. IP:CTL , control immunoprecipitation. D , distribution of raw reads from the TDP-43 library mapped to exonic and intronic genes regions. CDS , coding sequence. E , read density, number of reads per 1,000 mappable nucleotides per million reads ( mRPKM ) of gene regions from the TDP-43 library.

Techniques Used: Western Blot, CTL Assay, Immunoprecipitation, Sequencing

39) Product Images from "Modulation of HIV-like particle assembly in vitro by inositol phosphates"

Article Title: Modulation of HIV-like particle assembly in vitro by inositol phosphates

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

doi: 10.1073/pnas.191224698

Nuclease and salt resistance of particles assembled in vitro and of authentic, immature particles. Particles assembled in buffer B ( A ), buffer B plus 5% rabbit reticulocyte lysate ( B ), or buffer B plus 2 μM IP5 ( C ), were treated with RNase A (lanes 2) or NaCl (lanes 3). Gag proteins in particles pelleted after treatment were examined by SDS/PAGE and Coomassie blue staining. We estimate that > 90% of the Gag protein was solubilized in A (lanes 2 and 3), whereas > 90% remained pelletable in B and C (lanes 2 and 3). ( D ) Immature MoMuLV and HIV-1 virions produced in mammalian cells were analyzed separately (lanes 1 and 2, respectively) or were mixed together (lanes 3–9) and treated with RNase A (lanes 6 and 7) or NaCl (lanes 8 and 9). They were also digested with HIV-1 PR to confirm that the lipid envelope was removed by the detergent (lane 3), or sedimented without RNase or NaCl treatment to confirm that the particles are stable in buffer B (lanes 4 and 5). Gag proteins in the samples in D were detected by immunoblotting with antibodies against the CA proteins of both HIV-1 and MoMuLV. We estimate that > 90% of the HIV-1 Gag protein was still pelletable after NaCl or RNase treatment. P, pellet; S, supernatant.
Figure Legend Snippet: Nuclease and salt resistance of particles assembled in vitro and of authentic, immature particles. Particles assembled in buffer B ( A ), buffer B plus 5% rabbit reticulocyte lysate ( B ), or buffer B plus 2 μM IP5 ( C ), were treated with RNase A (lanes 2) or NaCl (lanes 3). Gag proteins in particles pelleted after treatment were examined by SDS/PAGE and Coomassie blue staining. We estimate that > 90% of the Gag protein was solubilized in A (lanes 2 and 3), whereas > 90% remained pelletable in B and C (lanes 2 and 3). ( D ) Immature MoMuLV and HIV-1 virions produced in mammalian cells were analyzed separately (lanes 1 and 2, respectively) or were mixed together (lanes 3–9) and treated with RNase A (lanes 6 and 7) or NaCl (lanes 8 and 9). They were also digested with HIV-1 PR to confirm that the lipid envelope was removed by the detergent (lane 3), or sedimented without RNase or NaCl treatment to confirm that the particles are stable in buffer B (lanes 4 and 5). Gag proteins in the samples in D were detected by immunoblotting with antibodies against the CA proteins of both HIV-1 and MoMuLV. We estimate that > 90% of the HIV-1 Gag protein was still pelletable after NaCl or RNase treatment. P, pellet; S, supernatant.

Techniques Used: In Vitro, SDS Page, Staining, Produced

40) Product Images from "Mesenchymal stem cell secretes microparticles enriched in pre-microRNAs"

Article Title: Mesenchymal stem cell secretes microparticles enriched in pre-microRNAs

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkp857

Secreted RNAs were sequestered in phospholipid vesicles. ( A ) Untreated CM or CM pretreated with SDS-based lysis buffer, cyclodextrin or phospholipase A2 was incubated with RNase A. After incubation, the CMs were extracted for RNA and the RNAs were resolved on a 15% TBE–urea gel. ( B ) RNA was extracted from CM without RNase inhibitor (lane 2), with RNase inhibitor (lane 3) or CM pretreated with cyclodextrin (lane 4), SDS-based lysis buffer (lane 5) or phospholipase A2 (lane 6).
Figure Legend Snippet: Secreted RNAs were sequestered in phospholipid vesicles. ( A ) Untreated CM or CM pretreated with SDS-based lysis buffer, cyclodextrin or phospholipase A2 was incubated with RNase A. After incubation, the CMs were extracted for RNA and the RNAs were resolved on a 15% TBE–urea gel. ( B ) RNA was extracted from CM without RNase inhibitor (lane 2), with RNase inhibitor (lane 3) or CM pretreated with cyclodextrin (lane 4), SDS-based lysis buffer (lane 5) or phospholipase A2 (lane 6).

Techniques Used: Lysis, Incubation

Related Articles

Incubation:

Article Title: Newly Synthesized APOBEC3G Is Incorporated into HIV Virions, Inhibited by HIV RNA, and Subsequently Activated by RNase H
Article Snippet: .. Labeled oligonucleotides and input samples were incubated in 20 μl of 50 mM Tris buffer (pH 7.4), with or without RNase A (1 μg) at 37 °C for 3 h unless otherwise indicated. .. For incubations under conditions stimulating endogenous reverse transcription, KCl (final concentration, 60 mM), MgCl2 (final concentration, 4 mM), and dNTPs (final concentration, 1 mM) were added.

other:

Article Title: Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats
Article Snippet: To better visualize any RNA:DNA hybrid complexes that may have formed, the transcription products were treated with RNase A, a ribonuclease that specifically digests single-stranded RNA.

Article Title: Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats
Article Snippet: This material was resistant to RNase A and RNase H but was sensitive to the double-stranded RNA-specific ribonuclease III ( , ) ( Supplementary Figure S6 ).

Article Title: Identification of Neuronal RNA Targets of TDP-43-containing Ribonucleoprotein Complexes *
Article Snippet: We show that with the addition of RNase A, TDP-43 elution in the high molecular mass range could be shifted to the lower molecular mass range ( A ).

Article Title: Evidence for a Posttranscriptional Role of a TFIIIC?-like Protein in Chironomus tentans
Article Snippet: To check whether any of the p2D10 complexes contained RNA, a nuclear extract was treated with RNase A, fractionated, and analyzed as above.

Article Title: Identification of Neuronal RNA Targets of TDP-43-containing Ribonucleoprotein Complexes *
Article Snippet: A similar pattern was observed for hnRNPA1 in control lysates as well as lysates treated with RNase A but not for lamin A/C ( A ).

Labeling:

Article Title: Newly Synthesized APOBEC3G Is Incorporated into HIV Virions, Inhibited by HIV RNA, and Subsequently Activated by RNase H
Article Snippet: .. Labeled oligonucleotides and input samples were incubated in 20 μl of 50 mM Tris buffer (pH 7.4), with or without RNase A (1 μg) at 37 °C for 3 h unless otherwise indicated. .. For incubations under conditions stimulating endogenous reverse transcription, KCl (final concentration, 60 mM), MgCl2 (final concentration, 4 mM), and dNTPs (final concentration, 1 mM) were added.

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    Chromatographic analysis of TFIIIC-α–containing complexes. Nuclear proteins from tissue culture cells of C. tentans were fractionated on a gel filtration Superose HR6 column. (A) The chromatogram, showing the fractionation of some molecular mass standards, in kDa, used for calibration. V O : void volume. (B) Fractions were pooled two by two (for example, lane 12 contains fractions 12 and 13), separated by SDS-PAGE, and analyzed by immunoblotting using mAb 2D10. The mobilities of molecular mass standards in SDS-PAGE are shown on the left. (C) Nuclear extract was preincubated with 25 μg/ml <t>RNase</t> A for 20 min before chromatography and Western blot analysis as in B.
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    Chromatographic analysis of TFIIIC-α–containing complexes. Nuclear proteins from tissue culture cells of C. tentans were fractionated on a gel filtration Superose HR6 column. (A) The chromatogram, showing the fractionation of some molecular mass standards, in kDa, used for calibration. V O : void volume. (B) Fractions were pooled two by two (for example, lane 12 contains fractions 12 and 13), separated by SDS-PAGE, and analyzed by immunoblotting using mAb 2D10. The mobilities of molecular mass standards in SDS-PAGE are shown on the left. (C) Nuclear extract was preincubated with 25 μg/ml RNase A for 20 min before chromatography and Western blot analysis as in B.

    Journal: Molecular Biology of the Cell

    Article Title: Evidence for a Posttranscriptional Role of a TFIIIC?-like Protein in Chironomus tentans

    doi: 10.1091/mbc.01-09-0436

    Figure Lengend Snippet: Chromatographic analysis of TFIIIC-α–containing complexes. Nuclear proteins from tissue culture cells of C. tentans were fractionated on a gel filtration Superose HR6 column. (A) The chromatogram, showing the fractionation of some molecular mass standards, in kDa, used for calibration. V O : void volume. (B) Fractions were pooled two by two (for example, lane 12 contains fractions 12 and 13), separated by SDS-PAGE, and analyzed by immunoblotting using mAb 2D10. The mobilities of molecular mass standards in SDS-PAGE are shown on the left. (C) Nuclear extract was preincubated with 25 μg/ml RNase A for 20 min before chromatography and Western blot analysis as in B.

    Article Snippet: To check whether any of the p2D10 complexes contained RNA, a nuclear extract was treated with RNase A, fractionated, and analyzed as above.

    Techniques: Filtration, Fractionation, SDS Page, Chromatography, Western Blot

    Virion-Incorporated HA-A3G Associates with Viral Genomic RNA (A) Viral genomic RNA, detected by RT-PCR, was detected in virions and virus-producing cells but not in lysates of uninfected cells. Genomic RNA was also detected in the IVAC derived from virions (fraction 7) and coimmunoprecipitated with HA-A3G from both virions and producer cell lysates. RT was performed using RNA derived from either whole lysates (L) or anti-HA immunoprecipitates (IP). Control reactions were performed in the absence of RT (–RT). Control PCRs were performed using proviral plasmid DNA, in the absence or presence of Taq, as indicated. (B) Viral genomic RNA, detected by RT-PCR, was assessed from size-fractionated virion lysates that lacked (HA) or contained HA-A3G. Amplicons generated probed across the TAR/Gag region or Pol/Vpu regions, as indicated. (C) Incorporation of HA-A3G into virions enhances the recruitment of NC into the IVAC. (D) HA-A3G from virus-producing cells is HMM and is converted to LMM form after RNase A treatment. “IB” indicates immunoblotting with the indicated antibody.

    Journal: PLoS Pathogens

    Article Title: Newly Synthesized APOBEC3G Is Incorporated into HIV Virions, Inhibited by HIV RNA, and Subsequently Activated by RNase H

    doi: 10.1371/journal.ppat.0030015

    Figure Lengend Snippet: Virion-Incorporated HA-A3G Associates with Viral Genomic RNA (A) Viral genomic RNA, detected by RT-PCR, was detected in virions and virus-producing cells but not in lysates of uninfected cells. Genomic RNA was also detected in the IVAC derived from virions (fraction 7) and coimmunoprecipitated with HA-A3G from both virions and producer cell lysates. RT was performed using RNA derived from either whole lysates (L) or anti-HA immunoprecipitates (IP). Control reactions were performed in the absence of RT (–RT). Control PCRs were performed using proviral plasmid DNA, in the absence or presence of Taq, as indicated. (B) Viral genomic RNA, detected by RT-PCR, was assessed from size-fractionated virion lysates that lacked (HA) or contained HA-A3G. Amplicons generated probed across the TAR/Gag region or Pol/Vpu regions, as indicated. (C) Incorporation of HA-A3G into virions enhances the recruitment of NC into the IVAC. (D) HA-A3G from virus-producing cells is HMM and is converted to LMM form after RNase A treatment. “IB” indicates immunoblotting with the indicated antibody.

    Article Snippet: Labeled oligonucleotides and input samples were incubated in 20 μl of 50 mM Tris buffer (pH 7.4), with or without RNase A (1 μg) at 37 °C for 3 h unless otherwise indicated.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Plasmid Preparation, Generated

    Intravirion A3G Enzymatic Activity Is Negatively Regulated by Binding to Genomic HIV RNA (A) HA-A3G was immunoprecipitated from IVAC fraction 7 (F7) of virion lysates ( Figure 3 A) or from a lower fraction, F17, generated by treatment of the virion lysates with RNase A ( Figure 3 B). Immunoprecipitates (IPs) were tested for enzymatic activity in an in vitro deoxycytidine deaminase assay with or without RNase A addition and contained equivalent amounts of HA-A3G as shown in the corresponding immunoblot. The generation of a shorter cleavage product from the input ssDNA substrate reveals A3G deoxycytidine deaminase activity. Data shown are representative of multiple experiments. (B) Lysates of virions containing or lacking A3G were assessed in the deaminase assay, with or without RNase A treatment. (C) Lysates of virions containing increasing amounts of HA-A3G (as shown in the corresponding immunoblot) were assessed in the deaminase assay, with or without RNase A treatment. The asterisk marks bleed-through of marker loaded to the left of the samples. The triangles represent the increasing dose of A3G relative to provirus and correspond to the sample numbers presented in Figure 1 A. (A–C) All deaminase reactions were carried out in 50 mM Tris (pH 7.4) with (+) or without (−) RNase A, as indicated. (D) IPs of HMM or LMM HA-A3G from producer cell lysates were similarly assessed in the deaminase assay, with (+) or without (−) added RNase A. The IPs contained equivalent amounts of HA-A3G as shown in the corresponding immunoblot (IB).

    Journal: PLoS Pathogens

    Article Title: Newly Synthesized APOBEC3G Is Incorporated into HIV Virions, Inhibited by HIV RNA, and Subsequently Activated by RNase H

    doi: 10.1371/journal.ppat.0030015

    Figure Lengend Snippet: Intravirion A3G Enzymatic Activity Is Negatively Regulated by Binding to Genomic HIV RNA (A) HA-A3G was immunoprecipitated from IVAC fraction 7 (F7) of virion lysates ( Figure 3 A) or from a lower fraction, F17, generated by treatment of the virion lysates with RNase A ( Figure 3 B). Immunoprecipitates (IPs) were tested for enzymatic activity in an in vitro deoxycytidine deaminase assay with or without RNase A addition and contained equivalent amounts of HA-A3G as shown in the corresponding immunoblot. The generation of a shorter cleavage product from the input ssDNA substrate reveals A3G deoxycytidine deaminase activity. Data shown are representative of multiple experiments. (B) Lysates of virions containing or lacking A3G were assessed in the deaminase assay, with or without RNase A treatment. (C) Lysates of virions containing increasing amounts of HA-A3G (as shown in the corresponding immunoblot) were assessed in the deaminase assay, with or without RNase A treatment. The asterisk marks bleed-through of marker loaded to the left of the samples. The triangles represent the increasing dose of A3G relative to provirus and correspond to the sample numbers presented in Figure 1 A. (A–C) All deaminase reactions were carried out in 50 mM Tris (pH 7.4) with (+) or without (−) RNase A, as indicated. (D) IPs of HMM or LMM HA-A3G from producer cell lysates were similarly assessed in the deaminase assay, with (+) or without (−) added RNase A. The IPs contained equivalent amounts of HA-A3G as shown in the corresponding immunoblot (IB).

    Article Snippet: Labeled oligonucleotides and input samples were incubated in 20 μl of 50 mM Tris buffer (pH 7.4), with or without RNase A (1 μg) at 37 °C for 3 h unless otherwise indicated.

    Techniques: Activity Assay, Binding Assay, Immunoprecipitation, Generated, In Vitro, Marker

    Enzymatically Inactive Virion-Incorporated HA-A3G Is Activated by Viral RNase H (A) Recombinant RTs containing either a WT or mutant (E478Q) RNase H catalytic domain were assessed for RNase H activity in vitro in the absence or presence of the RNase H inhibitor Compound I (final concentration of 1, 10, or 100 μM). The RNA of an RNA–DNA hybrid remains intact unless RNase H digests the RNA into a smaller cleavage product that is distinguishable from the more complete cleavage product generated by RNase A. WT RNase H cannot digest ssDNA or DNA of an RNA–DNA hybrid, or RNA–RNA hybrids (data not shown). RNase H assays were performed in RNase H buffer (50 mM Tris [pH 8.0], 60 mM KCl) with (+) or without (−) 5 mM MgCl 2 or RNase A, as indicated. (B) Viruses bearing the RNase H E478Q mutation are compromised for in vitro RNase H activity. RNase H assays were performed in RNase H buffer with (+) or without (−) 5 mM MgCl 2 or RNase A, as indicated. (C) Virion lysates were subjected to endogenous reverse transcription (enRT) conditions with or without Compound I (final concentration of 0.1, 1, 10, or 100 μM), and A3G activity in these samples assessed in the in vitro deoxycytidine deaminase assay. Deaminase assays were performed in RNase H buffer either supplemented (enRT:+) or not (enRT:−) with 4 mM MgCl 2 and 1 mM dNTPs. (D) Compound I does not inhibit the intrinsic deoxycytidine deaminase activity of A3G. HA-A3G from RNase A–treated virion lysates was assessed for in vitro deaminase activity in the presence of increasing doses of Compound I (0.1, 1, 10, and 100 μM). Deaminase assay was performed in RNase H buffer supplemented with RNase A only. (E) Virions containing WT RNase H or the E478Q mutation in the RNase H catalytic domain were subjected to the enRT reaction followed by assessment of A3G enzymatic activity. Deaminase assays were performed in RNase H buffer either supplemented (enRT:+) or not (enRT:−) with 4 mM MgCl 2 and 1 mM dNTPs. (F) WT and RNase H–compromised ΔVif virions containing WT or mutant RNase H displayed equivalent A3G activity when RNase A was added to the virion lysate. Deaminase assay was performed in RNase H buffer with (+) or without (−) RNase A, as indicated. All data are representative of multiple experiments.

    Journal: PLoS Pathogens

    Article Title: Newly Synthesized APOBEC3G Is Incorporated into HIV Virions, Inhibited by HIV RNA, and Subsequently Activated by RNase H

    doi: 10.1371/journal.ppat.0030015

    Figure Lengend Snippet: Enzymatically Inactive Virion-Incorporated HA-A3G Is Activated by Viral RNase H (A) Recombinant RTs containing either a WT or mutant (E478Q) RNase H catalytic domain were assessed for RNase H activity in vitro in the absence or presence of the RNase H inhibitor Compound I (final concentration of 1, 10, or 100 μM). The RNA of an RNA–DNA hybrid remains intact unless RNase H digests the RNA into a smaller cleavage product that is distinguishable from the more complete cleavage product generated by RNase A. WT RNase H cannot digest ssDNA or DNA of an RNA–DNA hybrid, or RNA–RNA hybrids (data not shown). RNase H assays were performed in RNase H buffer (50 mM Tris [pH 8.0], 60 mM KCl) with (+) or without (−) 5 mM MgCl 2 or RNase A, as indicated. (B) Viruses bearing the RNase H E478Q mutation are compromised for in vitro RNase H activity. RNase H assays were performed in RNase H buffer with (+) or without (−) 5 mM MgCl 2 or RNase A, as indicated. (C) Virion lysates were subjected to endogenous reverse transcription (enRT) conditions with or without Compound I (final concentration of 0.1, 1, 10, or 100 μM), and A3G activity in these samples assessed in the in vitro deoxycytidine deaminase assay. Deaminase assays were performed in RNase H buffer either supplemented (enRT:+) or not (enRT:−) with 4 mM MgCl 2 and 1 mM dNTPs. (D) Compound I does not inhibit the intrinsic deoxycytidine deaminase activity of A3G. HA-A3G from RNase A–treated virion lysates was assessed for in vitro deaminase activity in the presence of increasing doses of Compound I (0.1, 1, 10, and 100 μM). Deaminase assay was performed in RNase H buffer supplemented with RNase A only. (E) Virions containing WT RNase H or the E478Q mutation in the RNase H catalytic domain were subjected to the enRT reaction followed by assessment of A3G enzymatic activity. Deaminase assays were performed in RNase H buffer either supplemented (enRT:+) or not (enRT:−) with 4 mM MgCl 2 and 1 mM dNTPs. (F) WT and RNase H–compromised ΔVif virions containing WT or mutant RNase H displayed equivalent A3G activity when RNase A was added to the virion lysate. Deaminase assay was performed in RNase H buffer with (+) or without (−) RNase A, as indicated. All data are representative of multiple experiments.

    Article Snippet: Labeled oligonucleotides and input samples were incubated in 20 μl of 50 mM Tris buffer (pH 7.4), with or without RNase A (1 μg) at 37 °C for 3 h unless otherwise indicated.

    Techniques: Recombinant, Mutagenesis, Activity Assay, In Vitro, Concentration Assay, Generated

    Virion-Incorporated HA-A3G Resides in a Large RNase A–Sensitive Complex and Biochemically Fractionates with Viral RNP Proteins (A) Virions collected from cells expressing HIV-1ΔVif contain HA-A3G that predominantly fractionates in a large complex (fractions 6 to 8) as assessed by gel filtration. (B) The IVAC is sensitive to RNase A treatment which shifts HA-A3G into lower fractions (fractions 15 to 19). (C) Virion cores obtained in Figure 1 were subjected to further biochemical fractionation to generate viral RNPs. Shown are the viral RNPs from virions either lacking or containing A3G, as indicated, and containing viral RT, IN, and NC but not p24-CA, as detected by immunoblotting (IB). The triangles represent the increasing dose of A3G relative to provirus and correspond exactly to the sample numbers in Figure 1 A.

    Journal: PLoS Pathogens

    Article Title: Newly Synthesized APOBEC3G Is Incorporated into HIV Virions, Inhibited by HIV RNA, and Subsequently Activated by RNase H

    doi: 10.1371/journal.ppat.0030015

    Figure Lengend Snippet: Virion-Incorporated HA-A3G Resides in a Large RNase A–Sensitive Complex and Biochemically Fractionates with Viral RNP Proteins (A) Virions collected from cells expressing HIV-1ΔVif contain HA-A3G that predominantly fractionates in a large complex (fractions 6 to 8) as assessed by gel filtration. (B) The IVAC is sensitive to RNase A treatment which shifts HA-A3G into lower fractions (fractions 15 to 19). (C) Virion cores obtained in Figure 1 were subjected to further biochemical fractionation to generate viral RNPs. Shown are the viral RNPs from virions either lacking or containing A3G, as indicated, and containing viral RT, IN, and NC but not p24-CA, as detected by immunoblotting (IB). The triangles represent the increasing dose of A3G relative to provirus and correspond exactly to the sample numbers in Figure 1 A.

    Article Snippet: Labeled oligonucleotides and input samples were incubated in 20 μl of 50 mM Tris buffer (pH 7.4), with or without RNase A (1 μg) at 37 °C for 3 h unless otherwise indicated.

    Techniques: Expressing, Filtration, Fractionation

    Genomic distribution of reads from TDP-43 RNA library. A , Western blot ( IB ) of fractions from HeLa nuclear extracts (± RNase A) applied to a size exclusion column, blotted for TDP-43, hnRNPA1, and lamin A/C. Fraction 6 = blue dextran (2000 kDa), fraction 30 = apoferritin (443 kDa), fraction 40 = alcohol dehydrogenase (150 kDa), and fraction 54 = bovine serum albumin (54 kDa) (data not shown).  B , Western blot of fractions from rat brain nuclear extracts (± micrococcal nuclease (±  MNase )) blotted for TDP-43. Fraction 5 = blue dextran (2,000 kDa), fraction 45 = bovine serum albumin (54 kDa), and fraction 54 = carbonic anhydrase (29 kDa) (data not shown). Note that different size exclusion columns were used in  A  and  B. C ,  panel i , diagram of TDP-43 RIP method.  C ,  panel ii , representative Western blot of TDP-43 RIP.  IP:CTL , control immunoprecipitation.  D , distribution of raw reads from the TDP-43 library mapped to exonic and intronic genes regions.  CDS , coding sequence.  E , read density, number of reads per 1,000 mappable nucleotides per million reads ( mRPKM ) of gene regions from the TDP-43 library.

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Neuronal RNA Targets of TDP-43-containing Ribonucleoprotein Complexes *

    doi: 10.1074/jbc.M110.190884

    Figure Lengend Snippet: Genomic distribution of reads from TDP-43 RNA library. A , Western blot ( IB ) of fractions from HeLa nuclear extracts (± RNase A) applied to a size exclusion column, blotted for TDP-43, hnRNPA1, and lamin A/C. Fraction 6 = blue dextran (2000 kDa), fraction 30 = apoferritin (443 kDa), fraction 40 = alcohol dehydrogenase (150 kDa), and fraction 54 = bovine serum albumin (54 kDa) (data not shown). B , Western blot of fractions from rat brain nuclear extracts (± micrococcal nuclease (± MNase )) blotted for TDP-43. Fraction 5 = blue dextran (2,000 kDa), fraction 45 = bovine serum albumin (54 kDa), and fraction 54 = carbonic anhydrase (29 kDa) (data not shown). Note that different size exclusion columns were used in A and B. C , panel i , diagram of TDP-43 RIP method. C , panel ii , representative Western blot of TDP-43 RIP. IP:CTL , control immunoprecipitation. D , distribution of raw reads from the TDP-43 library mapped to exonic and intronic genes regions. CDS , coding sequence. E , read density, number of reads per 1,000 mappable nucleotides per million reads ( mRPKM ) of gene regions from the TDP-43 library.

    Article Snippet: We show that with the addition of RNase A, TDP-43 elution in the high molecular mass range could be shifted to the lower molecular mass range ( A ).

    Techniques: Western Blot, CTL Assay, Immunoprecipitation, Sequencing