rnase h Thermo Fisher Search Results


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    Thermo Fisher rnase h rt
    RT-PCR in the analyses of naturally occurring endogenous sense-antisense RNA pair expression .  (a)  Schematic of the cardiac MHC (MYH) gene locus and its transcription products. The upper strand transcribes the cardiac MYH7 and MYH6 sense RNA, the lower strand transcribes the antisense MYH7 RNA, which is abundant in normal control hearts [1].  (b) : representative gels obtained from RT-PCR targeting sense and antisense RNA corresponding to the MYH7 gene. RT used RNase H -  enzyme under manufacturer standard conditions (see methods) in presense of specific primers (+p) or in absence of primers (-p).  (c)  Bar graph depicting the net signal of MYH7 sense and antisense in each group, net consisting of the difference between +p and -p RT-PCR band intensity. Note that a normal control heart in the rat is associated with abundant relatively MYH7 gene expression (MYH6 gene expression is dominant). Under the PTU condition, MYH7sense RNA expression is increased. Antisense MYH7 RNA is strongly expressed in the normal control heart, based on strong net signal. In PTU heart, the antisense MYH7 RNA is decreased to a very low level. Note the +p product is similar to -p when targeting antisense MYH7 RNA in PTU hearts.  (d)  Bar graph depicting relative no-primer signal (NP) to the total signal in each group as determined by real time PCR methods.  (e)  Net MYH7 sense and antisense RNA copy numbers in NC and PTU hearts using real time PCR. Data are means ± SE. N = 6/group. See Additional file 4 for primer information. For both sense and antisense MYH7 targets, end-point PCR (b and c) used 0.2 μl of the cDNA and was performed for 28 cycles. For real time PCR, we used 320 nl cDNA for each sample, and the signal was compared to a standard curve established with a serial dilution of a standard consisting of purified PCR product as explained in the methods. See Additional file 4 for primer information. Based on standard curve linear regression analyses, copies for each target RNA were calculated.+p: a strand specific RT primer was included; -p: RT without primer. Sense is the amplification product of the sense target obtained when the reverse primer was added to the RT reaction. Antisense is the amplification product of the antisense target obtained when the forward primer was included in the RT reaction. In all these reactions, the presence of the no primer product depended on the presence of RNA and the RT enzyme, and was not formed in RT reactions that were carried out in the absence of the reverse transcriptase enzyme.
    Rnase H Rt, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Thermo Fisher v5 tagged rnase h1
    Both RNase H1 and P32 interact with mitochondrial DNA and pre-rRNA. ( A ) The positions of Probes and PCR primers for the human mitochondrial DNA. The DNA map was derived from published review   [65] . Two oligonucleotide probes specific to 12 S and 16 S mitochondria rRNA regions are shown in  Blue bars . Three sets of PCR probes corresponding to the A, B and C regions are indicated in  Green arrows . ( B ) RNase H1 and P32 bind mitochondrial DNA. Cell extracts were prepared from an HA-H1 stably expressing cell line (RNase H1), control HEK cells or HEK cells transfected with the HA-P32 expression plasmid (P32). Equal amounts of each extract were used for immunoprecipitation with anti-HA beads. Nucleic acids were extracted from the precipitated samples using phenol/chloroform and subjected to PCR analysis. The probe sets for PCR were shown in   Figure 6A . Genomic DNA from HEK cells that was used as a positive control. The PCR products were analyzed on 2% Agarose gels. ( C ) RNase H1 may interact with the mitochondrial rDNA region. The extracts from HA-H1 cell and control HEK cells were used for immunoprecipitation with HA-antibody. The precipitates were digested on beads with (+) or without (−) DNase I. The DNA associated with beads was then extracted and subjected to PCR analysis. The PCR products were separated in 2% agarose gel. ( D ) RNase H1 and P32 also co-immunoprecipitated with mitochondrial pre-rRNA. The same extracted nucleic acids from panel B were digested with DNase I. The RNA is used for reverse transcription with (+) or without (−) reverse transcriptase, followed by PCR amplification using different primer sets as indicated below the panels. PCR reaction using primers specific to U16 snoRNA was used as control.
    V5 Tagged Rnase H1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 77 stars, based on 2 article reviews
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    77
    Thermo Fisher rnase h reverse transcriptase first strand synthesis kit
    Transcription through a long GAA·TTC tract results in formation of an RNA·DNA hybrid. ( A ) The native gel mobility of supercoiled templates carrying 0, 11 or 44 GAA·TTC triplets is shown in the first three lanes and after transcription by T7 RNAP in the second three lanes. The RNA product partially obscures the templates. Gel mobilities of relaxed plasmids (gray arrowhead) and supercoiled plasmids (black arrowhead) are indicated. ( B ) Treatment with RNase H after transcription (first three lanes) returns the (GAA·TTC) 44  template to control mobility. Treatment with the single-strand-specific RNases A and T1 (last three lanes) reveals conformers of the (GAA·TTC) 44  template (small arrows) with mobilities approaching that of a fully relaxed template (gray arrowhead). The degree of relaxation reflects the length of the RNA·DNA hybrid, which unwinds negative supercoils as indicated in the schematic to the right of the arrows. In contrast, templates with 0 or 11 triplets retain the mobility of untranscribed controls, regardless of treatment.
    Rnase H Reverse Transcriptase First Strand Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    RT-PCR in the analyses of naturally occurring endogenous sense-antisense RNA pair expression .  (a)  Schematic of the cardiac MHC (MYH) gene locus and its transcription products. The upper strand transcribes the cardiac MYH7 and MYH6 sense RNA, the lower strand transcribes the antisense MYH7 RNA, which is abundant in normal control hearts [1].  (b) : representative gels obtained from RT-PCR targeting sense and antisense RNA corresponding to the MYH7 gene. RT used RNase H -  enzyme under manufacturer standard conditions (see methods) in presense of specific primers (+p) or in absence of primers (-p).  (c)  Bar graph depicting the net signal of MYH7 sense and antisense in each group, net consisting of the difference between +p and -p RT-PCR band intensity. Note that a normal control heart in the rat is associated with abundant relatively MYH7 gene expression (MYH6 gene expression is dominant). Under the PTU condition, MYH7sense RNA expression is increased. Antisense MYH7 RNA is strongly expressed in the normal control heart, based on strong net signal. In PTU heart, the antisense MYH7 RNA is decreased to a very low level. Note the +p product is similar to -p when targeting antisense MYH7 RNA in PTU hearts.  (d)  Bar graph depicting relative no-primer signal (NP) to the total signal in each group as determined by real time PCR methods.  (e)  Net MYH7 sense and antisense RNA copy numbers in NC and PTU hearts using real time PCR. Data are means ± SE. N = 6/group. See Additional file 4 for primer information. For both sense and antisense MYH7 targets, end-point PCR (b and c) used 0.2 μl of the cDNA and was performed for 28 cycles. For real time PCR, we used 320 nl cDNA for each sample, and the signal was compared to a standard curve established with a serial dilution of a standard consisting of purified PCR product as explained in the methods. See Additional file 4 for primer information. Based on standard curve linear regression analyses, copies for each target RNA were calculated.+p: a strand specific RT primer was included; -p: RT without primer. Sense is the amplification product of the sense target obtained when the reverse primer was added to the RT reaction. Antisense is the amplification product of the antisense target obtained when the forward primer was included in the RT reaction. In all these reactions, the presence of the no primer product depended on the presence of RNA and the RT enzyme, and was not formed in RT reactions that were carried out in the absence of the reverse transcriptase enzyme.

    Journal: BMC Biotechnology

    Article Title: Potential pitfalls in the accuracy of analysis of natural sense-antisense RNA pairs by reverse transcription-PCR

    doi: 10.1186/1472-6750-7-21

    Figure Lengend Snippet: RT-PCR in the analyses of naturally occurring endogenous sense-antisense RNA pair expression . (a) Schematic of the cardiac MHC (MYH) gene locus and its transcription products. The upper strand transcribes the cardiac MYH7 and MYH6 sense RNA, the lower strand transcribes the antisense MYH7 RNA, which is abundant in normal control hearts [1]. (b) : representative gels obtained from RT-PCR targeting sense and antisense RNA corresponding to the MYH7 gene. RT used RNase H - enzyme under manufacturer standard conditions (see methods) in presense of specific primers (+p) or in absence of primers (-p). (c) Bar graph depicting the net signal of MYH7 sense and antisense in each group, net consisting of the difference between +p and -p RT-PCR band intensity. Note that a normal control heart in the rat is associated with abundant relatively MYH7 gene expression (MYH6 gene expression is dominant). Under the PTU condition, MYH7sense RNA expression is increased. Antisense MYH7 RNA is strongly expressed in the normal control heart, based on strong net signal. In PTU heart, the antisense MYH7 RNA is decreased to a very low level. Note the +p product is similar to -p when targeting antisense MYH7 RNA in PTU hearts. (d) Bar graph depicting relative no-primer signal (NP) to the total signal in each group as determined by real time PCR methods. (e) Net MYH7 sense and antisense RNA copy numbers in NC and PTU hearts using real time PCR. Data are means ± SE. N = 6/group. See Additional file 4 for primer information. For both sense and antisense MYH7 targets, end-point PCR (b and c) used 0.2 μl of the cDNA and was performed for 28 cycles. For real time PCR, we used 320 nl cDNA for each sample, and the signal was compared to a standard curve established with a serial dilution of a standard consisting of purified PCR product as explained in the methods. See Additional file 4 for primer information. Based on standard curve linear regression analyses, copies for each target RNA were calculated.+p: a strand specific RT primer was included; -p: RT without primer. Sense is the amplification product of the sense target obtained when the reverse primer was added to the RT reaction. Antisense is the amplification product of the antisense target obtained when the forward primer was included in the RT reaction. In all these reactions, the presence of the no primer product depended on the presence of RNA and the RT enzyme, and was not formed in RT reactions that were carried out in the absence of the reverse transcriptase enzyme.

    Article Snippet: To answer the question on how the RNase H activity of the reverse transcriptase or the reaction temperature may affect the RNA strand-specificity of cDNA synthesis, we compared RNase H positive (+ ) RT (Omniscript, Qiagen) vs. RNase H- RT (Superscript II, Invitrogen) specificity at two different temperatures, 44°C and 50°C, basing the comparison on RT-PCR results when the RT is carried out in the absence of primers.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, RNA Expression, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Serial Dilution, Purification, Amplification

    Testing different RT enzyme properties and conditions .  (a)  In a two-step RT-PCR system, 2 μg total RNA from NC and PTU treated hearts were reverse transcribed in 20 μl reactions in absence (-p) or presence (+p) of RT primers. The RT primer targeted the MYH7 sense RNA, and the PCR primer set amplified a 284 bp product corresponding to the 3' end of the MYH7 gene. PCR used 1 μl cDNA and was carried out for either 28 or 30 cycles. Shown are results from using two different RT enzymes that differed by their RNase H properties. RNase H -  and RNase H + . For each enzyme, the RT reactions were carried out under two different temperatures: 44°C or 50°C for 30 minutes/ea.  (b)  RT-PCR targeting the antisense MYH7 RNA in total RNA mixes of known proportions of sense and antisense RNA. RNA template contained either only sense MYH7 RNA, or a mix of sense and antisense MYH7 RNA corresponding to 99 to1 or 90 to 10 sense to antisense ratios (S:AS). Soleus total RNA was used as a source of the sense MYH7 RNA in absence of antisense. Whereas, T3-treated heart total RNA was used as a source of the antisense MYH7 RNA without co-expression of the sense. Mixes of soleus and T3 treated heart RNA were used to achieve the noted S:AS amounts in 2 μg of total RNA per 20 μl reactions. Reverse transcriptions were carried out in absence of RT primers (-P), in presence of the forward primer (+F) targeting the antisense, and in presence of a non specific primer corresponding to the 3' untranslated region of the human MYH4 mRNA sequence (+N). RT reactions used RNase H -  RT (Invitrogen), performed at 44°C or at 50°C for 30 min. PCR was carried out on 1 μcDNA for 28 cycles targeting the 3' end of the MYH7 gene. See Additional file 4 for primers information.

    Journal: BMC Biotechnology

    Article Title: Potential pitfalls in the accuracy of analysis of natural sense-antisense RNA pairs by reverse transcription-PCR

    doi: 10.1186/1472-6750-7-21

    Figure Lengend Snippet: Testing different RT enzyme properties and conditions . (a) In a two-step RT-PCR system, 2 μg total RNA from NC and PTU treated hearts were reverse transcribed in 20 μl reactions in absence (-p) or presence (+p) of RT primers. The RT primer targeted the MYH7 sense RNA, and the PCR primer set amplified a 284 bp product corresponding to the 3' end of the MYH7 gene. PCR used 1 μl cDNA and was carried out for either 28 or 30 cycles. Shown are results from using two different RT enzymes that differed by their RNase H properties. RNase H - and RNase H + . For each enzyme, the RT reactions were carried out under two different temperatures: 44°C or 50°C for 30 minutes/ea. (b) RT-PCR targeting the antisense MYH7 RNA in total RNA mixes of known proportions of sense and antisense RNA. RNA template contained either only sense MYH7 RNA, or a mix of sense and antisense MYH7 RNA corresponding to 99 to1 or 90 to 10 sense to antisense ratios (S:AS). Soleus total RNA was used as a source of the sense MYH7 RNA in absence of antisense. Whereas, T3-treated heart total RNA was used as a source of the antisense MYH7 RNA without co-expression of the sense. Mixes of soleus and T3 treated heart RNA were used to achieve the noted S:AS amounts in 2 μg of total RNA per 20 μl reactions. Reverse transcriptions were carried out in absence of RT primers (-P), in presence of the forward primer (+F) targeting the antisense, and in presence of a non specific primer corresponding to the 3' untranslated region of the human MYH4 mRNA sequence (+N). RT reactions used RNase H - RT (Invitrogen), performed at 44°C or at 50°C for 30 min. PCR was carried out on 1 μcDNA for 28 cycles targeting the 3' end of the MYH7 gene. See Additional file 4 for primers information.

    Article Snippet: To answer the question on how the RNase H activity of the reverse transcriptase or the reaction temperature may affect the RNA strand-specificity of cDNA synthesis, we compared RNase H positive (+ ) RT (Omniscript, Qiagen) vs. RNase H- RT (Superscript II, Invitrogen) specificity at two different temperatures, 44°C and 50°C, basing the comparison on RT-PCR results when the RT is carried out in the absence of primers.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Expressing, Sequencing

    Both RNase H1 and P32 interact with mitochondrial DNA and pre-rRNA. ( A ) The positions of Probes and PCR primers for the human mitochondrial DNA. The DNA map was derived from published review   [65] . Two oligonucleotide probes specific to 12 S and 16 S mitochondria rRNA regions are shown in  Blue bars . Three sets of PCR probes corresponding to the A, B and C regions are indicated in  Green arrows . ( B ) RNase H1 and P32 bind mitochondrial DNA. Cell extracts were prepared from an HA-H1 stably expressing cell line (RNase H1), control HEK cells or HEK cells transfected with the HA-P32 expression plasmid (P32). Equal amounts of each extract were used for immunoprecipitation with anti-HA beads. Nucleic acids were extracted from the precipitated samples using phenol/chloroform and subjected to PCR analysis. The probe sets for PCR were shown in   Figure 6A . Genomic DNA from HEK cells that was used as a positive control. The PCR products were analyzed on 2% Agarose gels. ( C ) RNase H1 may interact with the mitochondrial rDNA region. The extracts from HA-H1 cell and control HEK cells were used for immunoprecipitation with HA-antibody. The precipitates were digested on beads with (+) or without (−) DNase I. The DNA associated with beads was then extracted and subjected to PCR analysis. The PCR products were separated in 2% agarose gel. ( D ) RNase H1 and P32 also co-immunoprecipitated with mitochondrial pre-rRNA. The same extracted nucleic acids from panel B were digested with DNase I. The RNA is used for reverse transcription with (+) or without (−) reverse transcriptase, followed by PCR amplification using different primer sets as indicated below the panels. PCR reaction using primers specific to U16 snoRNA was used as control.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: Both RNase H1 and P32 interact with mitochondrial DNA and pre-rRNA. ( A ) The positions of Probes and PCR primers for the human mitochondrial DNA. The DNA map was derived from published review [65] . Two oligonucleotide probes specific to 12 S and 16 S mitochondria rRNA regions are shown in Blue bars . Three sets of PCR probes corresponding to the A, B and C regions are indicated in Green arrows . ( B ) RNase H1 and P32 bind mitochondrial DNA. Cell extracts were prepared from an HA-H1 stably expressing cell line (RNase H1), control HEK cells or HEK cells transfected with the HA-P32 expression plasmid (P32). Equal amounts of each extract were used for immunoprecipitation with anti-HA beads. Nucleic acids were extracted from the precipitated samples using phenol/chloroform and subjected to PCR analysis. The probe sets for PCR were shown in Figure 6A . Genomic DNA from HEK cells that was used as a positive control. The PCR products were analyzed on 2% Agarose gels. ( C ) RNase H1 may interact with the mitochondrial rDNA region. The extracts from HA-H1 cell and control HEK cells were used for immunoprecipitation with HA-antibody. The precipitates were digested on beads with (+) or without (−) DNase I. The DNA associated with beads was then extracted and subjected to PCR analysis. The PCR products were separated in 2% agarose gel. ( D ) RNase H1 and P32 also co-immunoprecipitated with mitochondrial pre-rRNA. The same extracted nucleic acids from panel B were digested with DNase I. The RNA is used for reverse transcription with (+) or without (−) reverse transcriptase, followed by PCR amplification using different primer sets as indicated below the panels. PCR reaction using primers specific to U16 snoRNA was used as control.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Polymerase Chain Reaction, Derivative Assay, Hemagglutination Assay, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Positive Control, Agarose Gel Electrophoresis, Amplification

    Depletion of RNase H1 or P32 resulted in accumulation of mitochondrial pre-12S/16S rRNA. HeLa cells were treated with 2 nM or 20 nM of RNase H1-siRNA or P32 –siRNA for 24 or 48 hours. ( A ) The mRNA levels of RNase H1 and P32 were determined by qRT-PCR 24 hrs after siRNA treatment. ( B ) Protein levels of RNase H1 and P32 were analyzed by western analysis 24 hours post siRNA treatment. ( C ) Reduction of RNase H1 or P32 significantly increased the level of mitochondrial pre-rRNA. HeLa cells were treated with either RNase H1-siRNA (2 nM) or P32-siRNA (2 nM) for 24 hours. Total RNA was prepared and subjected to Northern analysis with  32 P labeled probes specific to 12S or 16S rRNAs. U3 snoRNA was detected and served as a control. The relative levels of pre-rRNA were measured from the results obtained with 12 S probe, normalized to U3, and plotted in the right panel. The error bars indicate standard error of the three replicates. (D) RT-PCR assay for the levels of pre-16 S and pre-ND3 RNAs. Total RNA prepared from HeLa cells treated for 24 hrs with corresponding siRNAs was analyzed by qRT-PCR, using primer probe sets specific to the tRNA Val -16 S rRNA junction (pre-16 S) or to the tRNA Gly -ND3 junction (pre-ND3). The error bars represent standard deviation of three replicates.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: Depletion of RNase H1 or P32 resulted in accumulation of mitochondrial pre-12S/16S rRNA. HeLa cells were treated with 2 nM or 20 nM of RNase H1-siRNA or P32 –siRNA for 24 or 48 hours. ( A ) The mRNA levels of RNase H1 and P32 were determined by qRT-PCR 24 hrs after siRNA treatment. ( B ) Protein levels of RNase H1 and P32 were analyzed by western analysis 24 hours post siRNA treatment. ( C ) Reduction of RNase H1 or P32 significantly increased the level of mitochondrial pre-rRNA. HeLa cells were treated with either RNase H1-siRNA (2 nM) or P32-siRNA (2 nM) for 24 hours. Total RNA was prepared and subjected to Northern analysis with 32 P labeled probes specific to 12S or 16S rRNAs. U3 snoRNA was detected and served as a control. The relative levels of pre-rRNA were measured from the results obtained with 12 S probe, normalized to U3, and plotted in the right panel. The error bars indicate standard error of the three replicates. (D) RT-PCR assay for the levels of pre-16 S and pre-ND3 RNAs. Total RNA prepared from HeLa cells treated for 24 hrs with corresponding siRNAs was analyzed by qRT-PCR, using primer probe sets specific to the tRNA Val -16 S rRNA junction (pre-16 S) or to the tRNA Gly -ND3 junction (pre-ND3). The error bars represent standard deviation of three replicates.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Quantitative RT-PCR, Western Blot, Northern Blot, Labeling, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

    Co-localization of P32 and RNase H1. ( A ) Immunofluorescence Staining of P32 and RNase H1. Upper panel: HeLa cells were stained for endogenous P32 and RNase H1 using mouse monoclonal anti-P32 antibody and rabbit anti-RNase H1 antibody, respectively, followed by FITC conjugated donkey anti-mouse ( green ) and TRITC conjugated anti-rabbit secondary antibodies ( red ). Nuclei were stained with DAP1 ( Blue ) and Mitochondria were stained with mitotracker ( white ). Lower panel: HeLa cells were infected with adenovirus expressing RNase H1. Cells were stained as described in upper panel. ( B ) Subcellular fractionation of P32 protein. The proteins from sub-cellular compartments (cytosol, mitochondrial and ER membranes, nucleus and cytoskeleton) were prepared from HEK cells using proteome cell compartment kit (Qiagen). About 10 µg protein samples from each fraction were analyzed by western for P32. The same blot was stripped and tubulin-γ was detected to serve as a control.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: Co-localization of P32 and RNase H1. ( A ) Immunofluorescence Staining of P32 and RNase H1. Upper panel: HeLa cells were stained for endogenous P32 and RNase H1 using mouse monoclonal anti-P32 antibody and rabbit anti-RNase H1 antibody, respectively, followed by FITC conjugated donkey anti-mouse ( green ) and TRITC conjugated anti-rabbit secondary antibodies ( red ). Nuclei were stained with DAP1 ( Blue ) and Mitochondria were stained with mitotracker ( white ). Lower panel: HeLa cells were infected with adenovirus expressing RNase H1. Cells were stained as described in upper panel. ( B ) Subcellular fractionation of P32 protein. The proteins from sub-cellular compartments (cytosol, mitochondrial and ER membranes, nucleus and cytoskeleton) were prepared from HEK cells using proteome cell compartment kit (Qiagen). About 10 µg protein samples from each fraction were analyzed by western for P32. The same blot was stripped and tubulin-γ was detected to serve as a control.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Immunofluorescence, Staining, Infection, Expressing, Fractionation, Western Blot

    Recombinant P32 binds to recombinant RNase H1, enhances its turnover rate, and reduces the binding affinity of the enzyme for the heteroduplex substrate. ( A ) Coomassie blue staining of the purified human His-H1, GST protein, and GST-P32 proteins separated by SDS-PAGE. The sizes for the standard protein markers are indicated. ( B ) RNase H1 but not P32 appears to bind the heteroduplex substrate. Gel shift assay was performed using 0.4 ug purified RNase H1, GST-P32, or GST proteins incubated at 4°C for 30 min with a non-cleavable heteroduplex containing  32 P labeled uniformly modified 2′-fluoro RNA annealed to DNA and subjected to native gel electrophoresis. ( C ) The interaction between RNase H1 and P32 appears to be equal molar. A fixed amount of GST-P32 was bound to GST affinity beads and then incubated with increasing amounts of RNase H1. Glutathione (GSH) eluted RNase H1 and P32 were quantified by Western blot as described in the Material and Methods. The amounts of bead-bound P32 and P32-associated RNase H1 were determined by loading known amounts of the respective proteins (left panel). The molecular ratio of bound RNase H1 relative to P32 was calculated and plotted in the right panel. ( D ) The effects of ionic strength on RNase H1/P32 interaction. Left panel: RNase H1 binds GST-P32 but not GST protein. GST or GST-P32 bound to anti-GST beads was incubated with RNase H1 in NaCl concentrations ranging from 0-950 mM as described in the Material and Methods. Middle panel: increasing NaCl concentration inhibits binding of RNase H1 to P32. Both unbound (flow through) and bound (affinity eluted) fractions were collected and the levels of RNase H1 and P32 evaluated by western blot. Right panel: Increasing pH reduced binding of RNase H1 to P32. ( E ) Michaelis-Menten kinetics and binding constants for RNase H1 cleavage of an RNA/DNA duplex in the presence or absence of P32. The K m , V max , and K d  were determined by incubating the Apo B RNA/DNA duplex with RNase H1 plus GST (as control) or RNase H1 plus different amounts of P32 resulting in an H1:P32 ratio = 1∶1 or 1∶5. An uncleavable competitive inhibitor (2′-fluororibonucleotide/DNA) was used to determine the binding to the RNA/DNA duplex, as described in the Material and Methods. The calculated constants are indicated in the right panel. The error bars indicate the standard error from three parallel experiments.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: Recombinant P32 binds to recombinant RNase H1, enhances its turnover rate, and reduces the binding affinity of the enzyme for the heteroduplex substrate. ( A ) Coomassie blue staining of the purified human His-H1, GST protein, and GST-P32 proteins separated by SDS-PAGE. The sizes for the standard protein markers are indicated. ( B ) RNase H1 but not P32 appears to bind the heteroduplex substrate. Gel shift assay was performed using 0.4 ug purified RNase H1, GST-P32, or GST proteins incubated at 4°C for 30 min with a non-cleavable heteroduplex containing 32 P labeled uniformly modified 2′-fluoro RNA annealed to DNA and subjected to native gel electrophoresis. ( C ) The interaction between RNase H1 and P32 appears to be equal molar. A fixed amount of GST-P32 was bound to GST affinity beads and then incubated with increasing amounts of RNase H1. Glutathione (GSH) eluted RNase H1 and P32 were quantified by Western blot as described in the Material and Methods. The amounts of bead-bound P32 and P32-associated RNase H1 were determined by loading known amounts of the respective proteins (left panel). The molecular ratio of bound RNase H1 relative to P32 was calculated and plotted in the right panel. ( D ) The effects of ionic strength on RNase H1/P32 interaction. Left panel: RNase H1 binds GST-P32 but not GST protein. GST or GST-P32 bound to anti-GST beads was incubated with RNase H1 in NaCl concentrations ranging from 0-950 mM as described in the Material and Methods. Middle panel: increasing NaCl concentration inhibits binding of RNase H1 to P32. Both unbound (flow through) and bound (affinity eluted) fractions were collected and the levels of RNase H1 and P32 evaluated by western blot. Right panel: Increasing pH reduced binding of RNase H1 to P32. ( E ) Michaelis-Menten kinetics and binding constants for RNase H1 cleavage of an RNA/DNA duplex in the presence or absence of P32. The K m , V max , and K d were determined by incubating the Apo B RNA/DNA duplex with RNase H1 plus GST (as control) or RNase H1 plus different amounts of P32 resulting in an H1:P32 ratio = 1∶1 or 1∶5. An uncleavable competitive inhibitor (2′-fluororibonucleotide/DNA) was used to determine the binding to the RNA/DNA duplex, as described in the Material and Methods. The calculated constants are indicated in the right panel. The error bars indicate the standard error from three parallel experiments.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Recombinant, Binding Assay, Staining, Purification, SDS Page, Electrophoretic Mobility Shift Assay, Incubation, Labeling, Modification, Nucleic Acid Electrophoresis, Western Blot, Concentration Assay, Flow Cytometry

    P32 appears to interact with the N-terminal duplex binding domain of RNase H1. ( A ) Expression and purification of RNase H1 deletion mutants. Left panel: Schematic depiction of the different human RNase H1 deletion mutants. DL1 deletes the hybrid binding domain (amino acid positions 1–73); DL2 deletes both the hybrid binding domain and the spacer domain (amino acid 1–129). The black bars at the N-terminus of each mutant represent a His tag. Right panel: Coomassie blue staining of the purified RNase H1 deletion mutants. The sizes of the standard markers are given. ( B ) Interaction of full length RNase H1 and its deletion mutants with P32. The full length or truncated RNase H1 proteins were incubated with GST-P32 bound to GST-beads under different NaCl concentrations ranging from 150–450 mM in both the binding and washing solutions. The P32 and RNase H1 or deletion mutants were eluted and analyzed by Western blot, using P32 or RNase H1 antibodies, respectively (right panel). Western blot to RNase H1 and deletion mutants DL1 and DL2 demonstrates that the mutant proteins are recognized by the RNase H1 antibody (left panel). ( C ) Michaelis-Menten Kinetics of DL-1 mutant in the presence or absence of P32. K m , V max , and k cat  for DL-1 plus GST or GST-P32 (DL-1:P32 = 1:5 in molecular ratio) were determined in 50 and 150 mM NaCl concentration with the Apo B RNA/DNA duplex as described in the Material and Methods.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: P32 appears to interact with the N-terminal duplex binding domain of RNase H1. ( A ) Expression and purification of RNase H1 deletion mutants. Left panel: Schematic depiction of the different human RNase H1 deletion mutants. DL1 deletes the hybrid binding domain (amino acid positions 1–73); DL2 deletes both the hybrid binding domain and the spacer domain (amino acid 1–129). The black bars at the N-terminus of each mutant represent a His tag. Right panel: Coomassie blue staining of the purified RNase H1 deletion mutants. The sizes of the standard markers are given. ( B ) Interaction of full length RNase H1 and its deletion mutants with P32. The full length or truncated RNase H1 proteins were incubated with GST-P32 bound to GST-beads under different NaCl concentrations ranging from 150–450 mM in both the binding and washing solutions. The P32 and RNase H1 or deletion mutants were eluted and analyzed by Western blot, using P32 or RNase H1 antibodies, respectively (right panel). Western blot to RNase H1 and deletion mutants DL1 and DL2 demonstrates that the mutant proteins are recognized by the RNase H1 antibody (left panel). ( C ) Michaelis-Menten Kinetics of DL-1 mutant in the presence or absence of P32. K m , V max , and k cat for DL-1 plus GST or GST-P32 (DL-1:P32 = 1:5 in molecular ratio) were determined in 50 and 150 mM NaCl concentration with the Apo B RNA/DNA duplex as described in the Material and Methods.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Binding Assay, Expressing, Purification, Mutagenesis, Staining, Incubation, Western Blot, Concentration Assay

    Human RNase H1 is associated with P32. ( A ) Western blot analysis of cell lysates and immunoprecipitated samples show Flag-tagged RNase H1 and H2 expression from cells stably transformed with RNase H1 (H1) or H2 (H2) or wild type (control) HEK cell lines. ( B ) Co-selection of RNase H1 binding proteins by immunoprecipitation. Extracts from cells expressing the Flag-H1, Flag-H2, or HA-H1 cell lines were immunoprecipitated with either anti-Flag or anti-HA antibody. Co-precipitated proteins were resolved by SDS-PAGE, and visualized by silver staining. Protein bands that were different from the co-precipitated proteins from control cells were subjected to mass spectrometry. The protein bands corresponding to the tagged RNase H1, H2 and the co-precipitated P32 proteins are indicated. The size marker was SeeBlue Plus2 Pre-Stained Standard (Invitrogen). ( C ) 2D gel electrophoresis of proteins co-precipitated with Flag-H1 or Flag-H2. About 5 mg cell lysates were prepared for immunoprecipitation with anti-flag beads from cell lines which stably express Flag-H1 or Flag-H2. The immunoprecipitates were washed four times with RIPA buffer and directly sent to Applied Biomics Inc. (San Francisco, CA) for 2D gel electrophoresis coupled with MS analysis. In brief, the co-precipitated proteins from Flag-H1 or Flag-H2 cells were labeled by fluorescent DIGE CyDyers, respectively, followed by 2D gel electrophoresis. The protein image was scanned with a fluorescence detector. The figure illustrates the proteins differentially associated with RNase H1 (green) or H2 (red). The P32 protein was confirmed with mass spectrum from the extracted gel sample. Circled spots were identified as RNase H1, H2 or P32 by mass spectrometric analysis. ( D ) Both endogenous and expressed RNase H1 are co-precipitated with the expressed P32. Left panel: western blots with P32, RNase H1, or H2 antibodies for proteins co-precipitated using anti-HA antibody from extracts of control HeLa cells or cells transfected with HA-P32 expression plasmid. Right panel: western blots for proteins co-selected using anti-HA antibody from extracts of Flag-H1, Flag-H2 stable cell lines and control cells, all of which were transfected with HA-P32 expression plasmid. ( E ) Confirmation of the specific interaction between RNase H1 and P32. RNase H cleavage activity indicates that the P32 co-immunoprecipitated material contains only RNase H1 enzyme activity. Upper panel: Cleavage patterns of human RNase H1 and H2 from IP-coupled enzyme activity assays. Immunoprecipitations were performed with either anti-flag, anti-RNase H1 or anti-H2 antibodies from extracts of Flag-H1, Flag-H2 expressing cells or control cells. The co-precipitated samples were incubated for the indicated times with a  32 P-labeled RNA/DNA-methoxyethyl (MOE) gapmer duplex and the cleavage products were separated using denaturing gel electrophoresis. The preferred cleavage sites of RNase H1 and H2 are indicated with * or #, respectively. The positions of the preferred cleavage sites in the heteroduplex are shown in the middle panel with the sequences of the RNA substrate (upper strand) and the oligonucleotide (lower strand). The bold nucleotides in the oligonucleotide strand indicate the position of the MOE substitutions. Lower panel: only the RNase H1 enzyme activity was detected in the co-precipitated material from lysates containing tagged P32. Immunoprecipitations were performed with anti-HA antibody from extracts of Flag-H1 or Flag-H2 stable cell lines or control HEK cells, which were all transfected or not transfected with HA-P32 expression plasmid. The precipitated samples were analyzed for cleavage patterns as described above. The position of the cleavage bands relative to the sequence of the cleavage products is shown on the left. A partial alkaline digestion of the same labeled RNA was used as a sequence ladder. The cleavage pattern of purified human RNase H1 is shown at the far right of the lower panel.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: Human RNase H1 is associated with P32. ( A ) Western blot analysis of cell lysates and immunoprecipitated samples show Flag-tagged RNase H1 and H2 expression from cells stably transformed with RNase H1 (H1) or H2 (H2) or wild type (control) HEK cell lines. ( B ) Co-selection of RNase H1 binding proteins by immunoprecipitation. Extracts from cells expressing the Flag-H1, Flag-H2, or HA-H1 cell lines were immunoprecipitated with either anti-Flag or anti-HA antibody. Co-precipitated proteins were resolved by SDS-PAGE, and visualized by silver staining. Protein bands that were different from the co-precipitated proteins from control cells were subjected to mass spectrometry. The protein bands corresponding to the tagged RNase H1, H2 and the co-precipitated P32 proteins are indicated. The size marker was SeeBlue Plus2 Pre-Stained Standard (Invitrogen). ( C ) 2D gel electrophoresis of proteins co-precipitated with Flag-H1 or Flag-H2. About 5 mg cell lysates were prepared for immunoprecipitation with anti-flag beads from cell lines which stably express Flag-H1 or Flag-H2. The immunoprecipitates were washed four times with RIPA buffer and directly sent to Applied Biomics Inc. (San Francisco, CA) for 2D gel electrophoresis coupled with MS analysis. In brief, the co-precipitated proteins from Flag-H1 or Flag-H2 cells were labeled by fluorescent DIGE CyDyers, respectively, followed by 2D gel electrophoresis. The protein image was scanned with a fluorescence detector. The figure illustrates the proteins differentially associated with RNase H1 (green) or H2 (red). The P32 protein was confirmed with mass spectrum from the extracted gel sample. Circled spots were identified as RNase H1, H2 or P32 by mass spectrometric analysis. ( D ) Both endogenous and expressed RNase H1 are co-precipitated with the expressed P32. Left panel: western blots with P32, RNase H1, or H2 antibodies for proteins co-precipitated using anti-HA antibody from extracts of control HeLa cells or cells transfected with HA-P32 expression plasmid. Right panel: western blots for proteins co-selected using anti-HA antibody from extracts of Flag-H1, Flag-H2 stable cell lines and control cells, all of which were transfected with HA-P32 expression plasmid. ( E ) Confirmation of the specific interaction between RNase H1 and P32. RNase H cleavage activity indicates that the P32 co-immunoprecipitated material contains only RNase H1 enzyme activity. Upper panel: Cleavage patterns of human RNase H1 and H2 from IP-coupled enzyme activity assays. Immunoprecipitations were performed with either anti-flag, anti-RNase H1 or anti-H2 antibodies from extracts of Flag-H1, Flag-H2 expressing cells or control cells. The co-precipitated samples were incubated for the indicated times with a 32 P-labeled RNA/DNA-methoxyethyl (MOE) gapmer duplex and the cleavage products were separated using denaturing gel electrophoresis. The preferred cleavage sites of RNase H1 and H2 are indicated with * or #, respectively. The positions of the preferred cleavage sites in the heteroduplex are shown in the middle panel with the sequences of the RNA substrate (upper strand) and the oligonucleotide (lower strand). The bold nucleotides in the oligonucleotide strand indicate the position of the MOE substitutions. Lower panel: only the RNase H1 enzyme activity was detected in the co-precipitated material from lysates containing tagged P32. Immunoprecipitations were performed with anti-HA antibody from extracts of Flag-H1 or Flag-H2 stable cell lines or control HEK cells, which were all transfected or not transfected with HA-P32 expression plasmid. The precipitated samples were analyzed for cleavage patterns as described above. The position of the cleavage bands relative to the sequence of the cleavage products is shown on the left. A partial alkaline digestion of the same labeled RNA was used as a sequence ladder. The cleavage pattern of purified human RNase H1 is shown at the far right of the lower panel.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Western Blot, Immunoprecipitation, Expressing, Stable Transfection, Transformation Assay, Selection, Binding Assay, Hemagglutination Assay, SDS Page, Silver Staining, Mass Spectrometry, Marker, Staining, Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, Fluorescence, Transfection, Plasmid Preparation, Activity Assay, Incubation, Nucleic Acid Electrophoresis, Sequencing, Purification

    Overexpression of rnh1 relieves replication pausing. A–D , 2DNAGE of four restriction fragments of Drosophila S2 cells mtDNA, probed as indicated, in material from control cells and cells overexpressing RNase H1 in the form of epitope-tagged RNase H1-V5 (denoted OE ), both treated with 500 μ m CuSO 4 for 48 h to induce expression. E , schematic map of Drosophila mtDNA, as also shown in Fig. 8 , indicating the location of relevant restriction sites ( open circles ), mTTF-binding sites (bs1 and bs2; filled circles ), the noncoding region ( bold ), and the probes used. The open arrowhead marks the location and direction of replication initiation (see Ref. 40 ). The directions of first- and second-dimension electrophoresis in all gels are as indicated by the arrows . The images show relatively low exposures to reveal fine details of the arcs of RIs.

    Journal: The Journal of Biological Chemistry

    Article Title: RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA

    doi: 10.1074/jbc.RA118.007015

    Figure Lengend Snippet: Overexpression of rnh1 relieves replication pausing. A–D , 2DNAGE of four restriction fragments of Drosophila S2 cells mtDNA, probed as indicated, in material from control cells and cells overexpressing RNase H1 in the form of epitope-tagged RNase H1-V5 (denoted OE ), both treated with 500 μ m CuSO 4 for 48 h to induce expression. E , schematic map of Drosophila mtDNA, as also shown in Fig. 8 , indicating the location of relevant restriction sites ( open circles ), mTTF-binding sites (bs1 and bs2; filled circles ), the noncoding region ( bold ), and the probes used. The open arrowhead marks the location and direction of replication initiation (see Ref. 40 ). The directions of first- and second-dimension electrophoresis in all gels are as indicated by the arrows . The images show relatively low exposures to reveal fine details of the arcs of RIs.

    Article Snippet: To establish cell clones stably expressing V5-tagged RNase H1 and variants, pCoBlast (Thermo Fisher Scientific) was included in transfections.

    Techniques: Over Expression, Expressing, Binding Assay, Electrophoresis

    Subcellular localization of epitope-tagged RNase H1. A , immunocytochemistry of cells transiently transfected with RNase H1-V5, probed for the V5 epitope tag ( red ), Cox4 ( green ), and DAPI ( blue ), showing examples of the three types of intracellular distribution of V5-tagged RNase H1: nucleus and mitochondria ( i ), mitochondria only ( ii ), and nucleus only ( iii ). B , subcellular distribution of RNase H1-V5 in 100 transfected cells as indicated (mean of three experiments, error bars denote S.D.). C , Western blots of subcellular fractions from cells transfected with RNase H1-V5, highly enriched for nuclei ( nuc ) or mitochondria ( mt ) as indicated, probed simultaneously for V5 and for the markers indicated. M , molecular mass markers.

    Journal: The Journal of Biological Chemistry

    Article Title: RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA

    doi: 10.1074/jbc.RA118.007015

    Figure Lengend Snippet: Subcellular localization of epitope-tagged RNase H1. A , immunocytochemistry of cells transiently transfected with RNase H1-V5, probed for the V5 epitope tag ( red ), Cox4 ( green ), and DAPI ( blue ), showing examples of the three types of intracellular distribution of V5-tagged RNase H1: nucleus and mitochondria ( i ), mitochondria only ( ii ), and nucleus only ( iii ). B , subcellular distribution of RNase H1-V5 in 100 transfected cells as indicated (mean of three experiments, error bars denote S.D.). C , Western blots of subcellular fractions from cells transfected with RNase H1-V5, highly enriched for nuclei ( nuc ) or mitochondria ( mt ) as indicated, probed simultaneously for V5 and for the markers indicated. M , molecular mass markers.

    Article Snippet: To establish cell clones stably expressing V5-tagged RNase H1 and variants, pCoBlast (Thermo Fisher Scientific) was included in transfections.

    Techniques: Immunocytochemistry, Transfection, Western Blot

    Subcellular targeting of RNase H1 variants. A , intracellular localization of RNase H1-V5 variants in cultures of stably transfected cells exemplified in B . M1V and M16V, N-terminal methionine variants (see Fig. S2 A ); ΔNLS, with the putative nuclear localization signal deleted (see Fig. S2 C ). C , intracellular localization of RNase H1-V5 in cells synchronized in G1 and G2 (see FACS profiles in Fig. S2 E ). All plotted values are means of three experiments. Error bars denote S.D. nuc , nuclei; mt , mitochondria.

    Journal: The Journal of Biological Chemistry

    Article Title: RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA

    doi: 10.1074/jbc.RA118.007015

    Figure Lengend Snippet: Subcellular targeting of RNase H1 variants. A , intracellular localization of RNase H1-V5 variants in cultures of stably transfected cells exemplified in B . M1V and M16V, N-terminal methionine variants (see Fig. S2 A ); ΔNLS, with the putative nuclear localization signal deleted (see Fig. S2 C ). C , intracellular localization of RNase H1-V5 in cells synchronized in G1 and G2 (see FACS profiles in Fig. S2 E ). All plotted values are means of three experiments. Error bars denote S.D. nuc , nuclei; mt , mitochondria.

    Article Snippet: To establish cell clones stably expressing V5-tagged RNase H1 and variants, pCoBlast (Thermo Fisher Scientific) was included in transfections.

    Techniques: Stable Transfection, Transfection, FACS

    Transcription through a long GAA·TTC tract results in formation of an RNA·DNA hybrid. ( A ) The native gel mobility of supercoiled templates carrying 0, 11 or 44 GAA·TTC triplets is shown in the first three lanes and after transcription by T7 RNAP in the second three lanes. The RNA product partially obscures the templates. Gel mobilities of relaxed plasmids (gray arrowhead) and supercoiled plasmids (black arrowhead) are indicated. ( B ) Treatment with RNase H after transcription (first three lanes) returns the (GAA·TTC) 44  template to control mobility. Treatment with the single-strand-specific RNases A and T1 (last three lanes) reveals conformers of the (GAA·TTC) 44  template (small arrows) with mobilities approaching that of a fully relaxed template (gray arrowhead). The degree of relaxation reflects the length of the RNA·DNA hybrid, which unwinds negative supercoils as indicated in the schematic to the right of the arrows. In contrast, templates with 0 or 11 triplets retain the mobility of untranscribed controls, regardless of treatment.

    Journal: Nucleic Acids Research

    Article Title: A persistent RNA?DNA hybrid formed by transcription of the Friedreich ataxia triplet repeat in live bacteria, and by T7 RNAP in vitro

    doi: 10.1093/nar/gkm589

    Figure Lengend Snippet: Transcription through a long GAA·TTC tract results in formation of an RNA·DNA hybrid. ( A ) The native gel mobility of supercoiled templates carrying 0, 11 or 44 GAA·TTC triplets is shown in the first three lanes and after transcription by T7 RNAP in the second three lanes. The RNA product partially obscures the templates. Gel mobilities of relaxed plasmids (gray arrowhead) and supercoiled plasmids (black arrowhead) are indicated. ( B ) Treatment with RNase H after transcription (first three lanes) returns the (GAA·TTC) 44 template to control mobility. Treatment with the single-strand-specific RNases A and T1 (last three lanes) reveals conformers of the (GAA·TTC) 44 template (small arrows) with mobilities approaching that of a fully relaxed template (gray arrowhead). The degree of relaxation reflects the length of the RNA·DNA hybrid, which unwinds negative supercoils as indicated in the schematic to the right of the arrows. In contrast, templates with 0 or 11 triplets retain the mobility of untranscribed controls, regardless of treatment.

    Article Snippet: The Superscript II, RNase H-reverse transcriptase first strand synthesis kit (Life Technologies) was then used following the manufacturers’ protocol.

    Techniques:

    RNA·DNA hybrids are associated with transcription arrest on TTC templates  in vitro . Pairs of transcription reactions were done in the absence (−) or presence (+) of RNase H on supercoiled templates containing the triplet repeat sequences (CTG·CAG) 88  (lanes 1 and 2), (GAA·TTC) 88  (lanes 3 and 4) and (TTC·GAA) 88  (lanes 5 and 6). The gel image shows the products of those reactions. To the right of the gel image the length in bases of select DNA size markers are indicated. At the far right of the figure is a scan of lanes 3 (dark line) and 4 (gray line) which highlights the RNase H mediated shift in truncation points from promoter distal (arrowhead) to promoter proximal within the GAA repeat. All the templates contain the sequence for a self-cleaving ribozyme that cuts the transcript 270 bases 3′ to the end of the repeat tract producing a full-length transcript of 590 bases. Transcription from the phage T7 promoter started 55 bases 5′ to the GAA·TTC insert. Transcripts were end-labeled by including gamma  32 P-GTP in the reaction.

    Journal: Nucleic Acids Research

    Article Title: A persistent RNA?DNA hybrid formed by transcription of the Friedreich ataxia triplet repeat in live bacteria, and by T7 RNAP in vitro

    doi: 10.1093/nar/gkm589

    Figure Lengend Snippet: RNA·DNA hybrids are associated with transcription arrest on TTC templates in vitro . Pairs of transcription reactions were done in the absence (−) or presence (+) of RNase H on supercoiled templates containing the triplet repeat sequences (CTG·CAG) 88 (lanes 1 and 2), (GAA·TTC) 88 (lanes 3 and 4) and (TTC·GAA) 88 (lanes 5 and 6). The gel image shows the products of those reactions. To the right of the gel image the length in bases of select DNA size markers are indicated. At the far right of the figure is a scan of lanes 3 (dark line) and 4 (gray line) which highlights the RNase H mediated shift in truncation points from promoter distal (arrowhead) to promoter proximal within the GAA repeat. All the templates contain the sequence for a self-cleaving ribozyme that cuts the transcript 270 bases 3′ to the end of the repeat tract producing a full-length transcript of 590 bases. Transcription from the phage T7 promoter started 55 bases 5′ to the GAA·TTC insert. Transcripts were end-labeled by including gamma 32 P-GTP in the reaction.

    Article Snippet: The Superscript II, RNase H-reverse transcriptase first strand synthesis kit (Life Technologies) was then used following the manufacturers’ protocol.

    Techniques: In Vitro, CTG Assay, Sequencing, Labeling

    A class of transcripts cleaved by RNase H can extend beyond the GAA·TTC repeat. ( A ) Primer extension provides high resolution mapping of the 3′ limit of the RNA·DNA hybrid within the transcript. The supercoiled (GAA·TTC) 88  template used in   Figure 4  was transcribed in the presence or absence of RNase H to provide RNA for reverse transcriptase primer extension of an end-labeled 30 base oligonucleotide that annealed from 72 to 42 bases beyond the 3′ end of the repeat in the transcript. Full-length extension to the 5′ end of the transcript yields a 390 base product in both samples. The major RNase H cleavage endpoints are clustered within a couple of triplets from the promoter distal end of the repeat sequence (arrow). ( B ) Experimental design and interpretation.

    Journal: Nucleic Acids Research

    Article Title: A persistent RNA?DNA hybrid formed by transcription of the Friedreich ataxia triplet repeat in live bacteria, and by T7 RNAP in vitro

    doi: 10.1093/nar/gkm589

    Figure Lengend Snippet: A class of transcripts cleaved by RNase H can extend beyond the GAA·TTC repeat. ( A ) Primer extension provides high resolution mapping of the 3′ limit of the RNA·DNA hybrid within the transcript. The supercoiled (GAA·TTC) 88 template used in Figure 4 was transcribed in the presence or absence of RNase H to provide RNA for reverse transcriptase primer extension of an end-labeled 30 base oligonucleotide that annealed from 72 to 42 bases beyond the 3′ end of the repeat in the transcript. Full-length extension to the 5′ end of the transcript yields a 390 base product in both samples. The major RNase H cleavage endpoints are clustered within a couple of triplets from the promoter distal end of the repeat sequence (arrow). ( B ) Experimental design and interpretation.

    Article Snippet: The Superscript II, RNase H-reverse transcriptase first strand synthesis kit (Life Technologies) was then used following the manufacturers’ protocol.

    Techniques: Labeling, Sequencing

    The RNA·DNA hybrid extends to the promoter proximal end of the repeat tract. Pairs of transcription reactions end-labeled by including gamma  32 P-GTP, were done in the absence (−) or presence (+) of RNase H to map the 5′ end of the RNA hybrid. Lanes 1–6 contain the end-labeled products derived from supercoiled templates with 0 (lanes 1 and 2), 11 (lanes 3 and 4) or 44 (lanes 5 and 6) GAA·TTC triplets. Lanes 7 and 8 contain the end-labeled products derived from a linear (GAA·TTC) 44  template. The most common end-labeled fragments generated by RNase H were at or near the start of the GAA tract in the templates with 44 triplets (lanes 6 and 12). Lane 13 contains a ‘G ladder’ from a partial RNase T1 digest of end-labeled (GAA) 44  transcript. The 3′ end of the (GAA) 44  tract at base 187 is indicated by a thin arrow, the first G of the triplet repeat sequence, at base 55 in the transcript 5′ end is indicated by a thick arrow near the bottom of the gel. Full-length transcripts are not resolved on this gel (but see   Figure 1 ).

    Journal: Nucleic Acids Research

    Article Title: A persistent RNA?DNA hybrid formed by transcription of the Friedreich ataxia triplet repeat in live bacteria, and by T7 RNAP in vitro

    doi: 10.1093/nar/gkm589

    Figure Lengend Snippet: The RNA·DNA hybrid extends to the promoter proximal end of the repeat tract. Pairs of transcription reactions end-labeled by including gamma 32 P-GTP, were done in the absence (−) or presence (+) of RNase H to map the 5′ end of the RNA hybrid. Lanes 1–6 contain the end-labeled products derived from supercoiled templates with 0 (lanes 1 and 2), 11 (lanes 3 and 4) or 44 (lanes 5 and 6) GAA·TTC triplets. Lanes 7 and 8 contain the end-labeled products derived from a linear (GAA·TTC) 44 template. The most common end-labeled fragments generated by RNase H were at or near the start of the GAA tract in the templates with 44 triplets (lanes 6 and 12). Lane 13 contains a ‘G ladder’ from a partial RNase T1 digest of end-labeled (GAA) 44 transcript. The 3′ end of the (GAA) 44 tract at base 187 is indicated by a thin arrow, the first G of the triplet repeat sequence, at base 55 in the transcript 5′ end is indicated by a thick arrow near the bottom of the gel. Full-length transcripts are not resolved on this gel (but see Figure 1 ).

    Article Snippet: The Superscript II, RNase H-reverse transcriptase first strand synthesis kit (Life Technologies) was then used following the manufacturers’ protocol.

    Techniques: Labeling, Derivative Assay, Generated, Sequencing

    Persistent RNA·DNA hybrids form during transcription of GAA·TTC repeats in bacteria. The gel pictured shows the mobility of templates isolated from bacteria in which transcription was repressed by glucose (first 4 lanes) or induced by arabinose (last 4 lanes). The plasmids were treated after isolation with the single-strand-specific RNases A and T1. Some aliquots were additionally treated with RNase H as indicated. The transcribed templates with 88 GAA·TTC triplets show a fully or partially relaxed mobility when treated only with single-strand-specific RNases (next to last lane). Treating an aliquot of the same sample with RNase H returns the bulk of the plasmid to supercoiled mobility in the last lane.

    Journal: Nucleic Acids Research

    Article Title: A persistent RNA?DNA hybrid formed by transcription of the Friedreich ataxia triplet repeat in live bacteria, and by T7 RNAP in vitro

    doi: 10.1093/nar/gkm589

    Figure Lengend Snippet: Persistent RNA·DNA hybrids form during transcription of GAA·TTC repeats in bacteria. The gel pictured shows the mobility of templates isolated from bacteria in which transcription was repressed by glucose (first 4 lanes) or induced by arabinose (last 4 lanes). The plasmids were treated after isolation with the single-strand-specific RNases A and T1. Some aliquots were additionally treated with RNase H as indicated. The transcribed templates with 88 GAA·TTC triplets show a fully or partially relaxed mobility when treated only with single-strand-specific RNases (next to last lane). Treating an aliquot of the same sample with RNase H returns the bulk of the plasmid to supercoiled mobility in the last lane.

    Article Snippet: The Superscript II, RNase H-reverse transcriptase first strand synthesis kit (Life Technologies) was then used following the manufacturers’ protocol.

    Techniques: Isolation, Plasmid Preparation

    Sequence preferences of  Escherichia coli, Homo sapiens  and HIV-1 RNase H ( A ) The heatmaps display the changes in nucleotide composition at different positions for the R7 construct (left) and the R4b construct (right) after cleavage with the three different RNase H enzymes. The intensity of the red and blue color indicates the  k rel  of having given nucleotide at a given position fixed relative to the average hydrolysis rate of the randomized pool. The barplots below the heatmaps show the overall information content at each position and the sequence logos are based on the 1% most downregulated pentamers. Note that only the randomized parts of the probed duplexes is displayed. ( B ) Cleavage of sequences predicted to be preferred (‘P’), avoided (‘A’) and neutral (‘N’) with respect to cleavage with human RNase H1 compared to the cleavage of a reference substrate. With respect to the reference substrate, the  k rel  of the preferred substrate is 3.7, of the avoided is 0.26 and of the neutral it is 1.4. ( C ) The design of the dumbbell substrate mimics. The gray box indicates the region having either the preferred (‘P’) or avoided (‘A’) sequence. ( D ) The cleavage of a reference substrate in the presence of increasing concentrations of a preferred or avoided dumbbell substrate mimic.

    Journal: Nucleic Acids Research

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

    doi: 10.1093/nar/gkx1073

    Figure Lengend Snippet: Sequence preferences of Escherichia coli, Homo sapiens and HIV-1 RNase H ( A ) The heatmaps display the changes in nucleotide composition at different positions for the R7 construct (left) and the R4b construct (right) after cleavage with the three different RNase H enzymes. The intensity of the red and blue color indicates the k rel of having given nucleotide at a given position fixed relative to the average hydrolysis rate of the randomized pool. The barplots below the heatmaps show the overall information content at each position and the sequence logos are based on the 1% most downregulated pentamers. Note that only the randomized parts of the probed duplexes is displayed. ( B ) Cleavage of sequences predicted to be preferred (‘P’), avoided (‘A’) and neutral (‘N’) with respect to cleavage with human RNase H1 compared to the cleavage of a reference substrate. With respect to the reference substrate, the k rel of the preferred substrate is 3.7, of the avoided is 0.26 and of the neutral it is 1.4. ( C ) The design of the dumbbell substrate mimics. The gray box indicates the region having either the preferred (‘P’) or avoided (‘A’) sequence. ( D ) The cleavage of a reference substrate in the presence of increasing concentrations of a preferred or avoided dumbbell substrate mimic.

    Article Snippet: Reactions with E. coli RNase H from Thermo Scientific (cat. EN0201) were performed at 37°C in a buffer composed of 50 mM Tris–HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol (DTT), 0.4 mg/ml bovine serum albumin and 0.1 mM EDTA using the same protocol as for the reactions with human-derived enzyme, but with a final enzyme concentration 0.5 mU/μl.

    Techniques: Sequencing, Construct

    Functional significance of predicted HIV-1 RNase H cleavage sites. ( A ) Predicted RNase H cleavage efficiency of the HIV-1 genome, shown as log 2 (fold change) (log 2 FC). ( B ) Schematic of the HIV reverse transcription. White scissors at the black circle indicate specific areas zoomed-in in subsequent panels. ( C ) Comparison of distances (in nucleotides) between well-cleaved sites in the HIV-1 genome and in the randomized HIV-1 genomes. The red rhombi shows the observed count of distances between positions predicted to be efficiently cleaved in HIV-1 genome that fall into the indicated distance intervals. The violin plots show the density of the distributions that resulted from the same analysis, but repeated 10 000× on HIV-1 genome sequences that were randomized with preserving the local dinucleotide content; Predicted cleavage efficiency of ( D ) the sequence surrounding the 3′PPT, ( E ) of the terminal 18 nt of the tRNA-Lys3 primer and ( F ) it is reverse complement (primer binding site). ( G ) Predicted cleavage efficiency of the best-cleaved site in the terminal 18 nt of the different human tRNAs (plus CCA) and of the corresponding reverse complement. The tRNA-Lys3 is indicated in red.

    Journal: Nucleic Acids Research

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

    doi: 10.1093/nar/gkx1073

    Figure Lengend Snippet: Functional significance of predicted HIV-1 RNase H cleavage sites. ( A ) Predicted RNase H cleavage efficiency of the HIV-1 genome, shown as log 2 (fold change) (log 2 FC). ( B ) Schematic of the HIV reverse transcription. White scissors at the black circle indicate specific areas zoomed-in in subsequent panels. ( C ) Comparison of distances (in nucleotides) between well-cleaved sites in the HIV-1 genome and in the randomized HIV-1 genomes. The red rhombi shows the observed count of distances between positions predicted to be efficiently cleaved in HIV-1 genome that fall into the indicated distance intervals. The violin plots show the density of the distributions that resulted from the same analysis, but repeated 10 000× on HIV-1 genome sequences that were randomized with preserving the local dinucleotide content; Predicted cleavage efficiency of ( D ) the sequence surrounding the 3′PPT, ( E ) of the terminal 18 nt of the tRNA-Lys3 primer and ( F ) it is reverse complement (primer binding site). ( G ) Predicted cleavage efficiency of the best-cleaved site in the terminal 18 nt of the different human tRNAs (plus CCA) and of the corresponding reverse complement. The tRNA-Lys3 is indicated in red.

    Article Snippet: Reactions with E. coli RNase H from Thermo Scientific (cat. EN0201) were performed at 37°C in a buffer composed of 50 mM Tris–HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol (DTT), 0.4 mg/ml bovine serum albumin and 0.1 mM EDTA using the same protocol as for the reactions with human-derived enzyme, but with a final enzyme concentration 0.5 mU/μl.

    Techniques: Functional Assay, Flow Cytometry, Preserving, Sequencing, Binding Assay

    RNase H Sequence Preferences correlate with gapmer efficiency. ( A ) Correlation between the log 2  fold changes of different hexamers observed for the R4b construct in the experiment and the corresponding log 2  fold changes as predicted by a single nucleotide model prepared from the data obtained in the R4a experiment. ( B ) As in (A), but with prediction with a dinucleotide model. ( C ) Prediction of RNase H1 mediated downregulation of the different 11-mers present in RNA sequence used for the RNase H RNA–DNA heteroduplex crystal structure [PDB: 2QK9]. Each bar corresponds to the cleavage site of a potential binding mode of RNase H1. The filled bar corresponds to the RNase H1 binding mode observed in the crystal structure and is also indicated in the drawing below the plot. ( D ) Correlation between the change of target RNA level for MAPT (  30 ) after treatment with 1518 different gapmers and the corresponding downregulation predicted by the dinucleotide model for the different binding modes of RNase H1 on each gapmer target duplex. The error bars show the 99% confidence intervals. The drawing below the plot indicates the RNase H1 binding mode associated with the best-observed correlation. ( E ) The same analysis as in (D), but with 1581 different gapmers targeted against ANGPTL3 (  31 ).

    Journal: Nucleic Acids Research

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

    doi: 10.1093/nar/gkx1073

    Figure Lengend Snippet: RNase H Sequence Preferences correlate with gapmer efficiency. ( A ) Correlation between the log 2 fold changes of different hexamers observed for the R4b construct in the experiment and the corresponding log 2 fold changes as predicted by a single nucleotide model prepared from the data obtained in the R4a experiment. ( B ) As in (A), but with prediction with a dinucleotide model. ( C ) Prediction of RNase H1 mediated downregulation of the different 11-mers present in RNA sequence used for the RNase H RNA–DNA heteroduplex crystal structure [PDB: 2QK9]. Each bar corresponds to the cleavage site of a potential binding mode of RNase H1. The filled bar corresponds to the RNase H1 binding mode observed in the crystal structure and is also indicated in the drawing below the plot. ( D ) Correlation between the change of target RNA level for MAPT ( 30 ) after treatment with 1518 different gapmers and the corresponding downregulation predicted by the dinucleotide model for the different binding modes of RNase H1 on each gapmer target duplex. The error bars show the 99% confidence intervals. The drawing below the plot indicates the RNase H1 binding mode associated with the best-observed correlation. ( E ) The same analysis as in (D), but with 1581 different gapmers targeted against ANGPTL3 ( 31 ).

    Article Snippet: Reactions with E. coli RNase H from Thermo Scientific (cat. EN0201) were performed at 37°C in a buffer composed of 50 mM Tris–HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol (DTT), 0.4 mg/ml bovine serum albumin and 0.1 mM EDTA using the same protocol as for the reactions with human-derived enzyme, but with a final enzyme concentration 0.5 mU/μl.

    Techniques: Sequencing, Construct, Binding Assay

    Refining the HIV-1 RNase H sequence preference model. ( A ) Distributions of the observed log 2  fold changes of RNA heptamers in R7 for human RNase H1 (right) and HIV-1 RNase H (left). ( B ) The observed log 2  fold changes after cleavage with HIV-1 RNase H for an efficiently cleaved hexamer (GCGCAA) located at different positions of R7. The position of the arrow indicates the cleavage site as aligned to the picture of scissors in the box and the arrow length represents the efficiency of cleavage. ( C ) Sequence logos of the best cleaved quartile of sets of heptamers predicted to have the same cleavage site. The arrows indicate the predicted cleavage site, with the length proportional to the observed cleavage efficiency.

    Journal: Nucleic Acids Research

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

    doi: 10.1093/nar/gkx1073

    Figure Lengend Snippet: Refining the HIV-1 RNase H sequence preference model. ( A ) Distributions of the observed log 2 fold changes of RNA heptamers in R7 for human RNase H1 (right) and HIV-1 RNase H (left). ( B ) The observed log 2 fold changes after cleavage with HIV-1 RNase H for an efficiently cleaved hexamer (GCGCAA) located at different positions of R7. The position of the arrow indicates the cleavage site as aligned to the picture of scissors in the box and the arrow length represents the efficiency of cleavage. ( C ) Sequence logos of the best cleaved quartile of sets of heptamers predicted to have the same cleavage site. The arrows indicate the predicted cleavage site, with the length proportional to the observed cleavage efficiency.

    Article Snippet: Reactions with E. coli RNase H from Thermo Scientific (cat. EN0201) were performed at 37°C in a buffer composed of 50 mM Tris–HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol (DTT), 0.4 mg/ml bovine serum albumin and 0.1 mM EDTA using the same protocol as for the reactions with human-derived enzyme, but with a final enzyme concentration 0.5 mU/μl.

    Techniques: Refining, Sequencing

    Second-strand transfer assay with model substrates. This illustrates the model strand transfer assay with the truncated substrate possessing only 12 (Δ6), 9 (Δ9), and 6 (Δ12) positions of the RNA sequence. Step 1 illustrates the input substrate for each truncated substrate, along with their respective input RNA-DNA sizes. Step 2 illustrates the polymerization reaction which can occur in the presence of RT and dNTPs and the size of the polymerization product for each substrate, 52-mer (Δ6), 49-mer (Δ9), and 46-mer (Δ12). DNA polymerization creates the RNA-DNA hybrid, which is a substrate for the RNase H domain (step 3). Once the RNA has been removed between the terminal ribo-A and ribo-C, the acceptor molecule can enter and produce a strand transfer product, 70-mer (step 4). In each step, the RNA portion is indicated in bold and the 5′ radiolabel is indicated by an asterisk. The size of the strand transfer product (70-mer) would be the same for each truncated substrate.

    Journal:

    Article Title: Comparison of Second-Strand Transfer Requirements and RNase H Cleavages Catalyzed by Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) and E478Q RT

    doi:

    Figure Lengend Snippet: Second-strand transfer assay with model substrates. This illustrates the model strand transfer assay with the truncated substrate possessing only 12 (Δ6), 9 (Δ9), and 6 (Δ12) positions of the RNA sequence. Step 1 illustrates the input substrate for each truncated substrate, along with their respective input RNA-DNA sizes. Step 2 illustrates the polymerization reaction which can occur in the presence of RT and dNTPs and the size of the polymerization product for each substrate, 52-mer (Δ6), 49-mer (Δ9), and 46-mer (Δ12). DNA polymerization creates the RNA-DNA hybrid, which is a substrate for the RNase H domain (step 3). Once the RNA has been removed between the terminal ribo-A and ribo-C, the acceptor molecule can enter and produce a strand transfer product, 70-mer (step 4). In each step, the RNA portion is indicated in bold and the 5′ radiolabel is indicated by an asterisk. The size of the strand transfer product (70-mer) would be the same for each truncated substrate.

    Article Snippet: E. coli RNase H was purchased from Gibco BRL.

    Techniques: Sequencing

    Model of WT RT and E478Q RT polymerase-dependent and polymerase-independent RNase H activities. The possible binding orientations for HIV-1 RT and E478Q RT are shown. (A to C) WT RT and E478Q RT are shown positioned on the various substrates, 50-mer substrate (32-mer DNA, 18-mer RNA) (A), 44-mer substrate (32-mer DNA, 12-mer RNA) (B), and 19-mer (7-mer DNA, 12-mer RNA) (C). The RNA portion is indicated by the thick line, and the DNA is indicated by the solid black line. The position of RNase H cleavage is indicated by a nick in the RNA strand. The WT and E478Q RT can be distinguished by the presence of an R (WT) or E/Q (E478Q RT) in the RNase H domain. Additionally, the thumb and polymerase domains are indicated by T and P, respectively. The 5′ phosphate is indicated, as well as the size of the RNA on each model substrate. (D) Models of substrates bound in the polymerase active site (left) and the RNase H active site (right) in HIV-1 RT. The electrostatic potential mapped on the molecular surface rendering of the HIV-1 RT (GRASP ) is shown with the template-primer as bound in the structure reported by Huang et al. (1rtd), and substrates are shown as stick models. Positively charged amino acids are shown in blue, and negatively charged amino acids are shown in red. In the right-hand panel, the substrate found in the 1rtd structure has been truncated to include only 12 bp of template-primer extending from the RNase H active site. This truncated substrate makes very limited interactions with the thumb. T, thumb; F, fingers; RH, RNase H active site; Pol, polymerase active site.

    Journal:

    Article Title: Comparison of Second-Strand Transfer Requirements and RNase H Cleavages Catalyzed by Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) and E478Q RT

    doi:

    Figure Lengend Snippet: Model of WT RT and E478Q RT polymerase-dependent and polymerase-independent RNase H activities. The possible binding orientations for HIV-1 RT and E478Q RT are shown. (A to C) WT RT and E478Q RT are shown positioned on the various substrates, 50-mer substrate (32-mer DNA, 18-mer RNA) (A), 44-mer substrate (32-mer DNA, 12-mer RNA) (B), and 19-mer (7-mer DNA, 12-mer RNA) (C). The RNA portion is indicated by the thick line, and the DNA is indicated by the solid black line. The position of RNase H cleavage is indicated by a nick in the RNA strand. The WT and E478Q RT can be distinguished by the presence of an R (WT) or E/Q (E478Q RT) in the RNase H domain. Additionally, the thumb and polymerase domains are indicated by T and P, respectively. The 5′ phosphate is indicated, as well as the size of the RNA on each model substrate. (D) Models of substrates bound in the polymerase active site (left) and the RNase H active site (right) in HIV-1 RT. The electrostatic potential mapped on the molecular surface rendering of the HIV-1 RT (GRASP ) is shown with the template-primer as bound in the structure reported by Huang et al. (1rtd), and substrates are shown as stick models. Positively charged amino acids are shown in blue, and negatively charged amino acids are shown in red. In the right-hand panel, the substrate found in the 1rtd structure has been truncated to include only 12 bp of template-primer extending from the RNase H active site. This truncated substrate makes very limited interactions with the thumb. T, thumb; F, fingers; RH, RNase H active site; Pol, polymerase active site.

    Article Snippet: E. coli RNase H was purchased from Gibco BRL.

    Techniques: Binding Assay

    RNase H cleavage analysis of truncated DNA substrates. (A) The substrates utilized are illustrated and are labeled A through F. The RNA portions are indicated in bold, and an asterisk indicates the radiolabel. The substrates were prepared as described in Materials and Methods. (B) Substrates B to F assayed with HIV-1 RT. Reactions were performed as described in Materials and Methods. Time course reactions are shown, and time points are indicated above each lane, along with the substrate utilized. The RNase H cleavage product is designated and indicated by an arrow. (C) Substrates B to F assayed with E478Q RT. Reactions were performed as described in Materials and Methods. Time course reactions are shown, and time points are indicated above each lane, along with the substrate utilized. The RNase H cleavage product is designated and indicated by an arrow.

    Journal:

    Article Title: Comparison of Second-Strand Transfer Requirements and RNase H Cleavages Catalyzed by Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) and E478Q RT

    doi:

    Figure Lengend Snippet: RNase H cleavage analysis of truncated DNA substrates. (A) The substrates utilized are illustrated and are labeled A through F. The RNA portions are indicated in bold, and an asterisk indicates the radiolabel. The substrates were prepared as described in Materials and Methods. (B) Substrates B to F assayed with HIV-1 RT. Reactions were performed as described in Materials and Methods. Time course reactions are shown, and time points are indicated above each lane, along with the substrate utilized. The RNase H cleavage product is designated and indicated by an arrow. (C) Substrates B to F assayed with E478Q RT. Reactions were performed as described in Materials and Methods. Time course reactions are shown, and time points are indicated above each lane, along with the substrate utilized. The RNase H cleavage product is designated and indicated by an arrow.

    Article Snippet: E. coli RNase H was purchased from Gibco BRL.

    Techniques: Labeling

    HPLC separation of products of miR191 cleavage by RNase H  E. coli . miR191 was hybridized with various AOs (Amir-0–Amir-4). The enzymatic reaction was stopped at specific times by the addition of EDTA to the solution. The numbers above selected peaks indicate products identified with MALDI TOF spectroscopy: ‘a’ indicates fragments of the same molecular mass, and any combination of them is possible. Sections of chromatograms corresponding to Amir and hybrid duplexes are shown in  Supplementary materials  ( Supplementary Figure S3 ).

    Journal: Nucleic Acids Research

    Article Title: 5?-O-Methylphosphonate nucleic acids--new modified DNAs that increase the Escherichia coli RNase H cleavage rate of hybrid duplexes

    doi: 10.1093/nar/gku125

    Figure Lengend Snippet: HPLC separation of products of miR191 cleavage by RNase H E. coli . miR191 was hybridized with various AOs (Amir-0–Amir-4). The enzymatic reaction was stopped at specific times by the addition of EDTA to the solution. The numbers above selected peaks indicate products identified with MALDI TOF spectroscopy: ‘a’ indicates fragments of the same molecular mass, and any combination of them is possible. Sections of chromatograms corresponding to Amir and hybrid duplexes are shown in Supplementary materials ( Supplementary Figure S3 ).

    Article Snippet: RNase H from E. coli and RNase H buffer were purchased from Invitrogen (Coralville, IA, USA).

    Techniques: High Performance Liquid Chromatography, Spectroscopy

    Design of the SPR experiment for the study of RNase H activity. Oligonucleotide immobilization, which gave the reference level (shown as a dashed line), was followed by hybridization of the injected AO and immobilized probe. The solution of RNase H and the AO was injected immediately after the AO hybridization. Hydrolysis of the probe was observed as a decrease in the sensor response. The difference between the initial level and level after the RNase H injection was proportional to the amount of cleaved substrate.

    Journal: Nucleic Acids Research

    Article Title: 5?-O-Methylphosphonate nucleic acids--new modified DNAs that increase the Escherichia coli RNase H cleavage rate of hybrid duplexes

    doi: 10.1093/nar/gku125

    Figure Lengend Snippet: Design of the SPR experiment for the study of RNase H activity. Oligonucleotide immobilization, which gave the reference level (shown as a dashed line), was followed by hybridization of the injected AO and immobilized probe. The solution of RNase H and the AO was injected immediately after the AO hybridization. Hydrolysis of the probe was observed as a decrease in the sensor response. The difference between the initial level and level after the RNase H injection was proportional to the amount of cleaved substrate.

    Article Snippet: RNase H from E. coli and RNase H buffer were purchased from Invitrogen (Coralville, IA, USA).

    Techniques: SPR Assay, Activity Assay, Hybridization, Injection

    SPR measurements of the RNase H activity on the Amir*miR-191 complex. The sensor response to the hybridization of Amir to the immobilized Pr191 probe containing the miR-191 RNA sequence and the resulting Pr191 cleavage by RNase H is shown. Arrows indicate the injection of the respective solutions.

    Journal: Nucleic Acids Research

    Article Title: 5?-O-Methylphosphonate nucleic acids--new modified DNAs that increase the Escherichia coli RNase H cleavage rate of hybrid duplexes

    doi: 10.1093/nar/gku125

    Figure Lengend Snippet: SPR measurements of the RNase H activity on the Amir*miR-191 complex. The sensor response to the hybridization of Amir to the immobilized Pr191 probe containing the miR-191 RNA sequence and the resulting Pr191 cleavage by RNase H is shown. Arrows indicate the injection of the respective solutions.

    Article Snippet: RNase H from E. coli and RNase H buffer were purchased from Invitrogen (Coralville, IA, USA).

    Techniques: SPR Assay, Activity Assay, Hybridization, Sequencing, Injection

    The extent of anti-terminator strand invasion is influenced by Mg 2+  concentration. ( a ) The RNase H cleavage assay (  Figure 4 ) uses a RNA/DNA chimera as an analog of the expression platform sequence. Partial formation of the anti-terminator helix creates a single site for RNase H cleavage. Full association creates a second site. ( b ) Denaturing poly-acrylamide gels showing the cleavage site selection on the aptamer domain. As Mg 2+  concentrations increase, the ability of the expression platform to fully form and become a substrate for RNase H increases. ( c ) Plot of fractional peak areas for the second site cleavage product [(area second site)/(area both sites)]. The curve represents the best fit of the data to the Hill equation (  equation 2 , methods). Errors are the standard fit errors for that parameter.

    Journal: Nucleic Acids Research

    Article Title: The expression platform and the aptamer: cooperativity between Mg2+ and ligand in the SAM-I riboswitch

    doi: 10.1093/nar/gks978

    Figure Lengend Snippet: The extent of anti-terminator strand invasion is influenced by Mg 2+ concentration. ( a ) The RNase H cleavage assay ( Figure 4 ) uses a RNA/DNA chimera as an analog of the expression platform sequence. Partial formation of the anti-terminator helix creates a single site for RNase H cleavage. Full association creates a second site. ( b ) Denaturing poly-acrylamide gels showing the cleavage site selection on the aptamer domain. As Mg 2+ concentrations increase, the ability of the expression platform to fully form and become a substrate for RNase H increases. ( c ) Plot of fractional peak areas for the second site cleavage product [(area second site)/(area both sites)]. The curve represents the best fit of the data to the Hill equation ( equation 2 , methods). Errors are the standard fit errors for that parameter.

    Article Snippet: Aptamer RNA (0.5 µM) was folded as outlined earlier, after which RNase H (Ambion, 0.02 U/µl) was added with the chimera (1 µM).

    Techniques: Concentration Assay, Cleavage Assay, Expressing, Sequencing, Selection

    Switching assay results. ( a ) Aptamer domain RNA is folded and challenged with a chimeric RNA/DNA oligomer based on the native expression platform sequence. Instability in the aptamer domain allows the expression platform sequence to compete for shared sequence in the aptamer domain. Formation of the anti-terminator helix produces a substrate RNA–DNA duplex for RNase H resulting in cleavage of the aptamer domain (right). ( b ) Example denaturing PAGE gels used to analyse the Mg 2+  titrations at various concentrations of SAM. Mg 2+  concentrations were chosen for each SAM concentration to best resolve the transition from destabilized (cleaved) to stable (uncleaved) aptamer. ( c ) After quantification of the bands representing the cleaved (sum of both cleavage products) and uncleaved fractions, the data [(fluorescence uncleaved aptamer)/(fluorescence cleaved + uncleaved aptamer)] were plotted versus [Mg 2+ ] and fit to the Hill equation (methods,   equation 2 ). Fits yielded the [Mg 2+ ] 1/2  (the concentration at which the transition was 50% complete) and Hill coefficients ( n H ) for the transitions at each concentration of SAM. Fits to the Hill model were performed on representative data sets, and errors represent the standard errors for the fitting of that parameter. Hill coefficients were not determined for the experiments with 200 µM SAM. High fit errors were caused by too few data points representing fully cleaved aptamer at low Mg 2+  concentrations (standard errors exceeded 100%).

    Journal: Nucleic Acids Research

    Article Title: The expression platform and the aptamer: cooperativity between Mg2+ and ligand in the SAM-I riboswitch

    doi: 10.1093/nar/gks978

    Figure Lengend Snippet: Switching assay results. ( a ) Aptamer domain RNA is folded and challenged with a chimeric RNA/DNA oligomer based on the native expression platform sequence. Instability in the aptamer domain allows the expression platform sequence to compete for shared sequence in the aptamer domain. Formation of the anti-terminator helix produces a substrate RNA–DNA duplex for RNase H resulting in cleavage of the aptamer domain (right). ( b ) Example denaturing PAGE gels used to analyse the Mg 2+ titrations at various concentrations of SAM. Mg 2+ concentrations were chosen for each SAM concentration to best resolve the transition from destabilized (cleaved) to stable (uncleaved) aptamer. ( c ) After quantification of the bands representing the cleaved (sum of both cleavage products) and uncleaved fractions, the data [(fluorescence uncleaved aptamer)/(fluorescence cleaved + uncleaved aptamer)] were plotted versus [Mg 2+ ] and fit to the Hill equation (methods, equation 2 ). Fits yielded the [Mg 2+ ] 1/2 (the concentration at which the transition was 50% complete) and Hill coefficients ( n H ) for the transitions at each concentration of SAM. Fits to the Hill model were performed on representative data sets, and errors represent the standard errors for the fitting of that parameter. Hill coefficients were not determined for the experiments with 200 µM SAM. High fit errors were caused by too few data points representing fully cleaved aptamer at low Mg 2+ concentrations (standard errors exceeded 100%).

    Article Snippet: Aptamer RNA (0.5 µM) was folded as outlined earlier, after which RNase H (Ambion, 0.02 U/µl) was added with the chimera (1 µM).

    Techniques: Expressing, Sequencing, Polyacrylamide Gel Electrophoresis, Concentration Assay, Fluorescence

    The expression platform switching assay was used as a selection screen in a phosphorothioate interference assay. A schematic detailing the selection methodology is available in  supplementary data  ( Supplementary Figure S5 ). Selection was performed using RNase H to cleave destabilized aptamers (see   Figure 4 ). Aptamer RNA is randomly incorporated to ∼5% with one of the four α-phosphorothioate-rNTPs. The RNA is 3′-end labeled with the Alexa-488 fluorophore. RNase H cleavage removes the label from aptamers unfit to compete for shared sequence. Populations of each phosphorothiate position are resolved by phosphorothioate cleavage with iodine after selection and before capillary electrophoresis. ( a ) Capillary electrophoresis traces of selected and unselected RNA incorporated with ATPαS. Experiments were performed at various concentrations of SAM; black (unselected control RNA), green (10 µM SAM), blue (30 µM SAM), cyan (100 µM SAM), red (rescue at 10 µM SAM with 1 mM Mn 2+ ) and brown (unselected control without iodine cleavage). Positions showing phophorothioate interference are indicated. As SAM concentrations increase, the population of phosphorothioate at that position returns to normal. ( b ) Electropherograms for UTPαS interference assay (colors the same as in a). ( c ) Traces are integrated and the areas normalized to peaks that display no selection. Bar graph color-code is the same as that for the cap-EP traces above. ( d ) Secondary structure plot showing the positions of interference with an inset showing the kink-turn element with residue numbering. Red and blue boxed nucleotides show important tertiary interaction (base-triple contacts) proximal to the central Mg 2+ -binding site formed by A10 and U71.

    Journal: Nucleic Acids Research

    Article Title: The expression platform and the aptamer: cooperativity between Mg2+ and ligand in the SAM-I riboswitch

    doi: 10.1093/nar/gks978

    Figure Lengend Snippet: The expression platform switching assay was used as a selection screen in a phosphorothioate interference assay. A schematic detailing the selection methodology is available in supplementary data ( Supplementary Figure S5 ). Selection was performed using RNase H to cleave destabilized aptamers (see Figure 4 ). Aptamer RNA is randomly incorporated to ∼5% with one of the four α-phosphorothioate-rNTPs. The RNA is 3′-end labeled with the Alexa-488 fluorophore. RNase H cleavage removes the label from aptamers unfit to compete for shared sequence. Populations of each phosphorothiate position are resolved by phosphorothioate cleavage with iodine after selection and before capillary electrophoresis. ( a ) Capillary electrophoresis traces of selected and unselected RNA incorporated with ATPαS. Experiments were performed at various concentrations of SAM; black (unselected control RNA), green (10 µM SAM), blue (30 µM SAM), cyan (100 µM SAM), red (rescue at 10 µM SAM with 1 mM Mn 2+ ) and brown (unselected control without iodine cleavage). Positions showing phophorothioate interference are indicated. As SAM concentrations increase, the population of phosphorothioate at that position returns to normal. ( b ) Electropherograms for UTPαS interference assay (colors the same as in a). ( c ) Traces are integrated and the areas normalized to peaks that display no selection. Bar graph color-code is the same as that for the cap-EP traces above. ( d ) Secondary structure plot showing the positions of interference with an inset showing the kink-turn element with residue numbering. Red and blue boxed nucleotides show important tertiary interaction (base-triple contacts) proximal to the central Mg 2+ -binding site formed by A10 and U71.

    Article Snippet: Aptamer RNA (0.5 µM) was folded as outlined earlier, after which RNase H (Ambion, 0.02 U/µl) was added with the chimera (1 µM).

    Techniques: Expressing, Selection, Labeling, Sequencing, Electrophoresis, Binding Assay

    Determination of the cleavage site by site-specific disconnection (SSD) . ( a ) Sequences of the fluorescein isothiocyanate (FITC)-labelled DNA/RNA chimeric oligomer. The red text indicates the DNA sequence, and the other text indicates the RNA portion that is complementary to the Aid-DNA (purple, italicized, underlined characters indicate the sequence of 2′-O-methyl-modified nucleotides). The expected cleavage site “UG” was shown by the green arrow. ( b ) Analysis of the cleavage product. Compared to the standard samples, the length of cleavage product was determined to be 17 nt. Other conditions used were 1.0 μmol/l substrate and Aid-DNA-16-1, 125 U/ml RNase H, 37 °C, 40 minutes, 20% denaturing urea polyacrylamide gel electrophoresis (PAGE).

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection

    doi: 10.1038/mtna.2014.66

    Figure Lengend Snippet: Determination of the cleavage site by site-specific disconnection (SSD) . ( a ) Sequences of the fluorescein isothiocyanate (FITC)-labelled DNA/RNA chimeric oligomer. The red text indicates the DNA sequence, and the other text indicates the RNA portion that is complementary to the Aid-DNA (purple, italicized, underlined characters indicate the sequence of 2′-O-methyl-modified nucleotides). The expected cleavage site “UG” was shown by the green arrow. ( b ) Analysis of the cleavage product. Compared to the standard samples, the length of cleavage product was determined to be 17 nt. Other conditions used were 1.0 μmol/l substrate and Aid-DNA-16-1, 125 U/ml RNase H, 37 °C, 40 minutes, 20% denaturing urea polyacrylamide gel electrophoresis (PAGE).

    Article Snippet: Then, the mixture with RNase H was kept at 37 °C for 40 minutes, followed by inactivation of the RNase H at 65 °C for 10 minutes.

    Techniques: Sequencing, Modification, Polyacrylamide Gel Electrophoresis

    Synthesis of the mir-16 oligomer using rolling circle transcription (RCT) site-specific disconnection (SSD) . ( a ) Ligation products of the cDNA and transcript of the RCT reaction on circular cDNA. C72, C66, and C60 are synthesized circular ssDNA oligomers used as markers; Lane L, RNA ladder. Samples were separated by 14% denaturing PAGE (8 mol/l urea). ( b ) Analysis of synthesized mir-16. Lane 1, chemically synthesized mir-16; lane 2, RNase H was absent during RCT; lane 3, RNase-H (2.5 U/ml) was present during RCT. Samples were separated by 20% denaturing PAGE (8 mol/l urea). Other conditions used for RCT were: 0.5 μmol/l cDNA, 2.5 U/ml RNA polymerase, 37 °C, 2 hours; other conditions used for SSD were, 125 U/ml RNase H, 1.0 μmol/l Aid-DNA, 37 °C, 2 hours.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection

    doi: 10.1038/mtna.2014.66

    Figure Lengend Snippet: Synthesis of the mir-16 oligomer using rolling circle transcription (RCT) site-specific disconnection (SSD) . ( a ) Ligation products of the cDNA and transcript of the RCT reaction on circular cDNA. C72, C66, and C60 are synthesized circular ssDNA oligomers used as markers; Lane L, RNA ladder. Samples were separated by 14% denaturing PAGE (8 mol/l urea). ( b ) Analysis of synthesized mir-16. Lane 1, chemically synthesized mir-16; lane 2, RNase H was absent during RCT; lane 3, RNase-H (2.5 U/ml) was present during RCT. Samples were separated by 20% denaturing PAGE (8 mol/l urea). Other conditions used for RCT were: 0.5 μmol/l cDNA, 2.5 U/ml RNA polymerase, 37 °C, 2 hours; other conditions used for SSD were, 125 U/ml RNase H, 1.0 μmol/l Aid-DNA, 37 °C, 2 hours.

    Article Snippet: Then, the mixture with RNase H was kept at 37 °C for 40 minutes, followed by inactivation of the RNase H at 65 °C for 10 minutes.

    Techniques: Ligation, Synthesized, Polyacrylamide Gel Electrophoresis

    Strategy for rolling circle transcription (RCT) site-specific disconnection (SSD) synthesis . cDNA is circularized to form a circular DNA template, and long RNA strands are generated by RCT consisting of tandem repeats of desired RNA. With the help of Aid-DNA, RNase H disconnects the transcript to generate thousands of copies of the desired RNA.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection

    doi: 10.1038/mtna.2014.66

    Figure Lengend Snippet: Strategy for rolling circle transcription (RCT) site-specific disconnection (SSD) synthesis . cDNA is circularized to form a circular DNA template, and long RNA strands are generated by RCT consisting of tandem repeats of desired RNA. With the help of Aid-DNA, RNase H disconnects the transcript to generate thousands of copies of the desired RNA.

    Article Snippet: Then, the mixture with RNase H was kept at 37 °C for 40 minutes, followed by inactivation of the RNase H at 65 °C for 10 minutes.

    Techniques: Generated

    LARP1 promotes PAT 3' length stabilization of non-5'TOP mRNAs. ( a ) Northern blot after transfection of HEK293 cells with expression plasmids indicated above the lanes, and the βG-TNFα-ARE reporter. ( b ) Same as in a but transfected with GFP followed by RNase H assay in presence or absence of oligo(dT) probed for GFP mRNA as indicated. The blot in b was probed for histone H2A. ( c ) Western blot showing LARP proteins expressed.

    Journal: eLife

    Article Title: LARP4 mRNA codon-tRNA match contributes to LARP4 activity for ribosomal protein mRNA poly(A) tail length protection

    doi: 10.7554/eLife.28889

    Figure Lengend Snippet: LARP1 promotes PAT 3' length stabilization of non-5'TOP mRNAs. ( a ) Northern blot after transfection of HEK293 cells with expression plasmids indicated above the lanes, and the βG-TNFα-ARE reporter. ( b ) Same as in a but transfected with GFP followed by RNase H assay in presence or absence of oligo(dT) probed for GFP mRNA as indicated. The blot in b was probed for histone H2A. ( c ) Western blot showing LARP proteins expressed.

    Article Snippet: 2 ul of 10X RNase H reaction buffer was added and 10 ul of RNase H (0.001 U/ul, Thermo Scientific) and incubated at 37°C for one hour.

    Techniques: Northern Blot, Transfection, Expressing, Rnase H Assay, Western Blot

    LARP4 gene-deleted knockout (KO) cells exhibit decreased 3' PAT length and stability of ribosome protein mRNAs. ( a ) Western blot of LARP4 from independent isolates of WT and LARP4 KO MEFs. ( b ) Northern blot from 4 MEF cell lines in a; probes as indicated to the left of the panels, Rps28, Rpl32, PPP1R14A, histone H2A mRNAs, and EtBr stained gel. Densitometric lane tracings for each lane of a Rps28 exposure is shown to the right as indicated. ( c ) RNase H assay in presence or absence of oligo(dT) as indicated. ( d e, ) Time course of mRNA decay in LARP4 KO and WT MEFs after transcription shut-off (in hrs) by actinomycin-D, probed for Rps28, Rpl32 and histone H2A mRNAs as indicated to the left; e contains the same RNA preparation as in d but run on a separate gel. ( f ) Northern blot of 12 hr act-D time courses for LARP4 KO and WT MEFs probed for the RNAs indicated to the left. ( g ) Graphs showing quantifications of duplicate experiments including panels in f, as indicated. The mRNA quantification at each time was normalized against 18S rRNA in the same lane. Error bars at each time point reflect the spread of the duplicates.

    Journal: eLife

    Article Title: LARP4 mRNA codon-tRNA match contributes to LARP4 activity for ribosomal protein mRNA poly(A) tail length protection

    doi: 10.7554/eLife.28889

    Figure Lengend Snippet: LARP4 gene-deleted knockout (KO) cells exhibit decreased 3' PAT length and stability of ribosome protein mRNAs. ( a ) Western blot of LARP4 from independent isolates of WT and LARP4 KO MEFs. ( b ) Northern blot from 4 MEF cell lines in a; probes as indicated to the left of the panels, Rps28, Rpl32, PPP1R14A, histone H2A mRNAs, and EtBr stained gel. Densitometric lane tracings for each lane of a Rps28 exposure is shown to the right as indicated. ( c ) RNase H assay in presence or absence of oligo(dT) as indicated. ( d e, ) Time course of mRNA decay in LARP4 KO and WT MEFs after transcription shut-off (in hrs) by actinomycin-D, probed for Rps28, Rpl32 and histone H2A mRNAs as indicated to the left; e contains the same RNA preparation as in d but run on a separate gel. ( f ) Northern blot of 12 hr act-D time courses for LARP4 KO and WT MEFs probed for the RNAs indicated to the left. ( g ) Graphs showing quantifications of duplicate experiments including panels in f, as indicated. The mRNA quantification at each time was normalized against 18S rRNA in the same lane. Error bars at each time point reflect the spread of the duplicates.

    Article Snippet: 2 ul of 10X RNase H reaction buffer was added and 10 ul of RNase H (0.001 U/ul, Thermo Scientific) and incubated at 37°C for one hour.

    Techniques: Knock-Out, Gene Knockout, Western Blot, Northern Blot, Staining, Rnase H Assay, Activated Clotting Time Assay

    CRD-mediated increase in LARP4 leads to heterologous mRNA 3' PAT lengthening and stabilization dependent on its PABP- and RNA- interaction domains. ( a ) Northern blot after RNase H ± oligo(dT) treatment of total RNA from HEK293 cells transfected with constructs indicated above the lanes; CS = CS R version of the CRD in full length LARP4. ( b ) Upper: northern blot for GFP mRNA mobility shift activity of LARP4 constructs some of which contain the CS-R version of the CRD. WT = wild type, CS = full length LARP4 with CS-R CRD, ΔPAM2 = PAM2 deleted w/CS R, ΔPBM = PBM/CRD deleted, ΔPAM2ΔPBM, mut LaM-RRM WT and mut LaM-RRM CS = previously described M3 point mutations in the LaM and RRM in the full length LARP4 WT and CS-R versions respectively. Lower: western blot. ( c ) Northern blot of mRNA decay time course of HeLa Tet-Off cells transfected with βG-TNFα-ARE, GFP and either empty vector, LARP4-WT, LARP4-CS-R or LARP6. Cells harvested after 0, 60, 120 and 240 mins after doxycycline. Numbers under lanes for t = 0 indicate quantification of βG mRNA divided by GFP mRNA in the same lane, with lane 1 set to 1.0. ( d ) Quantification of βG-TNFα-ARE mRNA from c; the t = 0 for each was set to 100%. ( e ) Western blot of proteins tested in c, d. ( f ) Northern blot of HEK293 cells after transfection with βG-TNFα-ARE (constitutive promoter), GFP and either empty pCMV2 or F-LARP4-WT or mutants indicated above lanes as described for b; three lower panels show western blot of extracts using antibodies as indicated.

    Journal: eLife

    Article Title: LARP4 mRNA codon-tRNA match contributes to LARP4 activity for ribosomal protein mRNA poly(A) tail length protection

    doi: 10.7554/eLife.28889

    Figure Lengend Snippet: CRD-mediated increase in LARP4 leads to heterologous mRNA 3' PAT lengthening and stabilization dependent on its PABP- and RNA- interaction domains. ( a ) Northern blot after RNase H ± oligo(dT) treatment of total RNA from HEK293 cells transfected with constructs indicated above the lanes; CS = CS R version of the CRD in full length LARP4. ( b ) Upper: northern blot for GFP mRNA mobility shift activity of LARP4 constructs some of which contain the CS-R version of the CRD. WT = wild type, CS = full length LARP4 with CS-R CRD, ΔPAM2 = PAM2 deleted w/CS R, ΔPBM = PBM/CRD deleted, ΔPAM2ΔPBM, mut LaM-RRM WT and mut LaM-RRM CS = previously described M3 point mutations in the LaM and RRM in the full length LARP4 WT and CS-R versions respectively. Lower: western blot. ( c ) Northern blot of mRNA decay time course of HeLa Tet-Off cells transfected with βG-TNFα-ARE, GFP and either empty vector, LARP4-WT, LARP4-CS-R or LARP6. Cells harvested after 0, 60, 120 and 240 mins after doxycycline. Numbers under lanes for t = 0 indicate quantification of βG mRNA divided by GFP mRNA in the same lane, with lane 1 set to 1.0. ( d ) Quantification of βG-TNFα-ARE mRNA from c; the t = 0 for each was set to 100%. ( e ) Western blot of proteins tested in c, d. ( f ) Northern blot of HEK293 cells after transfection with βG-TNFα-ARE (constitutive promoter), GFP and either empty pCMV2 or F-LARP4-WT or mutants indicated above lanes as described for b; three lower panels show western blot of extracts using antibodies as indicated.

    Article Snippet: 2 ul of 10X RNase H reaction buffer was added and 10 ul of RNase H (0.001 U/ul, Thermo Scientific) and incubated at 37°C for one hour.

    Techniques: Northern Blot, Transfection, Construct, Mobility Shift, Activity Assay, Laser Capture Microdissection, Western Blot, Plasmid Preparation

    HQ formation in transcribed plasmid containing a human mtDNA fragment starting from the light strand promoter (LSP) and containing the CSB II and CSB I. ( A ) Scheme of the plasmid and detection of G-quadruplex formation by ligand-induced photocleavage. Plasmid transcribed in 50 mM of K +  or Li +  solution in the presence or absence of 40% (w/v) PEG 200 with GTP or dzGTP was subjected to Zn-TTAPc-mediated photocleavage, then cut at the Mun I restriction site, and filled in at the recessive 3′ end with a dATP followed by a fluorescein-dUTP, before being resolved on a denaturing gel. The marker (M) was a single-stranded synthetic DNA equivalent to the fragment between the Mun I site and the 3′ end of the G 5 AG 7  motif. Filled and open bars indicate G-quadruplex-specific cleavage signals. ( B ) Detection of RNA in HQ by photo-crosslinking. Transcription was conducted using normal GTP or dzGTP and 4S-UTP in solution containing 50-mM K +  or Li + . With or without a prior RNase H digestion, transcribed plasmid was crosslinked and precipitated. Then a 5′-FAM-labeled primer (5′-CCAGCCTGCGG­CGAGTG-3′) was annealed to the non-template DNA strand downstream of CSB II, followed by extension with DNA sequenase. Extension products were resolved on a denaturing gel. G and T ladders were obtained by primer extension on the non-template DNA strand with ddCTP and ddATP, respectively. Filled and open bars indicate crosslinking sites. ( C ) Detection of G-quadruplex formation by RNA polymerase arrest assay. A plasmid containing convergent T7 and SP6 promoters and the correspondent terminators (top scheme) was transcribed by SP6 RNA polymerase in 50-mM K +  or Li +  solution without (lanes 1 and 2) or with (lanes 4 and 5) a prior transcription with T7 RNA polymerase in the same solution. The T7 transcription was stopped by competitive DNA specific to the T7 polymerase before the SP6 transcription was initiated. Fluorescein-UTP was supplied with SP6 RNA polymerase. RNA transcripts were resolved on a denaturing gel and visualized by the incorporated fluorescein-UTP. The marker represents SP6 transcript terminated right before the CSB II obtained by transcription of a linear DNA amplified from the plasmid.

    Journal: Nucleic Acids Research

    Article Title: A competitive formation of DNA:RNA hybrid G-quadruplex is responsible to the mitochondrial transcription termination at the DNA replication priming site

    doi: 10.1093/nar/gku764

    Figure Lengend Snippet: HQ formation in transcribed plasmid containing a human mtDNA fragment starting from the light strand promoter (LSP) and containing the CSB II and CSB I. ( A ) Scheme of the plasmid and detection of G-quadruplex formation by ligand-induced photocleavage. Plasmid transcribed in 50 mM of K + or Li + solution in the presence or absence of 40% (w/v) PEG 200 with GTP or dzGTP was subjected to Zn-TTAPc-mediated photocleavage, then cut at the Mun I restriction site, and filled in at the recessive 3′ end with a dATP followed by a fluorescein-dUTP, before being resolved on a denaturing gel. The marker (M) was a single-stranded synthetic DNA equivalent to the fragment between the Mun I site and the 3′ end of the G 5 AG 7 motif. Filled and open bars indicate G-quadruplex-specific cleavage signals. ( B ) Detection of RNA in HQ by photo-crosslinking. Transcription was conducted using normal GTP or dzGTP and 4S-UTP in solution containing 50-mM K + or Li + . With or without a prior RNase H digestion, transcribed plasmid was crosslinked and precipitated. Then a 5′-FAM-labeled primer (5′-CCAGCCTGCGG­CGAGTG-3′) was annealed to the non-template DNA strand downstream of CSB II, followed by extension with DNA sequenase. Extension products were resolved on a denaturing gel. G and T ladders were obtained by primer extension on the non-template DNA strand with ddCTP and ddATP, respectively. Filled and open bars indicate crosslinking sites. ( C ) Detection of G-quadruplex formation by RNA polymerase arrest assay. A plasmid containing convergent T7 and SP6 promoters and the correspondent terminators (top scheme) was transcribed by SP6 RNA polymerase in 50-mM K + or Li + solution without (lanes 1 and 2) or with (lanes 4 and 5) a prior transcription with T7 RNA polymerase in the same solution. The T7 transcription was stopped by competitive DNA specific to the T7 polymerase before the SP6 transcription was initiated. Fluorescein-UTP was supplied with SP6 RNA polymerase. RNA transcripts were resolved on a denaturing gel and visualized by the incorporated fluorescein-UTP. The marker represents SP6 transcript terminated right before the CSB II obtained by transcription of a linear DNA amplified from the plasmid.

    Article Snippet: They were then individually diluted to 0.15 μM into buffer of 40-mM Tris-HCl (pH 7.9), 6-mM MgCl2 , 10-mM dithiothreitol (DTT), 2-mM spermidine and 50-mM KCl and maintained at 37°C for 1 h. The dimeric HQ sample was then digested with 0.3-U/μl RNase H (Fermentas, Thermo Scientific) at 37°C for 20 min to remove the RNA at the duplex region.

    Techniques: Plasmid Preparation, Marker, Labeling, Amplification

    Identification of G-quadruplexes in plasmid at the wild and mutated CSB II by ( A ) ligand-induced photocleavage and ( B ) photo-crosslinking. (A) Plasmids transcribed in 50-mM K +  solution were treated with RNase A (A) or A and H (AH), incubated with Zn-TTAPc, and irradiated with UV light. The plasmids were then cut with Mun I, labeled at the 3′ recessive end with an FAM dye by a fill-in reaction using fluorescein-dUTP. Marker was prepared in the same way using a synthetic dsDNA that has the same sequence as the plasmid at the correspondent region (scheme at bottom). The labeling may add one or two Ts, resulting in two bands. Cleavage fragments were resolved on a denaturing gel. (B) Plasmids were transcribed in 50-mM K +  with 4-S-UTP and the other three NTPs, treated with RNase H, followed by UV irradiation. A 5′-FAM-labeled primer was extended on the non-template DNA strand that stalled at the crosslinking sites. Extension products were resolved on a denaturing gel.

    Journal: Nucleic Acids Research

    Article Title: A competitive formation of DNA:RNA hybrid G-quadruplex is responsible to the mitochondrial transcription termination at the DNA replication priming site

    doi: 10.1093/nar/gku764

    Figure Lengend Snippet: Identification of G-quadruplexes in plasmid at the wild and mutated CSB II by ( A ) ligand-induced photocleavage and ( B ) photo-crosslinking. (A) Plasmids transcribed in 50-mM K + solution were treated with RNase A (A) or A and H (AH), incubated with Zn-TTAPc, and irradiated with UV light. The plasmids were then cut with Mun I, labeled at the 3′ recessive end with an FAM dye by a fill-in reaction using fluorescein-dUTP. Marker was prepared in the same way using a synthetic dsDNA that has the same sequence as the plasmid at the correspondent region (scheme at bottom). The labeling may add one or two Ts, resulting in two bands. Cleavage fragments were resolved on a denaturing gel. (B) Plasmids were transcribed in 50-mM K + with 4-S-UTP and the other three NTPs, treated with RNase H, followed by UV irradiation. A 5′-FAM-labeled primer was extended on the non-template DNA strand that stalled at the crosslinking sites. Extension products were resolved on a denaturing gel.

    Article Snippet: They were then individually diluted to 0.15 μM into buffer of 40-mM Tris-HCl (pH 7.9), 6-mM MgCl2 , 10-mM dithiothreitol (DTT), 2-mM spermidine and 50-mM KCl and maintained at 37°C for 1 h. The dimeric HQ sample was then digested with 0.3-U/μl RNase H (Fermentas, Thermo Scientific) at 37°C for 20 min to remove the RNA at the duplex region.

    Techniques: Plasmid Preparation, Incubation, Irradiation, Labeling, Marker, Sequencing

    Stability of HQ and DQ formed in synthetic CSB II oligonucleotides in 50-mM K + . ( A ) Melting profile of intramolecular DNA DQ and chimeric DNA:RNA HQ. Sequences used are shown on the left. Each of them carried a fluorescent donor FAM at the 5′ end and an accepter TAMRA at the 3′ end. The curves in the graph show the first derivative of FAM fluorescence over temperature as a function of temperature. ( B ) Protection of DNA by the formation of DQ or HQ. DQ or HQ formed in the single-stranded DNA or dimeric DNA:RNA partial duplex protected the DNA from being hydrolyzed from the 3′ end by Exo I exonuclease (scheme at left). Three substrates (Wild, M3G and HQ) were treated with Exo I in a single tube and those survived the hydrolysis were resolved on a denaturing gel. The RNA in the duplex region of the HQ substrate was hydrolyzed by RNase H prior to the exonuclease digestion. The DNAs were visualized by the FAM dye covalently labeled at their 5′ end, digitized, and the results are given on the right. The numbers above the bars indicate the average of the two time points. The DNA oligomer or moiety is shown in uppercase and that of RNA in lowercase in (A,B).

    Journal: Nucleic Acids Research

    Article Title: A competitive formation of DNA:RNA hybrid G-quadruplex is responsible to the mitochondrial transcription termination at the DNA replication priming site

    doi: 10.1093/nar/gku764

    Figure Lengend Snippet: Stability of HQ and DQ formed in synthetic CSB II oligonucleotides in 50-mM K + . ( A ) Melting profile of intramolecular DNA DQ and chimeric DNA:RNA HQ. Sequences used are shown on the left. Each of them carried a fluorescent donor FAM at the 5′ end and an accepter TAMRA at the 3′ end. The curves in the graph show the first derivative of FAM fluorescence over temperature as a function of temperature. ( B ) Protection of DNA by the formation of DQ or HQ. DQ or HQ formed in the single-stranded DNA or dimeric DNA:RNA partial duplex protected the DNA from being hydrolyzed from the 3′ end by Exo I exonuclease (scheme at left). Three substrates (Wild, M3G and HQ) were treated with Exo I in a single tube and those survived the hydrolysis were resolved on a denaturing gel. The RNA in the duplex region of the HQ substrate was hydrolyzed by RNase H prior to the exonuclease digestion. The DNAs were visualized by the FAM dye covalently labeled at their 5′ end, digitized, and the results are given on the right. The numbers above the bars indicate the average of the two time points. The DNA oligomer or moiety is shown in uppercase and that of RNA in lowercase in (A,B).

    Article Snippet: They were then individually diluted to 0.15 μM into buffer of 40-mM Tris-HCl (pH 7.9), 6-mM MgCl2 , 10-mM dithiothreitol (DTT), 2-mM spermidine and 50-mM KCl and maintained at 37°C for 1 h. The dimeric HQ sample was then digested with 0.3-U/μl RNase H (Fermentas, Thermo Scientific) at 37°C for 20 min to remove the RNA at the duplex region.

    Techniques: Fluorescence, Labeling

    Base pairing-directed RNA primer ligation to RLuc-CVB3-CLΔ1−6 + 5 genomic RNA. ( A ) Schematic representation of the CLΔ1−6 + 5-modified CL structure. The 5-nt insertion in the 3′ strand of stem A is indicated in gray. Note that T7 RNA polymerase-transcribed RNA contains two additional guanine nucleotides ( italic ), which form base pairs with cytosine nucleotides of the 3′ strand of stem A. The 9-nt RNA primer used for base pairing-directed RNA ligation is depicted in light gray, and ‘R’ represents the different 5′ modifications. ( B ) RNA primer ligation efficiency to genomic RNA possessing the CLΔ1−6 + 5 structure was determined by urea–PAGE analysis of a 250-nt RNase H-digested 5′-terminal fragment. Note that ligation of the RNA primer reduces migration speed of the 250-nt RNase H-digested RNA fragment. ( C ) Translation of incoming genomic RLuc-CVB3 RNA (wt), RNA holding the mutated CL (Δ1−6 + 5) and RNA ligation products possessing different 5′ modifications (OH, BCN, GH[5], GH[9], GH[11]) were determined by transfection of RNA in HeLa cells in the presence of GuHCl. Eight hours post-transfection, HeLa cells were lysed and RLuc values were determined. Data from a representative experiment are presented as the mean of duplicate ±SD and analyzed using unpaired  t -test (* indicates significant difference  P

    Journal: Nucleic Acids Research

    Article Title: Modification of picornavirus genomic RNA using 'click' chemistry shows that unlinking of the VPg peptide is dispensable for translation and replication of the incoming viral RNA

    doi: 10.1093/nar/gkt1162

    Figure Lengend Snippet: Base pairing-directed RNA primer ligation to RLuc-CVB3-CLΔ1−6 + 5 genomic RNA. ( A ) Schematic representation of the CLΔ1−6 + 5-modified CL structure. The 5-nt insertion in the 3′ strand of stem A is indicated in gray. Note that T7 RNA polymerase-transcribed RNA contains two additional guanine nucleotides ( italic ), which form base pairs with cytosine nucleotides of the 3′ strand of stem A. The 9-nt RNA primer used for base pairing-directed RNA ligation is depicted in light gray, and ‘R’ represents the different 5′ modifications. ( B ) RNA primer ligation efficiency to genomic RNA possessing the CLΔ1−6 + 5 structure was determined by urea–PAGE analysis of a 250-nt RNase H-digested 5′-terminal fragment. Note that ligation of the RNA primer reduces migration speed of the 250-nt RNase H-digested RNA fragment. ( C ) Translation of incoming genomic RLuc-CVB3 RNA (wt), RNA holding the mutated CL (Δ1−6 + 5) and RNA ligation products possessing different 5′ modifications (OH, BCN, GH[5], GH[9], GH[11]) were determined by transfection of RNA in HeLa cells in the presence of GuHCl. Eight hours post-transfection, HeLa cells were lysed and RLuc values were determined. Data from a representative experiment are presented as the mean of duplicate ±SD and analyzed using unpaired t -test (* indicates significant difference P

    Article Snippet: RNA was incubated with 12.5 pmol of DNA primer (5′-GTAGTTGGCCGATAACGAACG-3′) and 5 U of RNase H (Fermentas) for 20 min at 50°C.

    Techniques: Ligation, Modification, Polyacrylamide Gel Electrophoresis, Migration, Transfection

    Base pairing-directed RNA primer ligation to RLuc-CVB3-CLΔ1−6 + 8 genomic RNA. ( A ) Schematic representation of the wt and the CLΔ1 − 6 + 8-modified CL structure. The 8-nt insertion in the 3′strand of stem A is indicated in gray. Note that T7 RNA polymerase-transcribed RNA contains two additional guanine nucleotides ( italic ), which form a wobble base pair with uracil nucleotides of the 3′strand of stem A in the Δ1 − 6 + 8-modified CL structure. The 12-nt RNA primer used for base pairing-directed RNA ligation is depicted in light gray, and ‘R’ represents the different 5′ modifications. ( B ) Urea–PAGE analysis of an RNA primer ligation to a 250-nt RNA fragment possessing the Δ1−6 + 8-mutated CL structure. RNA primer was ligated using either RNA Ligase 1 or RNA Ligase 2. Clearly, the RNA Ligase 2 was more efficient in ligating the RNA primer to the modified CLΔ1−6 + 8 structure. ( C ) RNA primer ligation efficiency to genomic RNA possessing the CLΔ1−6 + 8 structure was determined by urea–PAGE analysis of a 250-nt RNase H-digested 5′-terminal fragment. Note that ligation of the RNA primer reduces migration speed of the 250-nt RNase H-digested RNA fragment. ( D ) Translation of the incoming genomic RLuc-CVB3 RNA (wt), RNA holding the mutated CL (Δ1−6 + 8) and RNA ligation products with different 5′ ;modifications (OH, amine, biotin, Cy5) were determined by transfection of RNA in HeLa cells in the presence of GuHCl. Eight hours post-transfection, HeLa cells were lysed and RLuc values were determined. Data from a representative experiment are presented as the mean of duplicate ±SD and analyzed using unpaired t -test (** indicates significant difference P

    Journal: Nucleic Acids Research

    Article Title: Modification of picornavirus genomic RNA using 'click' chemistry shows that unlinking of the VPg peptide is dispensable for translation and replication of the incoming viral RNA

    doi: 10.1093/nar/gkt1162

    Figure Lengend Snippet: Base pairing-directed RNA primer ligation to RLuc-CVB3-CLΔ1−6 + 8 genomic RNA. ( A ) Schematic representation of the wt and the CLΔ1 − 6 + 8-modified CL structure. The 8-nt insertion in the 3′strand of stem A is indicated in gray. Note that T7 RNA polymerase-transcribed RNA contains two additional guanine nucleotides ( italic ), which form a wobble base pair with uracil nucleotides of the 3′strand of stem A in the Δ1 − 6 + 8-modified CL structure. The 12-nt RNA primer used for base pairing-directed RNA ligation is depicted in light gray, and ‘R’ represents the different 5′ modifications. ( B ) Urea–PAGE analysis of an RNA primer ligation to a 250-nt RNA fragment possessing the Δ1−6 + 8-mutated CL structure. RNA primer was ligated using either RNA Ligase 1 or RNA Ligase 2. Clearly, the RNA Ligase 2 was more efficient in ligating the RNA primer to the modified CLΔ1−6 + 8 structure. ( C ) RNA primer ligation efficiency to genomic RNA possessing the CLΔ1−6 + 8 structure was determined by urea–PAGE analysis of a 250-nt RNase H-digested 5′-terminal fragment. Note that ligation of the RNA primer reduces migration speed of the 250-nt RNase H-digested RNA fragment. ( D ) Translation of the incoming genomic RLuc-CVB3 RNA (wt), RNA holding the mutated CL (Δ1−6 + 8) and RNA ligation products with different 5′ ;modifications (OH, amine, biotin, Cy5) were determined by transfection of RNA in HeLa cells in the presence of GuHCl. Eight hours post-transfection, HeLa cells were lysed and RLuc values were determined. Data from a representative experiment are presented as the mean of duplicate ±SD and analyzed using unpaired t -test (** indicates significant difference P

    Article Snippet: RNA was incubated with 12.5 pmol of DNA primer (5′-GTAGTTGGCCGATAACGAACG-3′) and 5 U of RNase H (Fermentas) for 20 min at 50°C.

    Techniques: Ligation, Modification, Polyacrylamide Gel Electrophoresis, Migration, Transfection

    Modification of the 5′ terminus of picornavirus genomic RNA with VPg linked via a ‘non-cleavable’ bond. ( A ) Schematic representation of the SPAAC ‘click’ reaction that was used to couple the VPg peptides to the RNA primer ( B ) SPAAC ‘click’ reaction efficiency was determined by urea–PAGE analysis. The unmodified RNA primer (OH) and the BCN-modified RNA primer (BCN) migrated faster than the VPg-containing BCN primers (CVB3 VPg and PV VPg). ( C ) Structure of VPg-RNA linked either by the natural tyrosine phosphodiester bond or the triazole linkage. Arrow indicates the unlinkase site of the TDP2 enzyme. ( D ) RNA primer ligation efficiency to genomic RLuc-CVB3-Δ1-6 + 5 RNA was determined by urea–PAGE analysis of a 250-nt RNase H-digested 5′-terminal fragment. Note that ligation of the RNA primer reduces migration speed, especially in the case of the RNA primers containing VPg (CVB3 VPg and PV VPg). ( E ) The presence of VPg was determined by dot blot analysis. Equimolar amounts of the VPg peptide and RNA possessing VPg via a ‘non-cleavable’ bond were spotted on a membrane, and VPg presence was detected by a polyclonal antibody (  29 ). Note that CVB3 VPg is less reactive than PV VPg as the antibody is raised against the PV VPg. Importantly, the signals of the peptides correlated with the signal intensities from the RNA ligation products possessing VPg. ( F ) Unlinkase reaction using recombinant TDP2 was performed using the RNA ligation product containing the unmodified RNA primer (OH), the PV VPg peptide (PV VPg) linked via a ‘non-cleavable’ bond and genomic RNA isolated from PV virions (wt vRNA). Values were corrected for background signal (OH), and mean of two independent experiments are shown ±SD and analyzed using unpaired  t -test (* indicates significant difference P

    Journal: Nucleic Acids Research

    Article Title: Modification of picornavirus genomic RNA using 'click' chemistry shows that unlinking of the VPg peptide is dispensable for translation and replication of the incoming viral RNA

    doi: 10.1093/nar/gkt1162

    Figure Lengend Snippet: Modification of the 5′ terminus of picornavirus genomic RNA with VPg linked via a ‘non-cleavable’ bond. ( A ) Schematic representation of the SPAAC ‘click’ reaction that was used to couple the VPg peptides to the RNA primer ( B ) SPAAC ‘click’ reaction efficiency was determined by urea–PAGE analysis. The unmodified RNA primer (OH) and the BCN-modified RNA primer (BCN) migrated faster than the VPg-containing BCN primers (CVB3 VPg and PV VPg). ( C ) Structure of VPg-RNA linked either by the natural tyrosine phosphodiester bond or the triazole linkage. Arrow indicates the unlinkase site of the TDP2 enzyme. ( D ) RNA primer ligation efficiency to genomic RLuc-CVB3-Δ1-6 + 5 RNA was determined by urea–PAGE analysis of a 250-nt RNase H-digested 5′-terminal fragment. Note that ligation of the RNA primer reduces migration speed, especially in the case of the RNA primers containing VPg (CVB3 VPg and PV VPg). ( E ) The presence of VPg was determined by dot blot analysis. Equimolar amounts of the VPg peptide and RNA possessing VPg via a ‘non-cleavable’ bond were spotted on a membrane, and VPg presence was detected by a polyclonal antibody ( 29 ). Note that CVB3 VPg is less reactive than PV VPg as the antibody is raised against the PV VPg. Importantly, the signals of the peptides correlated with the signal intensities from the RNA ligation products possessing VPg. ( F ) Unlinkase reaction using recombinant TDP2 was performed using the RNA ligation product containing the unmodified RNA primer (OH), the PV VPg peptide (PV VPg) linked via a ‘non-cleavable’ bond and genomic RNA isolated from PV virions (wt vRNA). Values were corrected for background signal (OH), and mean of two independent experiments are shown ±SD and analyzed using unpaired t -test (* indicates significant difference P

    Article Snippet: RNA was incubated with 12.5 pmol of DNA primer (5′-GTAGTTGGCCGATAACGAACG-3′) and 5 U of RNase H (Fermentas) for 20 min at 50°C.

    Techniques: Modification, Polyacrylamide Gel Electrophoresis, Ligation, Migration, Dot Blot, Recombinant, Isolation

    Protection of the 5' splice site and branch point sequence of intron 10.  The templates were derived from minigenes A, C, K, and L, and either lacked the terminal 5' splice site of exon 11 (A, C, K,    L, left panel) or contained a consensus terminal splice site (Acon, Ccon, Kcon,    Lcon, right panel). The templates were incubated on ice or at 30°C for 30 min. An oligonucleotide complementary to the 5' splice site of exon 10 was added followed by 2U RNase H. Reactions were digested for 5 min at 37°C, the RNA extracted and separated on 5% sequencing gels. The time of incubation at 30°C is indicated above each panel along with the template added.  Panel A:  RNase H protection assay using the oligonucleotide complementary to the 5' splice site of exon 10.  Panel B:  RNase H protection assay using the oligonucleotide complementary to the branch point sequence of intron 10. Open arrowheads indicate cleavage products from each template. Spliced product and 5' exon can be seen in the right panel as the templates with the consensus splice site are spliced efficiently.

    Journal: BMC Molecular Biology

    Article Title: Assembly of splicing complexes on exon 11 of the human insulin receptor gene does not correlate with splicing efficiency in-vitro

    doi: 10.1186/1471-2199-5-7

    Figure Lengend Snippet: Protection of the 5' splice site and branch point sequence of intron 10. The templates were derived from minigenes A, C, K, and L, and either lacked the terminal 5' splice site of exon 11 (A, C, K, L, left panel) or contained a consensus terminal splice site (Acon, Ccon, Kcon, Lcon, right panel). The templates were incubated on ice or at 30°C for 30 min. An oligonucleotide complementary to the 5' splice site of exon 10 was added followed by 2U RNase H. Reactions were digested for 5 min at 37°C, the RNA extracted and separated on 5% sequencing gels. The time of incubation at 30°C is indicated above each panel along with the template added. Panel A: RNase H protection assay using the oligonucleotide complementary to the 5' splice site of exon 10. Panel B: RNase H protection assay using the oligonucleotide complementary to the branch point sequence of intron 10. Open arrowheads indicate cleavage products from each template. Spliced product and 5' exon can be seen in the right panel as the templates with the consensus splice site are spliced efficiently.

    Article Snippet: RNase H was from USB Corporation (Cleveland, OH).

    Techniques: Sequencing, Derivative Assay, Incubation

    Splicing complexes are assembled on all templates.  RNA templates were derived from minigenes A, C, K, and L and lacked the terminal 5' spice site of exon 11. The RNAs were incubated in 40 % HeLa nuclear extract on ice or for increasing times at 30°C to allow the formation of splicing complexes. Reactions were loaded directly onto 2% low-melting agarose gels (Panel A) or 4% native polyacrylamide gels (Panel B) and complexes separated by electrophoresis. Gels were dried under vacuum and exposed to film. The non-specific hnRNP complex H is indicated as well as the pre-spliceosomal complexes E and A. In Panel B, the higher order complexes B and C are indicated. In Panel C, the nuclear extract was pretreated with RNase H and an oligonucleotide complementary to the first 14 nucleotides of the U2 snRNA or a control oligonucleotide before complex assembly. In Panel D, the assembly reactions were performed in the absence or presence of ATP.

    Journal: BMC Molecular Biology

    Article Title: Assembly of splicing complexes on exon 11 of the human insulin receptor gene does not correlate with splicing efficiency in-vitro

    doi: 10.1186/1471-2199-5-7

    Figure Lengend Snippet: Splicing complexes are assembled on all templates. RNA templates were derived from minigenes A, C, K, and L and lacked the terminal 5' spice site of exon 11. The RNAs were incubated in 40 % HeLa nuclear extract on ice or for increasing times at 30°C to allow the formation of splicing complexes. Reactions were loaded directly onto 2% low-melting agarose gels (Panel A) or 4% native polyacrylamide gels (Panel B) and complexes separated by electrophoresis. Gels were dried under vacuum and exposed to film. The non-specific hnRNP complex H is indicated as well as the pre-spliceosomal complexes E and A. In Panel B, the higher order complexes B and C are indicated. In Panel C, the nuclear extract was pretreated with RNase H and an oligonucleotide complementary to the first 14 nucleotides of the U2 snRNA or a control oligonucleotide before complex assembly. In Panel D, the assembly reactions were performed in the absence or presence of ATP.

    Article Snippet: RNase H was from USB Corporation (Cleveland, OH).

    Techniques: Derivative Assay, Incubation, Electrophoresis

    Lack of protection by an oligonucleotide complementary to exon 10.  Panel A:  RNase H protection assay was performed using an oligonucleotide complementary to exon 10. The templates were derived from minigenes A, C, K, and L, and lacked the terminal 5' splice site of exon 11. The templates were incubated on ice or at 30°C for 30 min. An oligonucleotide complementary to exon 10 was added followed by 2U RNase H. Reactions were digested for 5 min at 37°C, the RNA extracted and separated on 5% sequencing gels. The time of incubation at 30°C is indicated above each panel along with the template added. Open arrowheads indicate cleavage products from each template. The ratio of cleaved to uncleaved RNA is unaltered with this oligonucleotide.  Panel B:  the HeLa nuclear extract was pretreated with RNase H and an oligonucleotide complementary to the first 14 nucleotides of the U1 snRNA to deplete functional U1 snRNP. RNaseH protection assays were run as before using the oligonucleotide complementary to the 5' splice site using control (HeLa) or depleted (HeLaΔU1) extracts. Open arrowheads indicate cleavage products.

    Journal: BMC Molecular Biology

    Article Title: Assembly of splicing complexes on exon 11 of the human insulin receptor gene does not correlate with splicing efficiency in-vitro

    doi: 10.1186/1471-2199-5-7

    Figure Lengend Snippet: Lack of protection by an oligonucleotide complementary to exon 10. Panel A: RNase H protection assay was performed using an oligonucleotide complementary to exon 10. The templates were derived from minigenes A, C, K, and L, and lacked the terminal 5' splice site of exon 11. The templates were incubated on ice or at 30°C for 30 min. An oligonucleotide complementary to exon 10 was added followed by 2U RNase H. Reactions were digested for 5 min at 37°C, the RNA extracted and separated on 5% sequencing gels. The time of incubation at 30°C is indicated above each panel along with the template added. Open arrowheads indicate cleavage products from each template. The ratio of cleaved to uncleaved RNA is unaltered with this oligonucleotide. Panel B: the HeLa nuclear extract was pretreated with RNase H and an oligonucleotide complementary to the first 14 nucleotides of the U1 snRNA to deplete functional U1 snRNP. RNaseH protection assays were run as before using the oligonucleotide complementary to the 5' splice site using control (HeLa) or depleted (HeLaΔU1) extracts. Open arrowheads indicate cleavage products.

    Article Snippet: RNase H was from USB Corporation (Cleveland, OH).

    Techniques: Derivative Assay, Incubation, Sequencing, Functional Assay

    RT-PCR in the analyses of naturally occurring endogenous sense-antisense RNA pair expression .  (a)  Schematic of the cardiac MHC (MYH) gene locus and its transcription products. The upper strand transcribes the cardiac MYH7 and MYH6 sense RNA, the lower strand transcribes the antisense MYH7 RNA, which is abundant in normal control hearts [1].  (b) : representative gels obtained from RT-PCR targeting sense and antisense RNA corresponding to the MYH7 gene. RT used RNase H -  enzyme under manufacturer standard conditions (see methods) in presense of specific primers (+p) or in absence of primers (-p).  (c)  Bar graph depicting the net signal of MYH7 sense and antisense in each group, net consisting of the difference between +p and -p RT-PCR band intensity. Note that a normal control heart in the rat is associated with abundant relatively MYH7 gene expression (MYH6 gene expression is dominant). Under the PTU condition, MYH7sense RNA expression is increased. Antisense MYH7 RNA is strongly expressed in the normal control heart, based on strong net signal. In PTU heart, the antisense MYH7 RNA is decreased to a very low level. Note the +p product is similar to -p when targeting antisense MYH7 RNA in PTU hearts.  (d)  Bar graph depicting relative no-primer signal (NP) to the total signal in each group as determined by real time PCR methods.  (e)  Net MYH7 sense and antisense RNA copy numbers in NC and PTU hearts using real time PCR. Data are means ± SE. N = 6/group. See Additional file 4 for primer information. For both sense and antisense MYH7 targets, end-point PCR (b and c) used 0.2 μl of the cDNA and was performed for 28 cycles. For real time PCR, we used 320 nl cDNA for each sample, and the signal was compared to a standard curve established with a serial dilution of a standard consisting of purified PCR product as explained in the methods. See Additional file 4 for primer information. Based on standard curve linear regression analyses, copies for each target RNA were calculated.+p: a strand specific RT primer was included; -p: RT without primer. Sense is the amplification product of the sense target obtained when the reverse primer was added to the RT reaction. Antisense is the amplification product of the antisense target obtained when the forward primer was included in the RT reaction. In all these reactions, the presence of the no primer product depended on the presence of RNA and the RT enzyme, and was not formed in RT reactions that were carried out in the absence of the reverse transcriptase enzyme.

    Journal: BMC Biotechnology

    Article Title: Potential pitfalls in the accuracy of analysis of natural sense-antisense RNA pairs by reverse transcription-PCR

    doi: 10.1186/1472-6750-7-21

    Figure Lengend Snippet: RT-PCR in the analyses of naturally occurring endogenous sense-antisense RNA pair expression . (a) Schematic of the cardiac MHC (MYH) gene locus and its transcription products. The upper strand transcribes the cardiac MYH7 and MYH6 sense RNA, the lower strand transcribes the antisense MYH7 RNA, which is abundant in normal control hearts [1]. (b) : representative gels obtained from RT-PCR targeting sense and antisense RNA corresponding to the MYH7 gene. RT used RNase H - enzyme under manufacturer standard conditions (see methods) in presense of specific primers (+p) or in absence of primers (-p). (c) Bar graph depicting the net signal of MYH7 sense and antisense in each group, net consisting of the difference between +p and -p RT-PCR band intensity. Note that a normal control heart in the rat is associated with abundant relatively MYH7 gene expression (MYH6 gene expression is dominant). Under the PTU condition, MYH7sense RNA expression is increased. Antisense MYH7 RNA is strongly expressed in the normal control heart, based on strong net signal. In PTU heart, the antisense MYH7 RNA is decreased to a very low level. Note the +p product is similar to -p when targeting antisense MYH7 RNA in PTU hearts. (d) Bar graph depicting relative no-primer signal (NP) to the total signal in each group as determined by real time PCR methods. (e) Net MYH7 sense and antisense RNA copy numbers in NC and PTU hearts using real time PCR. Data are means ± SE. N = 6/group. See Additional file 4 for primer information. For both sense and antisense MYH7 targets, end-point PCR (b and c) used 0.2 μl of the cDNA and was performed for 28 cycles. For real time PCR, we used 320 nl cDNA for each sample, and the signal was compared to a standard curve established with a serial dilution of a standard consisting of purified PCR product as explained in the methods. See Additional file 4 for primer information. Based on standard curve linear regression analyses, copies for each target RNA were calculated.+p: a strand specific RT primer was included; -p: RT without primer. Sense is the amplification product of the sense target obtained when the reverse primer was added to the RT reaction. Antisense is the amplification product of the antisense target obtained when the forward primer was included in the RT reaction. In all these reactions, the presence of the no primer product depended on the presence of RNA and the RT enzyme, and was not formed in RT reactions that were carried out in the absence of the reverse transcriptase enzyme.

    Article Snippet: These reverse transcriptions were carried out using RNase H- reverse transcriptase (Superscript II) according to the recommended protocol (Invitrogen).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, RNA Expression, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Serial Dilution, Purification, Amplification

    Testing different RT enzyme properties and conditions .  (a)  In a two-step RT-PCR system, 2 μg total RNA from NC and PTU treated hearts were reverse transcribed in 20 μl reactions in absence (-p) or presence (+p) of RT primers. The RT primer targeted the MYH7 sense RNA, and the PCR primer set amplified a 284 bp product corresponding to the 3' end of the MYH7 gene. PCR used 1 μl cDNA and was carried out for either 28 or 30 cycles. Shown are results from using two different RT enzymes that differed by their RNase H properties. RNase H -  and RNase H + . For each enzyme, the RT reactions were carried out under two different temperatures: 44°C or 50°C for 30 minutes/ea.  (b)  RT-PCR targeting the antisense MYH7 RNA in total RNA mixes of known proportions of sense and antisense RNA. RNA template contained either only sense MYH7 RNA, or a mix of sense and antisense MYH7 RNA corresponding to 99 to1 or 90 to 10 sense to antisense ratios (S:AS). Soleus total RNA was used as a source of the sense MYH7 RNA in absence of antisense. Whereas, T3-treated heart total RNA was used as a source of the antisense MYH7 RNA without co-expression of the sense. Mixes of soleus and T3 treated heart RNA were used to achieve the noted S:AS amounts in 2 μg of total RNA per 20 μl reactions. Reverse transcriptions were carried out in absence of RT primers (-P), in presence of the forward primer (+F) targeting the antisense, and in presence of a non specific primer corresponding to the 3' untranslated region of the human MYH4 mRNA sequence (+N). RT reactions used RNase H -  RT (Invitrogen), performed at 44°C or at 50°C for 30 min. PCR was carried out on 1 μcDNA for 28 cycles targeting the 3' end of the MYH7 gene. See Additional file 4 for primers information.

    Journal: BMC Biotechnology

    Article Title: Potential pitfalls in the accuracy of analysis of natural sense-antisense RNA pairs by reverse transcription-PCR

    doi: 10.1186/1472-6750-7-21

    Figure Lengend Snippet: Testing different RT enzyme properties and conditions . (a) In a two-step RT-PCR system, 2 μg total RNA from NC and PTU treated hearts were reverse transcribed in 20 μl reactions in absence (-p) or presence (+p) of RT primers. The RT primer targeted the MYH7 sense RNA, and the PCR primer set amplified a 284 bp product corresponding to the 3' end of the MYH7 gene. PCR used 1 μl cDNA and was carried out for either 28 or 30 cycles. Shown are results from using two different RT enzymes that differed by their RNase H properties. RNase H - and RNase H + . For each enzyme, the RT reactions were carried out under two different temperatures: 44°C or 50°C for 30 minutes/ea. (b) RT-PCR targeting the antisense MYH7 RNA in total RNA mixes of known proportions of sense and antisense RNA. RNA template contained either only sense MYH7 RNA, or a mix of sense and antisense MYH7 RNA corresponding to 99 to1 or 90 to 10 sense to antisense ratios (S:AS). Soleus total RNA was used as a source of the sense MYH7 RNA in absence of antisense. Whereas, T3-treated heart total RNA was used as a source of the antisense MYH7 RNA without co-expression of the sense. Mixes of soleus and T3 treated heart RNA were used to achieve the noted S:AS amounts in 2 μg of total RNA per 20 μl reactions. Reverse transcriptions were carried out in absence of RT primers (-P), in presence of the forward primer (+F) targeting the antisense, and in presence of a non specific primer corresponding to the 3' untranslated region of the human MYH4 mRNA sequence (+N). RT reactions used RNase H - RT (Invitrogen), performed at 44°C or at 50°C for 30 min. PCR was carried out on 1 μcDNA for 28 cycles targeting the 3' end of the MYH7 gene. See Additional file 4 for primers information.

    Article Snippet: These reverse transcriptions were carried out using RNase H- reverse transcriptase (Superscript II) according to the recommended protocol (Invitrogen).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Expressing, Sequencing