Structured Review

Thermo Fisher rnase v1
( A ) ‘G’ sequence determination of 3′ end-labeled 1–570 RNA in our standard conditions of cleavage by parallel running of RNA degradation with <t>RNase</t> T1 0.0005 µg/µl in denaturing conditions (lane 3), in standard buffer (lane 4) and in the presence of miR-122 at 15 nM (lane 5). Lanes 1 is an alkali ladder degradation and lane 2, the RNA incubated in standard buffer alone. Gs are identified at the right of the gel. ( B ) Secondary structure of stem loop VI and its boundary regions summarizing the position with differential reactivity for RNase T1. Increased resistance is indicated by solid triangles and increased sensitivity by blank triangles. Nucleotide numbering is used, as in Figure 1 . ( C ) Evaluation of the effect of increasing concentrations of probes on the T1 nuclease pattern of cleavage. Lane 1 is a MW marker. Control incubation of 1–570 RNA in the buffer (lane 2), or after addition 0.0005 µg/µl of RNase T1 (lane 3). In subsequent lanes, before addition of the T1 RNase, RNAs were pre-incubated for 1 h with increasing concentrations of ODN 22(−) of 15 nM, 150 nM and 1500 nM (lanes 4–6), or miR-122 at final concentrations of 1.5 nM, 15 nM and 150 nM (lanes 7–9) and a 10-mer oligoribonucleotide carrying the 7 nucleotides of the miR-122 seed sequence to a final concentration of 1.5 nM, 15 nM and 150 nM (lanes 10–12). ( D ) Same as panel B, but the RNase used was double-stranded RNase V1. Arrows indicate the changes in sensitivity to the nuclease.
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Images

1) Product Images from "In vitro characterization of a miR-122-sensitive double-helical switch element in the 5? region of hepatitis C virus RNA"

Article Title: In vitro characterization of a miR-122-sensitive double-helical switch element in the 5? region of hepatitis C virus RNA

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkp553

( A ) ‘G’ sequence determination of 3′ end-labeled 1–570 RNA in our standard conditions of cleavage by parallel running of RNA degradation with RNase T1 0.0005 µg/µl in denaturing conditions (lane 3), in standard buffer (lane 4) and in the presence of miR-122 at 15 nM (lane 5). Lanes 1 is an alkali ladder degradation and lane 2, the RNA incubated in standard buffer alone. Gs are identified at the right of the gel. ( B ) Secondary structure of stem loop VI and its boundary regions summarizing the position with differential reactivity for RNase T1. Increased resistance is indicated by solid triangles and increased sensitivity by blank triangles. Nucleotide numbering is used, as in Figure 1 . ( C ) Evaluation of the effect of increasing concentrations of probes on the T1 nuclease pattern of cleavage. Lane 1 is a MW marker. Control incubation of 1–570 RNA in the buffer (lane 2), or after addition 0.0005 µg/µl of RNase T1 (lane 3). In subsequent lanes, before addition of the T1 RNase, RNAs were pre-incubated for 1 h with increasing concentrations of ODN 22(−) of 15 nM, 150 nM and 1500 nM (lanes 4–6), or miR-122 at final concentrations of 1.5 nM, 15 nM and 150 nM (lanes 7–9) and a 10-mer oligoribonucleotide carrying the 7 nucleotides of the miR-122 seed sequence to a final concentration of 1.5 nM, 15 nM and 150 nM (lanes 10–12). ( D ) Same as panel B, but the RNase used was double-stranded RNase V1. Arrows indicate the changes in sensitivity to the nuclease.
Figure Legend Snippet: ( A ) ‘G’ sequence determination of 3′ end-labeled 1–570 RNA in our standard conditions of cleavage by parallel running of RNA degradation with RNase T1 0.0005 µg/µl in denaturing conditions (lane 3), in standard buffer (lane 4) and in the presence of miR-122 at 15 nM (lane 5). Lanes 1 is an alkali ladder degradation and lane 2, the RNA incubated in standard buffer alone. Gs are identified at the right of the gel. ( B ) Secondary structure of stem loop VI and its boundary regions summarizing the position with differential reactivity for RNase T1. Increased resistance is indicated by solid triangles and increased sensitivity by blank triangles. Nucleotide numbering is used, as in Figure 1 . ( C ) Evaluation of the effect of increasing concentrations of probes on the T1 nuclease pattern of cleavage. Lane 1 is a MW marker. Control incubation of 1–570 RNA in the buffer (lane 2), or after addition 0.0005 µg/µl of RNase T1 (lane 3). In subsequent lanes, before addition of the T1 RNase, RNAs were pre-incubated for 1 h with increasing concentrations of ODN 22(−) of 15 nM, 150 nM and 1500 nM (lanes 4–6), or miR-122 at final concentrations of 1.5 nM, 15 nM and 150 nM (lanes 7–9) and a 10-mer oligoribonucleotide carrying the 7 nucleotides of the miR-122 seed sequence to a final concentration of 1.5 nM, 15 nM and 150 nM (lanes 10–12). ( D ) Same as panel B, but the RNase used was double-stranded RNase V1. Arrows indicate the changes in sensitivity to the nuclease.

Techniques Used: Sequencing, Labeling, Incubation, Marker, Concentration Assay

2) Product Images from "A search for structurally similar cellular internal ribosome entry sites"

Article Title: A search for structurally similar cellular internal ribosome entry sites

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkm483

The proposed XIAP IRES RNA secondary structure model. The sites sensitive to RNase T1, which cuts after single-stranded guanine nucleotides, RNase T2, which cuts after single-stranded nucleotides, and RNase V1, which cuts within regions of double-stranded RNA, are shown on the derived structural model. These cut sites were used as constraints in the MFOLD ( 44 ) program to predict the secondary structure of the minimal XIAP IRES sequence. The full list of sensitive sites is shown in Supplementary Table 1.
Figure Legend Snippet: The proposed XIAP IRES RNA secondary structure model. The sites sensitive to RNase T1, which cuts after single-stranded guanine nucleotides, RNase T2, which cuts after single-stranded nucleotides, and RNase V1, which cuts within regions of double-stranded RNA, are shown on the derived structural model. These cut sites were used as constraints in the MFOLD ( 44 ) program to predict the secondary structure of the minimal XIAP IRES sequence. The full list of sensitive sites is shown in Supplementary Table 1.

Techniques Used: Derivative Assay, Sequencing

3) Product Images from "Recognition of tRNALeu by Aquifex aeolicus leucyl-tRNA synthetase during the aminoacylation and editing steps"

Article Title: Recognition of tRNALeu by Aquifex aeolicus leucyl-tRNA synthetase during the aminoacylation and editing steps

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkn028

Nuclease probing of Aa tRNA Leu CAA in the free form or in complex with Aa LeuRS. ( A ) tRNA Leu CAA was labeled at its 5′-end or 3′-end. Probing was done in the presence (+) or absence (−) of LeuRS. OH and T1 are ladders of the tRNA under the denaturing condition; Ctrl is the control without any probe. The probes comprised RNase T1, RNase T2 and RNase V1. Numbers refer to tRNA nucleotide positions. ( B ) Cloverleaf structure of tRNA summarizing the reactivity changes observed in the tRNA following Aa LeuRS binding. The symbols and color codes for the probes are indicated in the figure. Three intensities of cuts/modifications for each probe are shown (strong, medium and moderate).
Figure Legend Snippet: Nuclease probing of Aa tRNA Leu CAA in the free form or in complex with Aa LeuRS. ( A ) tRNA Leu CAA was labeled at its 5′-end or 3′-end. Probing was done in the presence (+) or absence (−) of LeuRS. OH and T1 are ladders of the tRNA under the denaturing condition; Ctrl is the control without any probe. The probes comprised RNase T1, RNase T2 and RNase V1. Numbers refer to tRNA nucleotide positions. ( B ) Cloverleaf structure of tRNA summarizing the reactivity changes observed in the tRNA following Aa LeuRS binding. The symbols and color codes for the probes are indicated in the figure. Three intensities of cuts/modifications for each probe are shown (strong, medium and moderate).

Techniques Used: Cellular Antioxidant Activity Assay, Labeling, Binding Assay

Nuclease probing of Aa tRNA Leu GAG in the free form or in complex with Aa LeuRS. tRNA Leu GAG was labeled at its 5′-end ( A ) or 3′-end ( B ). Probing was conducted in the presence (+) or absence (−) of LeuRS. OH and T1 are ladders of the tRNA under the denaturing condition; Ctrl is the control without any probe. The probes comprised RNase T1, RNase T2 and RNase V1. Numbers refer to tRNA nucleotide positions. ( C ) Cloverleaf structure of tRNA summarizing the reactivity changes observed in the tRNA following Aa LeuRS binding. The symbols and color codes for the probes are indicated in the figure. Three intensities of cuts/modifications for each probe are shown (strong, medium and moderate).
Figure Legend Snippet: Nuclease probing of Aa tRNA Leu GAG in the free form or in complex with Aa LeuRS. tRNA Leu GAG was labeled at its 5′-end ( A ) or 3′-end ( B ). Probing was conducted in the presence (+) or absence (−) of LeuRS. OH and T1 are ladders of the tRNA under the denaturing condition; Ctrl is the control without any probe. The probes comprised RNase T1, RNase T2 and RNase V1. Numbers refer to tRNA nucleotide positions. ( C ) Cloverleaf structure of tRNA summarizing the reactivity changes observed in the tRNA following Aa LeuRS binding. The symbols and color codes for the probes are indicated in the figure. Three intensities of cuts/modifications for each probe are shown (strong, medium and moderate).

Techniques Used: Labeling, Binding Assay

4) Product Images from "Two ribosome recruitment sites direct multiple translation events within HIV1 Gag open reading frame"

Article Title: Two ribosome recruitment sites direct multiple translation events within HIV1 Gag open reading frame

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx303

RNA secondary structure model of the HIV1 Gag-IRES and 40S ribosome subunit footprints and toeprints. ( A ) Schematic representation of the secondary structure model of Gag-IRES. Nucleotides are colored according to their reactivity toward 1M7 as indicated in the box. Red triangles and blue dots represent RNAse T1 and V1 cleavages. Pairings numbering (P n and N n ) are as described in the text. Nucleotide numbering is from the +1 of transcription (First nucleotide of TAR). Experimental values result from the mean of three independent experiments (see material and methods and supplementary material ). ( B ) Footprints and toeprints of the 40S ribosomal subunit on Gag-IRES. As indicated in the box, the colors and signs compare the reactivities without and with saturating concentration of 40S ribosomal subunit. Nucleotides in red are more reactive to 1M7, while nucleotides in blue are less. Red and Blue triangles indicate nucleotides exposed or protected to RNAse T1 respectively. Red and Blue dots indicate nucleotides exposed or protected to RNAse V1 respectively. Black Arrows indicate premature RT stops observed in the presence of 40S ribosomal subunits. Small and big arrows are respectively moderate and strong toeprints. Experimental values result from the mean of three independent experiments (see material and methods and supplementary material ).
Figure Legend Snippet: RNA secondary structure model of the HIV1 Gag-IRES and 40S ribosome subunit footprints and toeprints. ( A ) Schematic representation of the secondary structure model of Gag-IRES. Nucleotides are colored according to their reactivity toward 1M7 as indicated in the box. Red triangles and blue dots represent RNAse T1 and V1 cleavages. Pairings numbering (P n and N n ) are as described in the text. Nucleotide numbering is from the +1 of transcription (First nucleotide of TAR). Experimental values result from the mean of three independent experiments (see material and methods and supplementary material ). ( B ) Footprints and toeprints of the 40S ribosomal subunit on Gag-IRES. As indicated in the box, the colors and signs compare the reactivities without and with saturating concentration of 40S ribosomal subunit. Nucleotides in red are more reactive to 1M7, while nucleotides in blue are less. Red and Blue triangles indicate nucleotides exposed or protected to RNAse T1 respectively. Red and Blue dots indicate nucleotides exposed or protected to RNAse V1 respectively. Black Arrows indicate premature RT stops observed in the presence of 40S ribosomal subunits. Small and big arrows are respectively moderate and strong toeprints. Experimental values result from the mean of three independent experiments (see material and methods and supplementary material ).

Techniques Used: Concentration Assay

5) Product Images from "RNA secondary structure profiling in zebrafish reveals unique regulatory features"

Article Title: RNA secondary structure profiling in zebrafish reveals unique regulatory features

Journal: BMC Genomics

doi: 10.1186/s12864-018-4497-0

Schematic of RNA structure probing by PARS in zebrafish. Poly-A RNA from zebrafish is folded in-vitro. The folded RNA is cleaved by RNase V1 and S1 nuclease separately. The enzyme cut sites generate 5’P ends and 3’ OH ends at the cleaved sites. Long fragments generated by single-hit kinetics are further fragmented by alkaline hydrolysis, which blocks the 3′ site of the enzyme-cut fragments. Sequencing adapters are ligated to the 5′ end followed by alkaline phosphatase treatment to 3’ P group. Adapters are ligated to 3’ends followed cDNA synthesis and PCR purification of the library. Appropriate size of the library is maintained by purification by nucleic acid beads. Sequenced reads are aligned back to the genome and only unique reads with the correct read start positions are considered for PARS score calculation
Figure Legend Snippet: Schematic of RNA structure probing by PARS in zebrafish. Poly-A RNA from zebrafish is folded in-vitro. The folded RNA is cleaved by RNase V1 and S1 nuclease separately. The enzyme cut sites generate 5’P ends and 3’ OH ends at the cleaved sites. Long fragments generated by single-hit kinetics are further fragmented by alkaline hydrolysis, which blocks the 3′ site of the enzyme-cut fragments. Sequencing adapters are ligated to the 5′ end followed by alkaline phosphatase treatment to 3’ P group. Adapters are ligated to 3’ends followed cDNA synthesis and PCR purification of the library. Appropriate size of the library is maintained by purification by nucleic acid beads. Sequenced reads are aligned back to the genome and only unique reads with the correct read start positions are considered for PARS score calculation

Techniques Used: In Vitro, Generated, Sequencing, Polymerase Chain Reaction, Purification

Comparison of RNA structures of ubc 3’UTR as determined by PARS based pairing probability and enzymatic footprinting using RNase V1 and S1 Nuclease. a . Bar plot represents PARS scores of 3’UTR region of ubiquitin c (ubc) . Out of 105 positions, 87 positions are captured by PARS. b . Enzymatic footprinting of ubc 3’UTR probed by S1 Nuclease and RNase V1. Nucleotide positions are correlated with alkaline hydrolysis (AH) ladder and RNase T1 (G) ladder. Positions with similar structural pattern with PARS scores are highlighted. Red dots indicate unpaired positions; green indicates paired positions while yellow represents ambiguous regions. c . Heatmap representing secondary structure of 68 positions of ubc 3’UTR as determined by PARS and enzymatic footprinting (FP). Top panel represents PARS pairing probability; bottom panel indicates enzymatic footprinting pairing probability; middle panel represents the consensus between the two (PARS: FP). Red represents unpaired, green represents paired and yellow represents ambiguous regions
Figure Legend Snippet: Comparison of RNA structures of ubc 3’UTR as determined by PARS based pairing probability and enzymatic footprinting using RNase V1 and S1 Nuclease. a . Bar plot represents PARS scores of 3’UTR region of ubiquitin c (ubc) . Out of 105 positions, 87 positions are captured by PARS. b . Enzymatic footprinting of ubc 3’UTR probed by S1 Nuclease and RNase V1. Nucleotide positions are correlated with alkaline hydrolysis (AH) ladder and RNase T1 (G) ladder. Positions with similar structural pattern with PARS scores are highlighted. Red dots indicate unpaired positions; green indicates paired positions while yellow represents ambiguous regions. c . Heatmap representing secondary structure of 68 positions of ubc 3’UTR as determined by PARS and enzymatic footprinting (FP). Top panel represents PARS pairing probability; bottom panel indicates enzymatic footprinting pairing probability; middle panel represents the consensus between the two (PARS: FP). Red represents unpaired, green represents paired and yellow represents ambiguous regions

Techniques Used: Footprinting

6) Product Images from "Posttranscriptional Regulation of tnaA by Protein-RNA Interaction Mediated by Ribosomal Protein L4 in Escherichia coli"

Article Title: Posttranscriptional Regulation of tnaA by Protein-RNA Interaction Mediated by Ribosomal Protein L4 in Escherichia coli

Journal: Journal of Bacteriology

doi: 10.1128/JB.00799-19

Structural probing of the 5′-end-labeled tnaC-tnaA transcribed spacer in the absence and presence of FLAG-L4. (A) DNA template (239 bp) containing the tnaC-tnaA spacer was transcribed under the T7 promoter to generate a 220 nt final RNA product. The sequence of the final 220-nt RNA product (with addition of a G at the 5′ end by RNA polymerase during in vitro transcription) is shown. The Shine-Dalgarno (SD) sequence of TnaA is underlined. Affected regions I, II, and III in the footprint determination are indicated in the primary sequence. The numbers at the top of the primary sequence represent the RNase T1 cleavage sites between guanosine 3′-phosphate residues and the 5′-OH residues of adjacent nucleotides, as shown in panel B (lanes 3, 11 and 18). The gray font in the primary sequence from the 3′ end shows the beginning of the coding sequence for TnaA. (B) The RNA was further 5′ end labeled with [γ- 32 P]ATP and purified from the denatured acrylamide gel. The 5′-end-labeled RNA (30,000 cpm, ∼2 ng) was then incubated at room temperature in the absence (−) or presence (+) of L4 (5.5 μM) for binding, before conducting RNase V1 digestion (double strand-specific cleavage; lanes 5 to 7, 13 to 15, and 20 to 22) and separation on an 8% polyacrylamide–8 M urea gel. The 5′-end-labeled RNA fragments generated by alkaline and RNase T1 hydrolysis (OH and T1, respectively) are shown. The experiments were carried out on untreated RNA (un) or in the presence of increasing concentrations of purified FLAG-L4. Regions I, II, and III affected by L4 in the footprint determination are indicated (also in panel C). Regions a, b, and c used for quantification are indicated with colored lines. Bands (N) used for normalization of the phosphor image signals of regions a, b, and c are indicated. (C) The 220 nt RNA secondary structure predicted by RNAfold ( 56 ) (dG = −44.05). (D) Quantification of regions a, b, and c affected by L4 in the footprint determination.
Figure Legend Snippet: Structural probing of the 5′-end-labeled tnaC-tnaA transcribed spacer in the absence and presence of FLAG-L4. (A) DNA template (239 bp) containing the tnaC-tnaA spacer was transcribed under the T7 promoter to generate a 220 nt final RNA product. The sequence of the final 220-nt RNA product (with addition of a G at the 5′ end by RNA polymerase during in vitro transcription) is shown. The Shine-Dalgarno (SD) sequence of TnaA is underlined. Affected regions I, II, and III in the footprint determination are indicated in the primary sequence. The numbers at the top of the primary sequence represent the RNase T1 cleavage sites between guanosine 3′-phosphate residues and the 5′-OH residues of adjacent nucleotides, as shown in panel B (lanes 3, 11 and 18). The gray font in the primary sequence from the 3′ end shows the beginning of the coding sequence for TnaA. (B) The RNA was further 5′ end labeled with [γ- 32 P]ATP and purified from the denatured acrylamide gel. The 5′-end-labeled RNA (30,000 cpm, ∼2 ng) was then incubated at room temperature in the absence (−) or presence (+) of L4 (5.5 μM) for binding, before conducting RNase V1 digestion (double strand-specific cleavage; lanes 5 to 7, 13 to 15, and 20 to 22) and separation on an 8% polyacrylamide–8 M urea gel. The 5′-end-labeled RNA fragments generated by alkaline and RNase T1 hydrolysis (OH and T1, respectively) are shown. The experiments were carried out on untreated RNA (un) or in the presence of increasing concentrations of purified FLAG-L4. Regions I, II, and III affected by L4 in the footprint determination are indicated (also in panel C). Regions a, b, and c used for quantification are indicated with colored lines. Bands (N) used for normalization of the phosphor image signals of regions a, b, and c are indicated. (C) The 220 nt RNA secondary structure predicted by RNAfold ( 56 ) (dG = −44.05). (D) Quantification of regions a, b, and c affected by L4 in the footprint determination.

Techniques Used: Labeling, Sequencing, In Vitro, Purification, Acrylamide Gel Assay, Incubation, Binding Assay, Generated

7) Product Images from "Unstructured 5′-tails act through ribosome standby to override inhibitory structure at ribosome binding sites"

Article Title: Unstructured 5′-tails act through ribosome standby to override inhibitory structure at ribosome binding sites

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky073

Addition of CA-repeats does not change the secondary structure of the coat RBS stem–loop. ( A ) Enzymatic and chemical structure probing was conducted on control mRNA (00) or mRNAs with tails of 4, 6 or 8 (CA)-repeats, (see Materials and Methods), as indicated. The mRNAs were mock-treated (lanes ‘–’), partially digested with double-strand-specific RNase V1 (V1), or treated with lead(II) acetate (Pb 2+ ). UCGA: sequencing reactions on (CA)8 mRNA. The position of the SD and AUG start codon are indicated by red boxes. Regions of reactivity toward RNase V1 (red solid line) and lead(II) acetate (red dashed line) are indicated on the autoradiogram. ( B ) The localization of RNase V1 (filled triangles) and lead(II) acetate (black dots) cuts are shown on the secondary structure of the 5′-segment of (CA)8 mRNA. Black boxes: SD and AUG.
Figure Legend Snippet: Addition of CA-repeats does not change the secondary structure of the coat RBS stem–loop. ( A ) Enzymatic and chemical structure probing was conducted on control mRNA (00) or mRNAs with tails of 4, 6 or 8 (CA)-repeats, (see Materials and Methods), as indicated. The mRNAs were mock-treated (lanes ‘–’), partially digested with double-strand-specific RNase V1 (V1), or treated with lead(II) acetate (Pb 2+ ). UCGA: sequencing reactions on (CA)8 mRNA. The position of the SD and AUG start codon are indicated by red boxes. Regions of reactivity toward RNase V1 (red solid line) and lead(II) acetate (red dashed line) are indicated on the autoradiogram. ( B ) The localization of RNase V1 (filled triangles) and lead(II) acetate (black dots) cuts are shown on the secondary structure of the 5′-segment of (CA)8 mRNA. Black boxes: SD and AUG.

Techniques Used: Sequencing

8) Product Images from "Interaction of C5 protein with RNA aptamers selected by SELEX"

Article Title: Interaction of C5 protein with RNA aptamers selected by SELEX

Journal: Nucleic Acids Research

doi:

Predicted secondary structure of W2 and its minimal binding domain essential for interaction with C5 protein. ( A ) Boundary determination analysis. W2 was labeled with 32 P, either at the 5′ or 3′ end. Labeled W2 was partially digested by alkaline hydrolysis and incubated with MBP–C5 protein immobilized on amylose resin. RNA fragments binding to MBP–C5 protein were analyzed on an 8% denaturing polyacrylamide gel. The solid line to the right of the figure represents the essential domains in W2 for binding to C5 protein. P, protein-bound RNA fragments. OH, partial alkaline hydrolytic products of W2. G, RNase T1 digests of W2. ( B ) RNase mapping of W2. Nuclease S1, ribonuclease V1 and RNase T1 treatments are indicated by S1, V1 and T1, respectively. G, G-specific cleavage products by RNase T1. OH, alkaline ladders. ( C ) Footprinting of W2 using Fe(II)-EDTA/H 2 O 2 . 5′ End labeled W2 RNA was preincubated with the indicated concentrations of MBP–C5 protein at 20°C for 10 min. Cleavage products were separated on 8% denaturing polyacrylamide gels. A major protected region is indicated by the solid line to the right of the figure. ( D ) Predicted secondary structure of W2. The consensus nucleotides capable of forming a stem are represented by the rectangle. The nuclease S1 cleavage sites (representing single-stranded regions) are specified with either strong (open arrows) or weak (open arrowheads) bands. RNase V1 cleavage sites (representing double-stranded regions) are also specified with either strong (filled arrows) or weak (filled arrowheads) bands. End boundaries of the binding domain for interaction with C5 protein are indicated by thick arrows. Asterisks represent cleavage sites by Pb(II).
Figure Legend Snippet: Predicted secondary structure of W2 and its minimal binding domain essential for interaction with C5 protein. ( A ) Boundary determination analysis. W2 was labeled with 32 P, either at the 5′ or 3′ end. Labeled W2 was partially digested by alkaline hydrolysis and incubated with MBP–C5 protein immobilized on amylose resin. RNA fragments binding to MBP–C5 protein were analyzed on an 8% denaturing polyacrylamide gel. The solid line to the right of the figure represents the essential domains in W2 for binding to C5 protein. P, protein-bound RNA fragments. OH, partial alkaline hydrolytic products of W2. G, RNase T1 digests of W2. ( B ) RNase mapping of W2. Nuclease S1, ribonuclease V1 and RNase T1 treatments are indicated by S1, V1 and T1, respectively. G, G-specific cleavage products by RNase T1. OH, alkaline ladders. ( C ) Footprinting of W2 using Fe(II)-EDTA/H 2 O 2 . 5′ End labeled W2 RNA was preincubated with the indicated concentrations of MBP–C5 protein at 20°C for 10 min. Cleavage products were separated on 8% denaturing polyacrylamide gels. A major protected region is indicated by the solid line to the right of the figure. ( D ) Predicted secondary structure of W2. The consensus nucleotides capable of forming a stem are represented by the rectangle. The nuclease S1 cleavage sites (representing single-stranded regions) are specified with either strong (open arrows) or weak (open arrowheads) bands. RNase V1 cleavage sites (representing double-stranded regions) are also specified with either strong (filled arrows) or weak (filled arrowheads) bands. End boundaries of the binding domain for interaction with C5 protein are indicated by thick arrows. Asterisks represent cleavage sites by Pb(II).

Techniques Used: Binding Assay, Labeling, Incubation, Footprinting

9) Product Images from "Structural organization of a viral IRES depends on the integrity of the GNRA motif"

Article Title: Structural organization of a viral IRES depends on the integrity of the GNRA motif

Journal: RNA

doi: 10.1261/rna.5950603

( A ) RNase T1 probing of FMDV domain 3. [γ- 32 P]ATP-5′ end-labeled RNA corresponding to domain 3 was incubated with 0.03 U RNase T1 in native (N) or denaturing (D) conditions. Samples were analyzed on 6% acrylamide 7M urea gels run for different amount of time. Asterisks denote the residues with enhanced sensibility to T1; ss and ds stand for single-stranded or double-stranded, respectively. Bands with the same electrophoretic mobility than truncated fragments of the untreated transcript were not considered. ( B ) RNase A accessibility to domain 3 of the FMDV IRES. Domain 3 was incubated with diluted RNase A (5 × 10 −6 mg/mL) to obtain a partial RNA digestion, and then subjected to primer extension analysis. ( C ) RNase V1 digestion of domain 3, analyzed by primer extension.
Figure Legend Snippet: ( A ) RNase T1 probing of FMDV domain 3. [γ- 32 P]ATP-5′ end-labeled RNA corresponding to domain 3 was incubated with 0.03 U RNase T1 in native (N) or denaturing (D) conditions. Samples were analyzed on 6% acrylamide 7M urea gels run for different amount of time. Asterisks denote the residues with enhanced sensibility to T1; ss and ds stand for single-stranded or double-stranded, respectively. Bands with the same electrophoretic mobility than truncated fragments of the untreated transcript were not considered. ( B ) RNase A accessibility to domain 3 of the FMDV IRES. Domain 3 was incubated with diluted RNase A (5 × 10 −6 mg/mL) to obtain a partial RNA digestion, and then subjected to primer extension analysis. ( C ) RNase V1 digestion of domain 3, analyzed by primer extension.

Techniques Used: Labeling, Incubation

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

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

Journal: RNA

doi: 10.1261/rna.063073.117

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

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

11) Product Images from "Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1"

Article Title: Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0035031

Secondary structure model of the 5′UTR of selected HIV-1 VAR sequences. The HIV-1 5′leader recovered from the dl VAR constructs (followed by 58 nucleotides of fLuc gene) was probed using Dimethyl Sulfate (DMS), N-Cyclohexyl-N0- [N-Methylmorpholino)-ethyl]-Carbodiimide-4-Toluolsulfonate (CMCT) and RNAse V1 as previously described [20] . (A) Secondary structure model of the control (pNL4.3) HIV-1 5′UTR according to DMS, CMCT and RNAse V1 structure probing [6] , a key indicating the respective reactivity to the different probes is provided (box). Data included in the figure have been extracted from reference [20] . (B) Typical examples of DMS probing. The HIV-1 5′leader was probed using (+) DMS. Reverse transcription (RT) products were separated on a 8% gel as previously described [12] , [20] , [48] . Sequencing lanes were also included. Note that DMS induces a premature RT stop one nucleotide before the hit. Therefore the DMS induced stops migrate faster than the corresponding sequence product [12] , [20] , [48] . The RT pattern of the modified RNA was compared to the profile obtained with an unmodified RNA. Some hits are indicated in the figure. The asterisks on the gel denote the FLuc initiation codon (AUG). Results for VAR 1 (C), VAR 2 (D), VAR 3 (E), and VAR 4 (F) were fitted in a model of the HIV-1 5′ leader [6] as previously described [20] . Numbering in A-F is with respect to clone pNL4.3, here considered the wt sequence. Insertions are indicated in red as independent numbers (iN, were N is the number). Nucleotide changes with respect to clone pNL4.3 are indicated in green. The nucleotide located before a deletion is highlighted in yellow, in this case numbering with respect to pNL4.3 is not altered.
Figure Legend Snippet: Secondary structure model of the 5′UTR of selected HIV-1 VAR sequences. The HIV-1 5′leader recovered from the dl VAR constructs (followed by 58 nucleotides of fLuc gene) was probed using Dimethyl Sulfate (DMS), N-Cyclohexyl-N0- [N-Methylmorpholino)-ethyl]-Carbodiimide-4-Toluolsulfonate (CMCT) and RNAse V1 as previously described [20] . (A) Secondary structure model of the control (pNL4.3) HIV-1 5′UTR according to DMS, CMCT and RNAse V1 structure probing [6] , a key indicating the respective reactivity to the different probes is provided (box). Data included in the figure have been extracted from reference [20] . (B) Typical examples of DMS probing. The HIV-1 5′leader was probed using (+) DMS. Reverse transcription (RT) products were separated on a 8% gel as previously described [12] , [20] , [48] . Sequencing lanes were also included. Note that DMS induces a premature RT stop one nucleotide before the hit. Therefore the DMS induced stops migrate faster than the corresponding sequence product [12] , [20] , [48] . The RT pattern of the modified RNA was compared to the profile obtained with an unmodified RNA. Some hits are indicated in the figure. The asterisks on the gel denote the FLuc initiation codon (AUG). Results for VAR 1 (C), VAR 2 (D), VAR 3 (E), and VAR 4 (F) were fitted in a model of the HIV-1 5′ leader [6] as previously described [20] . Numbering in A-F is with respect to clone pNL4.3, here considered the wt sequence. Insertions are indicated in red as independent numbers (iN, were N is the number). Nucleotide changes with respect to clone pNL4.3 are indicated in green. The nucleotide located before a deletion is highlighted in yellow, in this case numbering with respect to pNL4.3 is not altered.

Techniques Used: Construct, Sequencing, Modification

12) Product Images from "The RNA Structure of cis-acting Translational Elements of the Chloroplast psbC mRNA in Chlamydomonas reinhardtii"

Article Title: The RNA Structure of cis-acting Translational Elements of the Chloroplast psbC mRNA in Chlamydomonas reinhardtii

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2016.00828

RNA structure mapping results are shown with respect to the psbC 5′ UTR sequence. Asterisks indicate bases methylated by DMS in vitro and in vivo . Arrowheads indicate residues that were cleaved by RNase V1. Arrows indicate G residues that were cleaved by RNase T1. Most frequently methylated or cleaved positions are indicated by black asterisks, arrows and arrowheads. Less frequently methylated or cleaved sites are indicated in gray. Brackets indicate results with high accuracy and resolution. The 5′ terminal residue is designated +1, the translation initiation codon (GUG) is in bold, and the predicted Shine–Dalgarno sequence is underlined. Black and gray dots represent strong and weak RT pause sites, respectively.
Figure Legend Snippet: RNA structure mapping results are shown with respect to the psbC 5′ UTR sequence. Asterisks indicate bases methylated by DMS in vitro and in vivo . Arrowheads indicate residues that were cleaved by RNase V1. Arrows indicate G residues that were cleaved by RNase T1. Most frequently methylated or cleaved positions are indicated by black asterisks, arrows and arrowheads. Less frequently methylated or cleaved sites are indicated in gray. Brackets indicate results with high accuracy and resolution. The 5′ terminal residue is designated +1, the translation initiation codon (GUG) is in bold, and the predicted Shine–Dalgarno sequence is underlined. Black and gray dots represent strong and weak RT pause sites, respectively.

Techniques Used: Sequencing, Methylation, In Vitro, In Vivo

In vitro analysis of stem-loop (SL) structures in the psbC 5′ UTR. Asterisks ( ∗ ) indicate A and C residues that are methylated by DMS in vitro . Arrows indicate G residues that are substrates for RNase T1 and arrowheads indicated cleavage sites for RNase V1. Most frequently methylated or cleaved positions are indicated by black asterisks, arrows and arrowheads. Less frequently methylated or cleaved sites are indicated in gray. Hexagons indicate locations of strong premature RT stops. Black dots indicate base-pairs that are strongly supported by the data, while gray dots are partially supported and could be present in a subpopulation of RNA substrate molecules. (A–C) DMS methylation and RNase cleavage sites on the three stem-loop structures (SL1, 2 and 3), which were seen in simulations using the mfold server ( Zuker, 2003 ). (D) Base-pairing of the nucleotides in the loop structure of SL2 to the sequences (367–383) 3′ to the SL2 structure giving rise to (E) pseudoknot tertiary structure. (F,G) DMS methylation, RNase cleavage and premature RT stop sites on the stem structure of SL2 from FuD34 and F34su1 mutant psbC 5′ UTR are shown. Note that DMS methylation could not be determined for the psbC-FuD34 mutant 5′ UTR and the absence of asterisks is due to this, not inaccessibility. (F,G) Mutant residues are shown in black boxes with white text.
Figure Legend Snippet: In vitro analysis of stem-loop (SL) structures in the psbC 5′ UTR. Asterisks ( ∗ ) indicate A and C residues that are methylated by DMS in vitro . Arrows indicate G residues that are substrates for RNase T1 and arrowheads indicated cleavage sites for RNase V1. Most frequently methylated or cleaved positions are indicated by black asterisks, arrows and arrowheads. Less frequently methylated or cleaved sites are indicated in gray. Hexagons indicate locations of strong premature RT stops. Black dots indicate base-pairs that are strongly supported by the data, while gray dots are partially supported and could be present in a subpopulation of RNA substrate molecules. (A–C) DMS methylation and RNase cleavage sites on the three stem-loop structures (SL1, 2 and 3), which were seen in simulations using the mfold server ( Zuker, 2003 ). (D) Base-pairing of the nucleotides in the loop structure of SL2 to the sequences (367–383) 3′ to the SL2 structure giving rise to (E) pseudoknot tertiary structure. (F,G) DMS methylation, RNase cleavage and premature RT stop sites on the stem structure of SL2 from FuD34 and F34su1 mutant psbC 5′ UTR are shown. Note that DMS methylation could not be determined for the psbC-FuD34 mutant 5′ UTR and the absence of asterisks is due to this, not inaccessibility. (F,G) Mutant residues are shown in black boxes with white text.

Techniques Used: In Vitro, Methylation, Mutagenesis

RNA structure analyses of the psbC 5′ UTR by enzymatic probing. In vitro transcribed RNA corresponding to the wild-type and mutant psbC 5′ UTR were untreated (0) or digested with RNase T1 (T1) or RNase V1 (V1), which cleave unpaired G residues or RNA helices, respectively. Digestion products were resolved by denaturing PAGE and revealed by autoradiography. The positions of cleavage sites are indicated with respect to their positions on the 5′ UTR, based on the motilities of molecular size markers (not shown). (A) The substrate RNA was 3′- 32 P-labeled wild-type psbC 5′ UTR. (B,C) The substrates were 5′- 32 P-end-labeled RNAs corresponding to the psb C 5′ UTR with wild-type sequence (wt), or carrying one of the mutations; psbC-FuD34 (m), or psbC-F34suI (s). The exact position of certain digestion products could not be determined with certainty ( ∗ ).
Figure Legend Snippet: RNA structure analyses of the psbC 5′ UTR by enzymatic probing. In vitro transcribed RNA corresponding to the wild-type and mutant psbC 5′ UTR were untreated (0) or digested with RNase T1 (T1) or RNase V1 (V1), which cleave unpaired G residues or RNA helices, respectively. Digestion products were resolved by denaturing PAGE and revealed by autoradiography. The positions of cleavage sites are indicated with respect to their positions on the 5′ UTR, based on the motilities of molecular size markers (not shown). (A) The substrate RNA was 3′- 32 P-labeled wild-type psbC 5′ UTR. (B,C) The substrates were 5′- 32 P-end-labeled RNAs corresponding to the psb C 5′ UTR with wild-type sequence (wt), or carrying one of the mutations; psbC-FuD34 (m), or psbC-F34suI (s). The exact position of certain digestion products could not be determined with certainty ( ∗ ).

Techniques Used: In Vitro, Mutagenesis, Polyacrylamide Gel Electrophoresis, Autoradiography, Labeling, Sequencing

13) Product Images from "Solution Structure of an Archaeal RNase P Binary Protein Complex. Formation of the 30-kDa Complex Between Pyrococcus furiosus RPP21 and RPP29 is Accompanied by Coupled Protein Folding, and Highlights Critical Features for Protein-Protein and Protein-RNA Interactions"

Article Title: Solution Structure of an Archaeal RNase P Binary Protein Complex. Formation of the 30-kDa Complex Between Pyrococcus furiosus RPP21 and RPP29 is Accompanied by Coupled Protein Folding, and Highlights Critical Features for Protein-Protein and Protein-RNA Interactions

Journal:

doi: 10.1016/j.jmb.2009.08.068

Footprinting using RNase V1 and RNase T1 to identify RPP-binding sites in Mja RPR. Mja RPR labeled at the 5'-end (a) or 3'-end (b) was incubated either without (lanes 1, 3, 5, 7 and 9) or with (lanes 2, 4, 6, 8 and 10) RNase V1 (panel a) or RNase T1 (panel
Figure Legend Snippet: Footprinting using RNase V1 and RNase T1 to identify RPP-binding sites in Mja RPR. Mja RPR labeled at the 5'-end (a) or 3'-end (b) was incubated either without (lanes 1, 3, 5, 7 and 9) or with (lanes 2, 4, 6, 8 and 10) RNase V1 (panel a) or RNase T1 (panel

Techniques Used: Footprinting, Binding Assay, Labeling, Incubation

14) Product Images from "The 5e motif of eukaryotic signal recognition particle RNA contains a conserved adenosine for the binding of SRP72"

Article Title: The 5e motif of eukaryotic signal recognition particle RNA contains a conserved adenosine for the binding of SRP72

Journal: RNA

doi: 10.1261/rna.979508

Enzymatic probing of 5ef RNA bound to the 72f polypeptide. Examples of sequencing gels displaying partial digests of 32 P-labeled 5ef RNA-72f polypeptide complexes with increasing concentrations of RNases A, T1, and V1 as indicated by the wedges. The peptide:RNA ratio was 2:1. Ten-microliter reaction mixtures contained 0.01, 0.02, 0.04 and 0.05 units of RNase T1, 0.05, 0.1, 0.15, and 0.2 units of RNase V1, or 0.00001, 0.0001, 0.0005, and 0.001 units of RNase A.
Figure Legend Snippet: Enzymatic probing of 5ef RNA bound to the 72f polypeptide. Examples of sequencing gels displaying partial digests of 32 P-labeled 5ef RNA-72f polypeptide complexes with increasing concentrations of RNases A, T1, and V1 as indicated by the wedges. The peptide:RNA ratio was 2:1. Ten-microliter reaction mixtures contained 0.01, 0.02, 0.04 and 0.05 units of RNase T1, 0.05, 0.1, 0.15, and 0.2 units of RNase V1, or 0.00001, 0.0001, 0.0005, and 0.001 units of RNase A.

Techniques Used: Sequencing, Labeling

15) Product Images from "Recognition and discrimination of target mRNAs by Sib RNAs, a cis-encoded sRNA family"

Article Title: Recognition and discrimination of target mRNAs by Sib RNAs, a cis-encoded sRNA family

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq292

Interaction of ibsC mRNA with SibC. ( A ) A reciprocal enzymatic and chemical footprint experiment was performed by labeling the 5′ end of ibsC mRNA, as described in Figure 6 . The RNase V1 ( B ), RNase T1 ( C ), lead(II) ( D ) and RNase III ( E ) protected or cleaved residues are shown in the secondary structure model of ibsC . The S/D sequence, start codon and stop codon are indicated by boxes. Increasing and decreasing cleavages are indicated by solid and open symbols, respectively.
Figure Legend Snippet: Interaction of ibsC mRNA with SibC. ( A ) A reciprocal enzymatic and chemical footprint experiment was performed by labeling the 5′ end of ibsC mRNA, as described in Figure 6 . The RNase V1 ( B ), RNase T1 ( C ), lead(II) ( D ) and RNase III ( E ) protected or cleaved residues are shown in the secondary structure model of ibsC . The S/D sequence, start codon and stop codon are indicated by boxes. Increasing and decreasing cleavages are indicated by solid and open symbols, respectively.

Techniques Used: Labeling, Sequencing

Time course of the interactions between SibC and ibsC RNA. ( A ) 32 P-labeled ibsC RNA (10 nM) was incubated with 50 nM of unlabeled SibC RNA at 37°C for 1, 30, 90 and 360 s. Samples were withdrawn from each time point and digested with RNase V1 for 15 min. The reaction was stopped by ethanol precipitation. Lanes C and OH correspond to untreated ibsC RNA and alkaline hydrolysis ladders, respectively. Lane T1 corresponds to G ladders generated by RNase T1 under denaturing conditions. The regions showing significant changes in the intensities of bands are denoted as a to e . ( B ) The regions showing significant changes during the time course of incubation are also shown in the secondary structure model of ibsC mRNA.
Figure Legend Snippet: Time course of the interactions between SibC and ibsC RNA. ( A ) 32 P-labeled ibsC RNA (10 nM) was incubated with 50 nM of unlabeled SibC RNA at 37°C for 1, 30, 90 and 360 s. Samples were withdrawn from each time point and digested with RNase V1 for 15 min. The reaction was stopped by ethanol precipitation. Lanes C and OH correspond to untreated ibsC RNA and alkaline hydrolysis ladders, respectively. Lane T1 corresponds to G ladders generated by RNase T1 under denaturing conditions. The regions showing significant changes in the intensities of bands are denoted as a to e . ( B ) The regions showing significant changes during the time course of incubation are also shown in the secondary structure model of ibsC mRNA.

Techniques Used: Labeling, Incubation, Ethanol Precipitation, Generated

Interaction of SibC with ibsC mRNA. ( A ) 32 P-labeled SibC RNA (10 nM) was partially digested with RNase V1, RNase T1, lead(II) and RNase III in the absence (lanes 1, 5, 9 and 13) and presence of increasing amounts of unlabeled ibsC mRNA (25 nM final concentration: lanes 2, 6, 10 and 14; 100 nM final concentration: lanes 3, 7, 11 and 15). The fully paired SibC– ibsC mRNA complex was generated by heating and annealing with 100 nM ibsC . The complex was then used for digestion (lanes 4, 8, 12 and 16). Untreated SibC and alkaline ladders are shown in lanes C and OH, respectively. Lane T1 corresponds to the RNase T1 ladders of denatured SibC RNA. The positions of the cleaved G residues are shown on the left side of the gel. The altered sites of RNase V1 ( B ), RNase T1 ( C ), lead(II) ( D ) and RNase III ( E ) cleavage/protection in the presence of ibsC mRNA are shown in the secondary structure of SibC. The complementary sequences corresponding to the S/D region, start codon and stop codon of ibsC mRNA are indicated by boxes. Increasing and decreasing cleavages are indicated by solid and open symbols, respectively.
Figure Legend Snippet: Interaction of SibC with ibsC mRNA. ( A ) 32 P-labeled SibC RNA (10 nM) was partially digested with RNase V1, RNase T1, lead(II) and RNase III in the absence (lanes 1, 5, 9 and 13) and presence of increasing amounts of unlabeled ibsC mRNA (25 nM final concentration: lanes 2, 6, 10 and 14; 100 nM final concentration: lanes 3, 7, 11 and 15). The fully paired SibC– ibsC mRNA complex was generated by heating and annealing with 100 nM ibsC . The complex was then used for digestion (lanes 4, 8, 12 and 16). Untreated SibC and alkaline ladders are shown in lanes C and OH, respectively. Lane T1 corresponds to the RNase T1 ladders of denatured SibC RNA. The positions of the cleaved G residues are shown on the left side of the gel. The altered sites of RNase V1 ( B ), RNase T1 ( C ), lead(II) ( D ) and RNase III ( E ) cleavage/protection in the presence of ibsC mRNA are shown in the secondary structure of SibC. The complementary sequences corresponding to the S/D region, start codon and stop codon of ibsC mRNA are indicated by boxes. Increasing and decreasing cleavages are indicated by solid and open symbols, respectively.

Techniques Used: Labeling, Concentration Assay, Generated

16) Product Images from "sRNA-mediated activation of gene expression by inhibition of 5'-3’ exonucleolytic mRNA degradation"

Article Title: sRNA-mediated activation of gene expression by inhibition of 5'-3’ exonucleolytic mRNA degradation

Journal: eLife

doi: 10.7554/eLife.23602

Enzymatic probing of the yflS mRNA secondary structure in the presence and absence of RoxS. The 5′-end-labeled yflS mRNA fragment (nts + 1 to+94) was digested with RNase T1, RNase T2 and RNase V1 and run on a 10% polyacrylamide–urea gel. The enzymatic digestions were performed in the absence (−) or in the presence of increasing concentrations of RoxS (100 or 250 nM). Incubation controls (in the absence of RNase) were done in the absence (−) and in the presence (+) of RoxS (250 nM). Arrows indicate the nucleotides protected by RoxS from the attack by RNase T1 or T2. The Shine–Dalgarno sequence (SD) and the AUG codon (Met), are indicated to the right of the gel. The probing experiment was performed two times (technical replicates). DOI: http://dx.doi.org/10.7554/eLife.23602.014
Figure Legend Snippet: Enzymatic probing of the yflS mRNA secondary structure in the presence and absence of RoxS. The 5′-end-labeled yflS mRNA fragment (nts + 1 to+94) was digested with RNase T1, RNase T2 and RNase V1 and run on a 10% polyacrylamide–urea gel. The enzymatic digestions were performed in the absence (−) or in the presence of increasing concentrations of RoxS (100 or 250 nM). Incubation controls (in the absence of RNase) were done in the absence (−) and in the presence (+) of RoxS (250 nM). Arrows indicate the nucleotides protected by RoxS from the attack by RNase T1 or T2. The Shine–Dalgarno sequence (SD) and the AUG codon (Met), are indicated to the right of the gel. The probing experiment was performed two times (technical replicates). DOI: http://dx.doi.org/10.7554/eLife.23602.014

Techniques Used: Labeling, Incubation, Sequencing

RoxS uses C-rich region 3 (CRR3) to interact with the extreme 5’-end of yflS mRNA. ( A ) Enzymatic probing of the RoxS sRNA secondary structure in the presence and absence of yflS . The 5’ end-labeled RoxS sRNA was digested with RNase V1 and RNase T2 and run on an 8% polyacrylamide–urea gel. The enzymatic digestions were performed in the absence (−) or in the presence of increasing concentrations of yflS mRNA (50, 100 and 400 nM) for digestion by RNase T2, and 50 and 100 nM for the experiment with RNase V1. Incubation controls (in the absence of RNase) were done in the absence (−) and in the presence (+) of yflS (400 nM). The positions of CRR1, 2 and 3 are indicated to the right of the gel. The probing experiment was performed twice (technical replicates). ( B ) Secondary structure of the RoxS sRNA and effect of yflS binding. Summary of the cleavage patterns generated by RNase V1 (red arrows) and RNase T2 (green arrows). Increased cleavages upon addition of yflS are indicated by asterisks, increased protection by circles. DOI: http://dx.doi.org/10.7554/eLife.23602.012
Figure Legend Snippet: RoxS uses C-rich region 3 (CRR3) to interact with the extreme 5’-end of yflS mRNA. ( A ) Enzymatic probing of the RoxS sRNA secondary structure in the presence and absence of yflS . The 5’ end-labeled RoxS sRNA was digested with RNase V1 and RNase T2 and run on an 8% polyacrylamide–urea gel. The enzymatic digestions were performed in the absence (−) or in the presence of increasing concentrations of yflS mRNA (50, 100 and 400 nM) for digestion by RNase T2, and 50 and 100 nM for the experiment with RNase V1. Incubation controls (in the absence of RNase) were done in the absence (−) and in the presence (+) of yflS (400 nM). The positions of CRR1, 2 and 3 are indicated to the right of the gel. The probing experiment was performed twice (technical replicates). ( B ) Secondary structure of the RoxS sRNA and effect of yflS binding. Summary of the cleavage patterns generated by RNase V1 (red arrows) and RNase T2 (green arrows). Increased cleavages upon addition of yflS are indicated by asterisks, increased protection by circles. DOI: http://dx.doi.org/10.7554/eLife.23602.012

Techniques Used: Labeling, Incubation, Binding Assay, Generated

17) Product Images from "RNA secondary structure profiling in zebrafish reveals unique regulatory features"

Article Title: RNA secondary structure profiling in zebrafish reveals unique regulatory features

Journal: BMC Genomics

doi: 10.1186/s12864-018-4497-0

Schematic of RNA structure probing by PARS in zebrafish. Poly-A RNA from zebrafish is folded in-vitro. The folded RNA is cleaved by RNase V1 and S1 nuclease separately. The enzyme cut sites generate 5’P ends and 3’ OH ends at the cleaved sites. Long fragments generated by single-hit kinetics are further fragmented by alkaline hydrolysis, which blocks the 3′ site of the enzyme-cut fragments. Sequencing adapters are ligated to the 5′ end followed by alkaline phosphatase treatment to 3’ P group. Adapters are ligated to 3’ends followed cDNA synthesis and PCR purification of the library. Appropriate size of the library is maintained by purification by nucleic acid beads. Sequenced reads are aligned back to the genome and only unique reads with the correct read start positions are considered for PARS score calculation
Figure Legend Snippet: Schematic of RNA structure probing by PARS in zebrafish. Poly-A RNA from zebrafish is folded in-vitro. The folded RNA is cleaved by RNase V1 and S1 nuclease separately. The enzyme cut sites generate 5’P ends and 3’ OH ends at the cleaved sites. Long fragments generated by single-hit kinetics are further fragmented by alkaline hydrolysis, which blocks the 3′ site of the enzyme-cut fragments. Sequencing adapters are ligated to the 5′ end followed by alkaline phosphatase treatment to 3’ P group. Adapters are ligated to 3’ends followed cDNA synthesis and PCR purification of the library. Appropriate size of the library is maintained by purification by nucleic acid beads. Sequenced reads are aligned back to the genome and only unique reads with the correct read start positions are considered for PARS score calculation

Techniques Used: In Vitro, Generated, Sequencing, Polymerase Chain Reaction, Purification

Comparison of RNA structures of ubc 3’UTR as determined by PARS based pairing probability and enzymatic footprinting using RNase V1 and S1 Nuclease. a . Bar plot represents PARS scores of 3’UTR region of ubiquitin c (ubc) . Out of 105 positions, 87 positions are captured by PARS. b . Enzymatic footprinting of ubc 3’UTR probed by S1 Nuclease and RNase V1. Nucleotide positions are correlated with alkaline hydrolysis (AH) ladder and RNase T1 (G) ladder. Positions with similar structural pattern with PARS scores are highlighted. Red dots indicate unpaired positions; green indicates paired positions while yellow represents ambiguous regions. c . Heatmap representing secondary structure of 68 positions of ubc 3’UTR as determined by PARS and enzymatic footprinting (FP). Top panel represents PARS pairing probability; bottom panel indicates enzymatic footprinting pairing probability; middle panel represents the consensus between the two (PARS: FP). Red represents unpaired, green represents paired and yellow represents ambiguous regions
Figure Legend Snippet: Comparison of RNA structures of ubc 3’UTR as determined by PARS based pairing probability and enzymatic footprinting using RNase V1 and S1 Nuclease. a . Bar plot represents PARS scores of 3’UTR region of ubiquitin c (ubc) . Out of 105 positions, 87 positions are captured by PARS. b . Enzymatic footprinting of ubc 3’UTR probed by S1 Nuclease and RNase V1. Nucleotide positions are correlated with alkaline hydrolysis (AH) ladder and RNase T1 (G) ladder. Positions with similar structural pattern with PARS scores are highlighted. Red dots indicate unpaired positions; green indicates paired positions while yellow represents ambiguous regions. c . Heatmap representing secondary structure of 68 positions of ubc 3’UTR as determined by PARS and enzymatic footprinting (FP). Top panel represents PARS pairing probability; bottom panel indicates enzymatic footprinting pairing probability; middle panel represents the consensus between the two (PARS: FP). Red represents unpaired, green represents paired and yellow represents ambiguous regions

Techniques Used: Footprinting

18) Product Images from "Tetrahymena Telomerase Protein p65 Induces Conformational Changes throughout Telomerase RNA (TER) and Rescues Telomerase Reverse Transcriptase and TER Assembly Mutants ▿ Telomerase Protein p65 Induces Conformational Changes throughout Telomerase RNA (TER) and Rescues Telomerase Reverse Transcriptase and TER Assembly Mutants ▿ †"

Article Title: Tetrahymena Telomerase Protein p65 Induces Conformational Changes throughout Telomerase RNA (TER) and Rescues Telomerase Reverse Transcriptase and TER Assembly Mutants ▿ Telomerase Protein p65 Induces Conformational Changes throughout Telomerase RNA (TER) and Rescues Telomerase Reverse Transcriptase and TER Assembly Mutants ▿ †

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00827-10

Nuclease protection assays of TER in binary and ternary complexes reveal p65 and TERT* footprints and the induction of more single strandedness between stems II and III. (A) RNase V1 data. (B) RNase ONE data. (C) RNase T1 data. Regions of sequencing
Figure Legend Snippet: Nuclease protection assays of TER in binary and ternary complexes reveal p65 and TERT* footprints and the induction of more single strandedness between stems II and III. (A) RNase V1 data. (B) RNase ONE data. (C) RNase T1 data. Regions of sequencing

Techniques Used: Sequencing

19) Product Images from "Genome-wide identification of natural RNA aptamers in prokaryotes and eukaryotes"

Article Title: Genome-wide identification of natural RNA aptamers in prokaryotes and eukaryotes

Journal: Nature Communications

doi: 10.1038/s41467-018-03675-1

PARCEL identifies new RNA aptamers in Candida albicans . a Pie chart of the number of C . albicans RNA aptamers that are located in 5′ UTR, CDS, and 3′ UTR. The majority of C . albicans RNA aptamers are found in CDSs. b Comparison of the distribution of Alifoldz scores for RNA aptamers vs. shuffled counterpart. The upper, middle, and lower bounds of the boxplot represent the 75, 50, and 25th percentile of the values, respectively. A negative score indicates a stable, conserved consensus structure. P -value was obtained using the non-parametric Kolmogorov–Smirnov test. c Nucleotide substitution rates, calculated as the number of substitutions per base-pair, for RNA aptamers (Kr), 3′ UTR (K 3UTR ), 5′ UTR(K 5UTR ), synonymous sites (Ks), and non-synonymous sites (Ka). The upper, middle, and lower bounds of the boxplot represent the 75, 50, and 25th percentile of the values, respectively. C . albicans SC5314 was compared to Candida dubliniensis for the calculation. p -value was obtained using the non-parametric Kolmogorov–Smirnov test. d Gel analysis of RPS31 mRNA using in-line probing (left) and RNase V1 (right) in the presence (lane 3) and absence (lane 2) of 100 µM FMN. The A ladder (A, lane 1) is also shown. The black arrows indicate positions along the RNA that changed in the presence of FMN. e A representative Western blot showing RPS31::FLAG (top) and loading (bottom) protein levels in RPS31::FLAG knock-in strains with WT (left) and fmn1∆ (right) backgrounds cultured at different FMN concentrations (mM). Using t -test ( n = 8), significant p -values of 0.009 and 0.01 (for 2.5 and 5.0 mM against 10.0 mM, respectively) were determined for fmn1∆ , but not WT ( p -values of 0.2 and 0.5). f Gel analysis of RPS31 mRNA using in-line probing in the presence of 20, 100, or 500 µM of FMN, FAD or riboflavin. In-line probing of RNA in the absence of metabolite (H 2 O, lane 2) and A ladder (A, lane 1) are also shown. g SAFA analysis of WT RPS31 (top) and codon-optimized RPS31 (bottom) in the presence (red line) and absence (black line) of 100 µM FMN. The beige box indicates the region of structural change in WT RPS31 when it interacts with FMN. This structural change is absent in the codon-optimized RPS31. h A representative Western blot showing codon-optimized RPS31::FLAG (top) and loading (bottom) protein levels in codon-optimized RPS31::FLAG knock-in strains with WT (left) and fmn1∆ (right) backgrounds cultured at different FMN concentrations (mM). Using t -test ( n = 3), calculated p -values for 2.5 and 5.0 mM were insignificant for both fmn1∆ (both 0.7) and WT (0.9 and 0.09)
Figure Legend Snippet: PARCEL identifies new RNA aptamers in Candida albicans . a Pie chart of the number of C . albicans RNA aptamers that are located in 5′ UTR, CDS, and 3′ UTR. The majority of C . albicans RNA aptamers are found in CDSs. b Comparison of the distribution of Alifoldz scores for RNA aptamers vs. shuffled counterpart. The upper, middle, and lower bounds of the boxplot represent the 75, 50, and 25th percentile of the values, respectively. A negative score indicates a stable, conserved consensus structure. P -value was obtained using the non-parametric Kolmogorov–Smirnov test. c Nucleotide substitution rates, calculated as the number of substitutions per base-pair, for RNA aptamers (Kr), 3′ UTR (K 3UTR ), 5′ UTR(K 5UTR ), synonymous sites (Ks), and non-synonymous sites (Ka). The upper, middle, and lower bounds of the boxplot represent the 75, 50, and 25th percentile of the values, respectively. C . albicans SC5314 was compared to Candida dubliniensis for the calculation. p -value was obtained using the non-parametric Kolmogorov–Smirnov test. d Gel analysis of RPS31 mRNA using in-line probing (left) and RNase V1 (right) in the presence (lane 3) and absence (lane 2) of 100 µM FMN. The A ladder (A, lane 1) is also shown. The black arrows indicate positions along the RNA that changed in the presence of FMN. e A representative Western blot showing RPS31::FLAG (top) and loading (bottom) protein levels in RPS31::FLAG knock-in strains with WT (left) and fmn1∆ (right) backgrounds cultured at different FMN concentrations (mM). Using t -test ( n = 8), significant p -values of 0.009 and 0.01 (for 2.5 and 5.0 mM against 10.0 mM, respectively) were determined for fmn1∆ , but not WT ( p -values of 0.2 and 0.5). f Gel analysis of RPS31 mRNA using in-line probing in the presence of 20, 100, or 500 µM of FMN, FAD or riboflavin. In-line probing of RNA in the absence of metabolite (H 2 O, lane 2) and A ladder (A, lane 1) are also shown. g SAFA analysis of WT RPS31 (top) and codon-optimized RPS31 (bottom) in the presence (red line) and absence (black line) of 100 µM FMN. The beige box indicates the region of structural change in WT RPS31 when it interacts with FMN. This structural change is absent in the codon-optimized RPS31. h A representative Western blot showing codon-optimized RPS31::FLAG (top) and loading (bottom) protein levels in codon-optimized RPS31::FLAG knock-in strains with WT (left) and fmn1∆ (right) backgrounds cultured at different FMN concentrations (mM). Using t -test ( n = 3), calculated p -values for 2.5 and 5.0 mM were insignificant for both fmn1∆ (both 0.7) and WT (0.9 and 0.09)

Techniques Used: Western Blot, Knock-In, Cell Culture

Measuring RNA-ligand binding by structure probing and deep sequencing. a RNA undergoes structure changes upon ligand binding. This structural change is detected by the double-strand specific nuclease, RNase V1, which cuts at different double-stranded places along the RNA in the presence and absence of the ligand. The cleavage sites are then captured and cloned into a cDNA library for deep sequencing. After mapping the reads to the transcriptome, we can identify which bases have undergone changes in structuredness upon ligand binding (highlighted in beige boxes). b Deep sequencing reveals structure changes of a known TPP riboswitch, thiM, using RNase V1 (top), S1 nuclease (middle), and in-line probing (bottom). The red and black lines indicate the structure profiles of thiM treated with and without 100 µM TPP, respectively. The beige regions highlight regions of structural changes upon ligand binding. c PARCEL identified 85% of known TPP, FMN, and SAM riboswitches in B . subtilis and P . aeruginosa . The black and the white bars indicate the number of known riboswitches that were captured and missed in our study, respectively. d PARCEL sequencing data for the B . subtilis . e The plots show normalized V1 read counts of the thiC TPP riboswitch under increasing concentrations of TPP. PARCEL was performed on the B . subtilis transcriptome
Figure Legend Snippet: Measuring RNA-ligand binding by structure probing and deep sequencing. a RNA undergoes structure changes upon ligand binding. This structural change is detected by the double-strand specific nuclease, RNase V1, which cuts at different double-stranded places along the RNA in the presence and absence of the ligand. The cleavage sites are then captured and cloned into a cDNA library for deep sequencing. After mapping the reads to the transcriptome, we can identify which bases have undergone changes in structuredness upon ligand binding (highlighted in beige boxes). b Deep sequencing reveals structure changes of a known TPP riboswitch, thiM, using RNase V1 (top), S1 nuclease (middle), and in-line probing (bottom). The red and black lines indicate the structure profiles of thiM treated with and without 100 µM TPP, respectively. The beige regions highlight regions of structural changes upon ligand binding. c PARCEL identified 85% of known TPP, FMN, and SAM riboswitches in B . subtilis and P . aeruginosa . The black and the white bars indicate the number of known riboswitches that were captured and missed in our study, respectively. d PARCEL sequencing data for the B . subtilis . e The plots show normalized V1 read counts of the thiC TPP riboswitch under increasing concentrations of TPP. PARCEL was performed on the B . subtilis transcriptome

Techniques Used: Ligand Binding Assay, Sequencing, Clone Assay, cDNA Library Assay

20) Product Images from "A cis-replication element functions in both orientations to enhance replication of Turnip crinkle virus"

Article Title: A cis-replication element functions in both orientations to enhance replication of Turnip crinkle virus

Journal:

doi: 10.1016/j.virol.2006.03.051

Chemical and enzymatic probing of H4(+) and H4(−). TCV plus-strand transcripts (A) or minus-strand transcripts (B) were treated with DMS for 10 or 20 minutes or with RNase T1, RNase A and RNase V1 for 5 or 10 minutes. The modified or cleaved RNAs
Figure Legend Snippet: Chemical and enzymatic probing of H4(+) and H4(−). TCV plus-strand transcripts (A) or minus-strand transcripts (B) were treated with DMS for 10 or 20 minutes or with RNase T1, RNase A and RNase V1 for 5 or 10 minutes. The modified or cleaved RNAs

Techniques Used: Modification

21) Product Images from "Characterization of the frameshift stimulatory signal controlling a programmed -1 ribosomal frameshift in the human immunodeficiency virus type 1"

Article Title: Characterization of the frameshift stimulatory signal controlling a programmed -1 ribosomal frameshift in the human immunodeficiency virus type 1

Journal: Nucleic Acids Research

doi:

Novel structure proposed for the frameshift stimulatory signal of HIV-1. ( A ) Structure probing of the frameshift stimulatory signal by RNase V1 attack. An RNA transcript encompassing the HIV-1 gag/pol frameshift region was 5′ end-labeled with [γ- 32 P] and digested with RNase V1. Digestion products were analyzed on a 20% acrylamide–7 M urea gel. The sites of cleavage were identified by comparison with a ladder of bands created by limited alkaline hydrolysis of the RNA (OH – ) and by the position of RNase T1 cuts (not shown). Uniquely cleaved nucleotides were identified by their absence in the untreated control lane (0). The amount of units of enzyme added to each reaction is also indicated. ( B ) Description of the novel two-stem model for the frameshift stimulatory signal as suggested by structure probing. The upper stem corresponds to the classic stem–loop and the lower stem is formed by pairing the spacer to a segment downstream of this stem–loop. The sensitivity of nucleotides in the HIV-1 frameshift region to RNaseV1 is shown. The size of the arrows is approximately proportional to the intensity of the cleavage at that site. Bases in bold originate from HIV-1.
Figure Legend Snippet: Novel structure proposed for the frameshift stimulatory signal of HIV-1. ( A ) Structure probing of the frameshift stimulatory signal by RNase V1 attack. An RNA transcript encompassing the HIV-1 gag/pol frameshift region was 5′ end-labeled with [γ- 32 P] and digested with RNase V1. Digestion products were analyzed on a 20% acrylamide–7 M urea gel. The sites of cleavage were identified by comparison with a ladder of bands created by limited alkaline hydrolysis of the RNA (OH – ) and by the position of RNase T1 cuts (not shown). Uniquely cleaved nucleotides were identified by their absence in the untreated control lane (0). The amount of units of enzyme added to each reaction is also indicated. ( B ) Description of the novel two-stem model for the frameshift stimulatory signal as suggested by structure probing. The upper stem corresponds to the classic stem–loop and the lower stem is formed by pairing the spacer to a segment downstream of this stem–loop. The sensitivity of nucleotides in the HIV-1 frameshift region to RNaseV1 is shown. The size of the arrows is approximately proportional to the intensity of the cleavage at that site. Bases in bold originate from HIV-1.

Techniques Used: Labeling

22) Product Images from "Genome-wide identification of natural RNA aptamers in prokaryotes and eukaryotes"

Article Title: Genome-wide identification of natural RNA aptamers in prokaryotes and eukaryotes

Journal: Nature Communications

doi: 10.1038/s41467-018-03675-1

Measuring RNA-ligand binding by structure probing and deep sequencing. a RNA undergoes structure changes upon ligand binding. This structural change is detected by the double-strand specific nuclease, RNase V1, which cuts at different double-stranded places along the RNA in the presence and absence of the ligand. The cleavage sites are then captured and cloned into a cDNA library for deep sequencing. After mapping the reads to the transcriptome, we can identify which bases have undergone changes in structuredness upon ligand binding (highlighted in beige boxes). b Deep sequencing reveals structure changes of a known TPP riboswitch, thiM, using RNase V1 (top), S1 nuclease (middle), and in-line probing (bottom). The red and black lines indicate the structure profiles of thiM treated with and without 100 µM TPP, respectively. The beige regions highlight regions of structural changes upon ligand binding. c PARCEL identified 85% of known TPP, FMN, and SAM riboswitches in B . subtilis and P . aeruginosa . The black and the white bars indicate the number of known riboswitches that were captured and missed in our study, respectively. d PARCEL sequencing data for the B . subtilis TPP riboswitch, thiT, in the presence and absence of 100 µM TPP (top), 100 µM thiamine (middle), and 100 µM oxythiamine (bottom). PARCEL detected strongest structural change in thiT in the presence of TPP, followed by thiamine and then oxythiamine, which corresponds to the binding affinities of TPP riboswitches for these metabolites 9 . e The plots show normalized V1 read counts of the thiC TPP riboswitch under increasing concentrations of TPP. PARCEL was performed on the B . subtilis transcriptome
Figure Legend Snippet: Measuring RNA-ligand binding by structure probing and deep sequencing. a RNA undergoes structure changes upon ligand binding. This structural change is detected by the double-strand specific nuclease, RNase V1, which cuts at different double-stranded places along the RNA in the presence and absence of the ligand. The cleavage sites are then captured and cloned into a cDNA library for deep sequencing. After mapping the reads to the transcriptome, we can identify which bases have undergone changes in structuredness upon ligand binding (highlighted in beige boxes). b Deep sequencing reveals structure changes of a known TPP riboswitch, thiM, using RNase V1 (top), S1 nuclease (middle), and in-line probing (bottom). The red and black lines indicate the structure profiles of thiM treated with and without 100 µM TPP, respectively. The beige regions highlight regions of structural changes upon ligand binding. c PARCEL identified 85% of known TPP, FMN, and SAM riboswitches in B . subtilis and P . aeruginosa . The black and the white bars indicate the number of known riboswitches that were captured and missed in our study, respectively. d PARCEL sequencing data for the B . subtilis TPP riboswitch, thiT, in the presence and absence of 100 µM TPP (top), 100 µM thiamine (middle), and 100 µM oxythiamine (bottom). PARCEL detected strongest structural change in thiT in the presence of TPP, followed by thiamine and then oxythiamine, which corresponds to the binding affinities of TPP riboswitches for these metabolites 9 . e The plots show normalized V1 read counts of the thiC TPP riboswitch under increasing concentrations of TPP. PARCEL was performed on the B . subtilis transcriptome

Techniques Used: Ligand Binding Assay, Sequencing, Clone Assay, cDNA Library Assay, Binding Assay

PARCEL identifies new RNA aptamers in Candida albicans . a Pie chart of the number of C . albicans RNA aptamers that are located in 5′ UTR, CDS, and 3′ UTR. The majority of C . albicans RNA aptamers are found in CDSs. b Comparison of the distribution of Alifoldz scores for RNA aptamers vs. shuffled counterpart. The upper, middle, and lower bounds of the boxplot represent the 75, 50, and 25th percentile of the values, respectively. A negative score indicates a stable, conserved consensus structure. P -value was obtained using the non-parametric Kolmogorov–Smirnov test. c Nucleotide substitution rates, calculated as the number of substitutions per base-pair, for RNA aptamers (Kr), 3′ UTR (K 3UTR ), 5′ UTR(K 5UTR ), synonymous sites (Ks), and non-synonymous sites (Ka). The upper, middle, and lower bounds of the boxplot represent the 75, 50, and 25th percentile of the values, respectively. C . albicans SC5314 was compared to Candida dubliniensis for the calculation. p -value was obtained using the non-parametric Kolmogorov–Smirnov test. d Gel analysis of RPS31 mRNA using in-line probing (left) and RNase V1 (right) in the presence (lane 3) and absence (lane 2) of 100 µM FMN. The A ladder (A, lane 1) is also shown. The black arrows indicate positions along the RNA that changed in the presence of FMN. e A representative Western blot showing RPS31::FLAG (top) and loading (bottom) protein levels in RPS31::FLAG knock-in strains with WT (left) and fmn1∆ (right) backgrounds cultured at different FMN concentrations (mM). Using t -test ( n = 8), significant p -values of 0.009 and 0.01 (for 2.5 and 5.0 mM against 10.0 mM, respectively) were determined for fmn1∆ , but not WT ( p -values of 0.2 and 0.5). f Gel analysis of RPS31 mRNA using in-line probing in the presence of 20, 100, or 500 µM of FMN, FAD or riboflavin. In-line probing of RNA in the absence of metabolite (H 2 O, lane 2) and A ladder (A, lane 1) are also shown. g SAFA analysis of WT RPS31 (top) and codon-optimized RPS31 (bottom) in the presence (red line) and absence (black line) of 100 µM FMN. The beige box indicates the region of structural change in WT RPS31 when it interacts with FMN. This structural change is absent in the codon-optimized RPS31. h A representative Western blot showing codon-optimized RPS31::FLAG (top) and loading (bottom) protein levels in codon-optimized RPS31::FLAG knock-in strains with WT (left) and fmn1∆ (right) backgrounds cultured at different FMN concentrations (mM). Using t -test ( n = 3), calculated p -values for 2.5 and 5.0 mM were insignificant for both fmn1∆ (both 0.7) and WT (0.9 and 0.09)
Figure Legend Snippet: PARCEL identifies new RNA aptamers in Candida albicans . a Pie chart of the number of C . albicans RNA aptamers that are located in 5′ UTR, CDS, and 3′ UTR. The majority of C . albicans RNA aptamers are found in CDSs. b Comparison of the distribution of Alifoldz scores for RNA aptamers vs. shuffled counterpart. The upper, middle, and lower bounds of the boxplot represent the 75, 50, and 25th percentile of the values, respectively. A negative score indicates a stable, conserved consensus structure. P -value was obtained using the non-parametric Kolmogorov–Smirnov test. c Nucleotide substitution rates, calculated as the number of substitutions per base-pair, for RNA aptamers (Kr), 3′ UTR (K 3UTR ), 5′ UTR(K 5UTR ), synonymous sites (Ks), and non-synonymous sites (Ka). The upper, middle, and lower bounds of the boxplot represent the 75, 50, and 25th percentile of the values, respectively. C . albicans SC5314 was compared to Candida dubliniensis for the calculation. p -value was obtained using the non-parametric Kolmogorov–Smirnov test. d Gel analysis of RPS31 mRNA using in-line probing (left) and RNase V1 (right) in the presence (lane 3) and absence (lane 2) of 100 µM FMN. The A ladder (A, lane 1) is also shown. The black arrows indicate positions along the RNA that changed in the presence of FMN. e A representative Western blot showing RPS31::FLAG (top) and loading (bottom) protein levels in RPS31::FLAG knock-in strains with WT (left) and fmn1∆ (right) backgrounds cultured at different FMN concentrations (mM). Using t -test ( n = 8), significant p -values of 0.009 and 0.01 (for 2.5 and 5.0 mM against 10.0 mM, respectively) were determined for fmn1∆ , but not WT ( p -values of 0.2 and 0.5). f Gel analysis of RPS31 mRNA using in-line probing in the presence of 20, 100, or 500 µM of FMN, FAD or riboflavin. In-line probing of RNA in the absence of metabolite (H 2 O, lane 2) and A ladder (A, lane 1) are also shown. g SAFA analysis of WT RPS31 (top) and codon-optimized RPS31 (bottom) in the presence (red line) and absence (black line) of 100 µM FMN. The beige box indicates the region of structural change in WT RPS31 when it interacts with FMN. This structural change is absent in the codon-optimized RPS31. h A representative Western blot showing codon-optimized RPS31::FLAG (top) and loading (bottom) protein levels in codon-optimized RPS31::FLAG knock-in strains with WT (left) and fmn1∆ (right) backgrounds cultured at different FMN concentrations (mM). Using t -test ( n = 3), calculated p -values for 2.5 and 5.0 mM were insignificant for both fmn1∆ (both 0.7) and WT (0.9 and 0.09)

Techniques Used: Western Blot, Knock-In, Cell Culture

23) Product Images from "A shared RNA-binding site in the Pet54 protein is required for translational activation and group I intron splicing in yeast mitochondria"

Article Title: A shared RNA-binding site in the Pet54 protein is required for translational activation and group I intron splicing in yeast mitochondria

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkn045

Visualization of the Pet54p-binding sites by RNase footprinting. 5′ end-labeled aI5β 3′TR RNA ( A ) or COX3 5′ UTL ( B ) were incubated with buffer (−), RNase V1 or 1 in the absence (first lane of each set) presence of saturating Pet54p (1.25 or 2.5 μM, second and third lanes of each set). Cleavage products were separated by denaturing gel electrophoresis and the data quantified as described in Materials and Methods section. Only those sites that showed 2-fold or higher reduction phosphorimager counts under both protein concentrations were identified as ‘protected’ (gray bars). Such sites were only observed in samples digested with RNase 1. The asterisk in (A) refers to bands of site-specific degradation caused by incubation with Pet54p. Below each gel image is a schematic of the relevant sequence protected from RNase1 digestion. Gray shading represents protection and the red shaded residues demarcate the 5′ binding boundary as defined in Figure 5 . In (A), a secondary structure of the first 21 nt defining the P1 helix is also shown. Note that residues g −3 to U3 are also cleaved by the double-stranded specific V1 RNase, but Pet54p binding does not protect these residues from V1 cleavage. ( C ) Alignment of the RNase 1 protected sequence. The sequence from positions A4 to A69 of aI5β intron and positions A −590 to U −512 of the COX3 5′ UTL were aligned using the EBOSS program ( 32 ). Gray-shaded residues are protected from RNase 1 digestion in the presence of Pet54p [see (A) and (B) above].
Figure Legend Snippet: Visualization of the Pet54p-binding sites by RNase footprinting. 5′ end-labeled aI5β 3′TR RNA ( A ) or COX3 5′ UTL ( B ) were incubated with buffer (−), RNase V1 or 1 in the absence (first lane of each set) presence of saturating Pet54p (1.25 or 2.5 μM, second and third lanes of each set). Cleavage products were separated by denaturing gel electrophoresis and the data quantified as described in Materials and Methods section. Only those sites that showed 2-fold or higher reduction phosphorimager counts under both protein concentrations were identified as ‘protected’ (gray bars). Such sites were only observed in samples digested with RNase 1. The asterisk in (A) refers to bands of site-specific degradation caused by incubation with Pet54p. Below each gel image is a schematic of the relevant sequence protected from RNase1 digestion. Gray shading represents protection and the red shaded residues demarcate the 5′ binding boundary as defined in Figure 5 . In (A), a secondary structure of the first 21 nt defining the P1 helix is also shown. Note that residues g −3 to U3 are also cleaved by the double-stranded specific V1 RNase, but Pet54p binding does not protect these residues from V1 cleavage. ( C ) Alignment of the RNase 1 protected sequence. The sequence from positions A4 to A69 of aI5β intron and positions A −590 to U −512 of the COX3 5′ UTL were aligned using the EBOSS program ( 32 ). Gray-shaded residues are protected from RNase 1 digestion in the presence of Pet54p [see (A) and (B) above].

Techniques Used: Binding Assay, Footprinting, Labeling, Incubation, Nucleic Acid Electrophoresis, Sequencing

24) Product Images from "The RNA strands of the plus and minus polarities of peach latent mosaic viroid fold into different structures"

Article Title: The RNA strands of the plus and minus polarities of peach latent mosaic viroid fold into different structures

Journal: RNA

doi: 10.1261/rna.1826710

Typical autoradiogram of an 8% PAGE gel performed for the enzymatic probing of a 5′-end labeled PLMVd transcripts of minus polarity. The lanes labeled L on both sides are identical ladders obtained by alkaline hydrolysis of PLMVd. Lanes 2 , 3 , and 4 contain PLMVd transcripts hydrolyzed by RNase T1, RNase TA, and RNase A, respectively, under denaturing conditions in order to provide guanosine, adenosine, and cytosine/uridine ladders. Lane 5 is an unhydrolyzed RNA sample. The remaining lanes contained samples obtained from hydrolyzes performed using two dilutions of RNase T1 (lanes 6 , 7 ), RNase TA (lanes 8 , 9 ), RNase A (lanes 10 , 11 ), and one dilution of RNase V1 (lane 12 ). The inset on the right is a schematic compilation of the RNase probing of the P10 stem–loop.
Figure Legend Snippet: Typical autoradiogram of an 8% PAGE gel performed for the enzymatic probing of a 5′-end labeled PLMVd transcripts of minus polarity. The lanes labeled L on both sides are identical ladders obtained by alkaline hydrolysis of PLMVd. Lanes 2 , 3 , and 4 contain PLMVd transcripts hydrolyzed by RNase T1, RNase TA, and RNase A, respectively, under denaturing conditions in order to provide guanosine, adenosine, and cytosine/uridine ladders. Lane 5 is an unhydrolyzed RNA sample. The remaining lanes contained samples obtained from hydrolyzes performed using two dilutions of RNase T1 (lanes 6 , 7 ), RNase TA (lanes 8 , 9 ), RNase A (lanes 10 , 11 ), and one dilution of RNase V1 (lane 12 ). The inset on the right is a schematic compilation of the RNase probing of the P10 stem–loop.

Techniques Used: Polyacrylamide Gel Electrophoresis, Labeling

25) Product Images from "LGP2 virus sensor regulates gene expression network mediated by TRBP-bound microRNAs"

Article Title: LGP2 virus sensor regulates gene expression network mediated by TRBP-bound microRNAs

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky575

LGP2 interacts with the dsRNA-binding site of TRBP. ( A ) Domain architecture of TRBP-WT and its mutant proteins. The lysines (K) at positions 80 and 81 in dsRBD1 were substituted with alanines (A) in the TRBP-dsRBDmt1 and TRBP-dsRBDmt1+2 mutants, and the lysines at positions 210 and 211 in dsRBD2 were substituted with alanines in the TRBP-dsRBDmt2 and TRBP-dsRBDmt1+2 mutants. The previously reported dsRNA binding affinities are summarized on the right. ( B ) Immunoprecipitation of LGP2 with TRBP-WT protein and its mutants. The anti-Myc antibody was used for immunoprecipitation of TRBP, and LGP2 was detected with anti-FLAG antibody. The bar graph shows the quantified signal intensities of the immunoprecipitates. ( C ) Immunoprecipitation of LGP2 with TRBP-WT protein and its mutants. RNase V1 was added to the immunoprecipitation buffer to remove dsRNA. The bar graph shows the quantified signal intensities of the immunoprecipitates.
Figure Legend Snippet: LGP2 interacts with the dsRNA-binding site of TRBP. ( A ) Domain architecture of TRBP-WT and its mutant proteins. The lysines (K) at positions 80 and 81 in dsRBD1 were substituted with alanines (A) in the TRBP-dsRBDmt1 and TRBP-dsRBDmt1+2 mutants, and the lysines at positions 210 and 211 in dsRBD2 were substituted with alanines in the TRBP-dsRBDmt2 and TRBP-dsRBDmt1+2 mutants. The previously reported dsRNA binding affinities are summarized on the right. ( B ) Immunoprecipitation of LGP2 with TRBP-WT protein and its mutants. The anti-Myc antibody was used for immunoprecipitation of TRBP, and LGP2 was detected with anti-FLAG antibody. The bar graph shows the quantified signal intensities of the immunoprecipitates. ( C ) Immunoprecipitation of LGP2 with TRBP-WT protein and its mutants. RNase V1 was added to the immunoprecipitation buffer to remove dsRNA. The bar graph shows the quantified signal intensities of the immunoprecipitates.

Techniques Used: Binding Assay, Mutagenesis, Immunoprecipitation

26) Product Images from "RNA secondary structure profiling in zebrafish reveals unique regulatory features"

Article Title: RNA secondary structure profiling in zebrafish reveals unique regulatory features

Journal: BMC Genomics

doi: 10.1186/s12864-018-4497-0

Schematic of RNA structure probing by PARS in zebrafish. Poly-A RNA from zebrafish is folded in-vitro. The folded RNA is cleaved by RNase V1 and S1 nuclease separately. The enzyme cut sites generate 5’P ends and 3’ OH ends at the cleaved sites. Long fragments generated by single-hit kinetics are further fragmented by alkaline hydrolysis, which blocks the 3′ site of the enzyme-cut fragments. Sequencing adapters are ligated to the 5′ end followed by alkaline phosphatase treatment to 3’ P group. Adapters are ligated to 3’ends followed cDNA synthesis and PCR purification of the library. Appropriate size of the library is maintained by purification by nucleic acid beads. Sequenced reads are aligned back to the genome and only unique reads with the correct read start positions are considered for PARS score calculation
Figure Legend Snippet: Schematic of RNA structure probing by PARS in zebrafish. Poly-A RNA from zebrafish is folded in-vitro. The folded RNA is cleaved by RNase V1 and S1 nuclease separately. The enzyme cut sites generate 5’P ends and 3’ OH ends at the cleaved sites. Long fragments generated by single-hit kinetics are further fragmented by alkaline hydrolysis, which blocks the 3′ site of the enzyme-cut fragments. Sequencing adapters are ligated to the 5′ end followed by alkaline phosphatase treatment to 3’ P group. Adapters are ligated to 3’ends followed cDNA synthesis and PCR purification of the library. Appropriate size of the library is maintained by purification by nucleic acid beads. Sequenced reads are aligned back to the genome and only unique reads with the correct read start positions are considered for PARS score calculation

Techniques Used: In Vitro, Generated, Sequencing, Polymerase Chain Reaction, Purification

Comparison of RNA structures of ubc 3’UTR as determined by PARS based pairing probability and enzymatic footprinting using RNase V1 and S1 Nuclease. a . Bar plot represents PARS scores of 3’UTR region of ubiquitin c (ubc) . Out of 105 positions, 87 positions are captured by PARS. b . Enzymatic footprinting of ubc 3’UTR probed by S1 Nuclease and RNase V1. Nucleotide positions are correlated with alkaline hydrolysis (AH) ladder and RNase T1 (G) ladder. Positions with similar structural pattern with PARS scores are highlighted. Red dots indicate unpaired positions; green indicates paired positions while yellow represents ambiguous regions. c . Heatmap representing secondary structure of 68 positions of ubc 3’UTR as determined by PARS and enzymatic footprinting (FP). Top panel represents PARS pairing probability; bottom panel indicates enzymatic footprinting pairing probability; middle panel represents the consensus between the two (PARS: FP). Red represents unpaired, green represents paired and yellow represents ambiguous regions
Figure Legend Snippet: Comparison of RNA structures of ubc 3’UTR as determined by PARS based pairing probability and enzymatic footprinting using RNase V1 and S1 Nuclease. a . Bar plot represents PARS scores of 3’UTR region of ubiquitin c (ubc) . Out of 105 positions, 87 positions are captured by PARS. b . Enzymatic footprinting of ubc 3’UTR probed by S1 Nuclease and RNase V1. Nucleotide positions are correlated with alkaline hydrolysis (AH) ladder and RNase T1 (G) ladder. Positions with similar structural pattern with PARS scores are highlighted. Red dots indicate unpaired positions; green indicates paired positions while yellow represents ambiguous regions. c . Heatmap representing secondary structure of 68 positions of ubc 3’UTR as determined by PARS and enzymatic footprinting (FP). Top panel represents PARS pairing probability; bottom panel indicates enzymatic footprinting pairing probability; middle panel represents the consensus between the two (PARS: FP). Red represents unpaired, green represents paired and yellow represents ambiguous regions

Techniques Used: Footprinting

27) Product Images from "Mapping of the Functional Boundaries and Secondary Structure of the Mouse Mammary Tumor Virus Rem-responsive Element *"

Article Title: Mapping of the Functional Boundaries and Secondary Structure of the Mouse Mammary Tumor Virus Rem-responsive Element *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.012476

Secondary structure prediction with experimental constraints for the MMTV RmRE. Cleavage sites with RNase V1, A, and T1 are highlighted in yellow , green , and blue , respectively. Those sites indicated by colored circles were used as constraints in a web-based
Figure Legend Snippet: Secondary structure prediction with experimental constraints for the MMTV RmRE. Cleavage sites with RNase V1, A, and T1 are highlighted in yellow , green , and blue , respectively. Those sites indicated by colored circles were used as constraints in a web-based

Techniques Used:

28) Product Images from "Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function"

Article Title: Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1005613

PARS analysis. (A) Overview of modified PARS approach. RNase V1 cleaves double-stranded RNA and combination of RNases A/T1 the single stranded RNA with optimal activities at physiological pH (7.0). RNAse A/T1 usage requires an additional phosphorylation step prior to library generation. (B) The PARS score of the rpoS leader sequence (inset) was overlaid with the experimentally determined structure [ 64 ]. Double-stranded nucleotides with positive PARS score are colored red, single-stranded nucleotides with negative PARS score–blue, nucleotides with missing PARS score or equal to zero–green. The color intensity of the rpoS nucleotides reflects the PARS scores (rainbow legend). (C) Metagene analysis of protein-coding transcripts. Average PARS score for each nucleotide (top) and GC content (bottom) across the 5’UTRs, CDS and 3’UTRs of all protein-coding transcripts, aligned at the start or stop codon, respectively. For the shaded areas the average PARS scores or GC content is calculated; thus note the deviations from the total GC content of 51% in E . coli . Unstructured region upstream of the start codon and structured sequence preceding the stop codon are marked by arrows with filled and open arrow heads, respectively.
Figure Legend Snippet: PARS analysis. (A) Overview of modified PARS approach. RNase V1 cleaves double-stranded RNA and combination of RNases A/T1 the single stranded RNA with optimal activities at physiological pH (7.0). RNAse A/T1 usage requires an additional phosphorylation step prior to library generation. (B) The PARS score of the rpoS leader sequence (inset) was overlaid with the experimentally determined structure [ 64 ]. Double-stranded nucleotides with positive PARS score are colored red, single-stranded nucleotides with negative PARS score–blue, nucleotides with missing PARS score or equal to zero–green. The color intensity of the rpoS nucleotides reflects the PARS scores (rainbow legend). (C) Metagene analysis of protein-coding transcripts. Average PARS score for each nucleotide (top) and GC content (bottom) across the 5’UTRs, CDS and 3’UTRs of all protein-coding transcripts, aligned at the start or stop codon, respectively. For the shaded areas the average PARS scores or GC content is calculated; thus note the deviations from the total GC content of 51% in E . coli . Unstructured region upstream of the start codon and structured sequence preceding the stop codon are marked by arrows with filled and open arrow heads, respectively.

Techniques Used: Modification, Sequencing

29) Product Images from "A novel structural rearrangement of hepatitis delta virus antigenomic ribozyme"

Article Title: A novel structural rearrangement of hepatitis delta virus antigenomic ribozyme

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkm674

Chemical and enzymatic probing of the bottom of the P2 stem. ( A ) Autoradiogram of a 10% PAGE gel of in-line probing performed on 5′-end-labelled wild-type and mutated trans -acting ribozymes. Both alkaline and RNase T1 hydrolyses of the wild-type ribozyme were performed in order to determine the location of each position (lanes OH and T1, respectively). In-line probing of the wild-type ribozyme (1, 2), the RzC 24 U,C 25 U,G 40 U,G 41 U (3, 4) and the RzA 78 U,A 79 U (5, 6) mutants are shown. The experiments were performed either in the absence (−) or the presence (+) of the SdA4 analogue. The secondary structure motifs are identified on the left. ( B ) Autoradiogram of a 10% PAGE gel of RNase V1 probing performed on 5′-end-labelled wild-type and mutated cis -acting ribozymes. Both alkaline and RNase T1 hydrolyses of the wild-type sequence were performed in order to determine the location of each position (lanes OH and T1, respectively). Lanes 1 to 4 correspond to the wild-type sequence (C 19 G 81 G 80 ) and the mutants RzC 19 ,G 81 A,G 80 A, RzC 19 ,G 81 AG 80 and RzC 19 G 81 G 80 A, respectively. The positions of the C 19 and C 24 (used to establish the relative level of hydrolysis) are indicated on the right. ( C ) Histogram of the relative levels of RNase V1 hydrolysis of C 19 for each ribozyme.
Figure Legend Snippet: Chemical and enzymatic probing of the bottom of the P2 stem. ( A ) Autoradiogram of a 10% PAGE gel of in-line probing performed on 5′-end-labelled wild-type and mutated trans -acting ribozymes. Both alkaline and RNase T1 hydrolyses of the wild-type ribozyme were performed in order to determine the location of each position (lanes OH and T1, respectively). In-line probing of the wild-type ribozyme (1, 2), the RzC 24 U,C 25 U,G 40 U,G 41 U (3, 4) and the RzA 78 U,A 79 U (5, 6) mutants are shown. The experiments were performed either in the absence (−) or the presence (+) of the SdA4 analogue. The secondary structure motifs are identified on the left. ( B ) Autoradiogram of a 10% PAGE gel of RNase V1 probing performed on 5′-end-labelled wild-type and mutated cis -acting ribozymes. Both alkaline and RNase T1 hydrolyses of the wild-type sequence were performed in order to determine the location of each position (lanes OH and T1, respectively). Lanes 1 to 4 correspond to the wild-type sequence (C 19 G 81 G 80 ) and the mutants RzC 19 ,G 81 A,G 80 A, RzC 19 ,G 81 AG 80 and RzC 19 G 81 G 80 A, respectively. The positions of the C 19 and C 24 (used to establish the relative level of hydrolysis) are indicated on the right. ( C ) Histogram of the relative levels of RNase V1 hydrolysis of C 19 for each ribozyme.

Techniques Used: Polyacrylamide Gel Electrophoresis, Sequencing

30) Product Images from "A comprehensive analysis of precursor microRNA cleavage by human Dicer"

Article Title: A comprehensive analysis of precursor microRNA cleavage by human Dicer

Journal: RNA

doi: 10.1261/rna.033688.112

Nuclease protection assays to probe hDicer interactions with pre-miRNAs. ( A ) RNase V1 protection assays. (Lanes 1 ) 5′-end 32 P-labeled RNA substrates without any treatment. (Lanes 2 ) RNAs treated with hDicer for 3 min at room temperature. (Lanes
Figure Legend Snippet: Nuclease protection assays to probe hDicer interactions with pre-miRNAs. ( A ) RNase V1 protection assays. (Lanes 1 ) 5′-end 32 P-labeled RNA substrates without any treatment. (Lanes 2 ) RNAs treated with hDicer for 3 min at room temperature. (Lanes

Techniques Used: Labeling

31) Product Images from "A comprehensive analysis of precursor microRNA cleavage by human Dicer"

Article Title: A comprehensive analysis of precursor microRNA cleavage by human Dicer

Journal: RNA

doi: 10.1261/rna.033688.112

Nuclease protection assays to probe hDicer interactions with pre-miRNAs. ( A ) RNase V1 protection assays. (Lanes 1 ) 5′-end 32 P-labeled RNA substrates without any treatment. (Lanes 2 ) RNAs treated with hDicer for 3 min at room temperature. (Lanes
Figure Legend Snippet: Nuclease protection assays to probe hDicer interactions with pre-miRNAs. ( A ) RNase V1 protection assays. (Lanes 1 ) 5′-end 32 P-labeled RNA substrates without any treatment. (Lanes 2 ) RNAs treated with hDicer for 3 min at room temperature. (Lanes

Techniques Used: Labeling

32) Product Images from "Genome-wide Measurement of RNA Secondary Structure in Yeast"

Article Title: Genome-wide Measurement of RNA Secondary Structure in Yeast

Journal: Nature

doi: 10.1038/nature09322

PARS correctly recapitulates results of RNA footprinting and known structures (a) The PARS signal obtained for bases 50-110 of the yeast gene CCW12 using the double-stranded cutter RNase V1 (red bars) or single-stranded cutter RNase S1 (green bars) accurately matches the signals obtained by traditional footprinting of that same transcript domain (black lines). PARS signal is shown as the number of sequence reads which mapped to each nucleotide; footprinting results are obtained by semi-automated quantification of the RNase lanes shown in (b). The red arrows indicate RNase V1 cleavages and the green arrows indicate RNase S1 cleavages as shown in the gel (b). (b) Gel analysis of RNase V1 (lanes 5,6) and S1 (lanes 3,4) probing of CCW12. Additionally, RNase T1 ladder (lanes 2,8), alkaline hydrolysis (lanes 1,9), and no RNase treatment (lane 7) are shown. (c) The PARS signal obtained from bases 50-120 of the yeast gene RPL41A matches the signals obtained by traditional footprinting. (d) RNase V1 (lanes 5,6) and S1 (lanes 7,8) probing of RPL41A, RNase T1 ladder (lane 2), alkaline hydrolysis (lanes 1,9), and no RNase treatment (lane 4). (e-f) Raw number of reads obtained using RNase V1 (red bars) or RNase S1 (green bars) and the resulting PARS score (blue bars) along one inspected domain of ASH1 (e) and URE2 ( f ). Also shown are the known structures of the inspected domains with nucleotides color-coded according to their computed PARS score.
Figure Legend Snippet: PARS correctly recapitulates results of RNA footprinting and known structures (a) The PARS signal obtained for bases 50-110 of the yeast gene CCW12 using the double-stranded cutter RNase V1 (red bars) or single-stranded cutter RNase S1 (green bars) accurately matches the signals obtained by traditional footprinting of that same transcript domain (black lines). PARS signal is shown as the number of sequence reads which mapped to each nucleotide; footprinting results are obtained by semi-automated quantification of the RNase lanes shown in (b). The red arrows indicate RNase V1 cleavages and the green arrows indicate RNase S1 cleavages as shown in the gel (b). (b) Gel analysis of RNase V1 (lanes 5,6) and S1 (lanes 3,4) probing of CCW12. Additionally, RNase T1 ladder (lanes 2,8), alkaline hydrolysis (lanes 1,9), and no RNase treatment (lane 7) are shown. (c) The PARS signal obtained from bases 50-120 of the yeast gene RPL41A matches the signals obtained by traditional footprinting. (d) RNase V1 (lanes 5,6) and S1 (lanes 7,8) probing of RPL41A, RNase T1 ladder (lane 2), alkaline hydrolysis (lanes 1,9), and no RNase treatment (lane 4). (e-f) Raw number of reads obtained using RNase V1 (red bars) or RNase S1 (green bars) and the resulting PARS score (blue bars) along one inspected domain of ASH1 (e) and URE2 ( f ). Also shown are the known structures of the inspected domains with nucleotides color-coded according to their computed PARS score.

Techniques Used: Footprinting, Sequencing

Measuring structural properties of RNA by deep sequencing (a) RNA molecules are cleaved by RNase V1, which cuts 3′ of double-stranded RNA, leaving a 5′ phosphate (5′P). One such cut is illustrated by a red arrow. Following random fragmentation, V1-generated fragments are specifically captured and subjected to deep sequencing. Each aligned sequence provides structural evidence about a single base. The marked red square illustrates the evidence obtained from one mapped sequence (red). Additional evidence (gray boxes) is collected by mapping more sequences (gray horizontal bars). A large number of reads aligned to the same base indicates that the base is cleaved multiple times by RNase V1 and is thus more likely to be in double stranded conformation. (b) Same as (a), but when the RNA sample is treated with RNase S1, which cuts 3′ of single-stranded RNA. Collected reads in this case suggest that the base was unpaired in the original RNA structure. (c) By combining the data extracted from the two complementary experiments (a) and (b), we obtain a nucleotide-resolution score representing the likelihood that the inspected base was in a double- or single-stranded conformation.
Figure Legend Snippet: Measuring structural properties of RNA by deep sequencing (a) RNA molecules are cleaved by RNase V1, which cuts 3′ of double-stranded RNA, leaving a 5′ phosphate (5′P). One such cut is illustrated by a red arrow. Following random fragmentation, V1-generated fragments are specifically captured and subjected to deep sequencing. Each aligned sequence provides structural evidence about a single base. The marked red square illustrates the evidence obtained from one mapped sequence (red). Additional evidence (gray boxes) is collected by mapping more sequences (gray horizontal bars). A large number of reads aligned to the same base indicates that the base is cleaved multiple times by RNase V1 and is thus more likely to be in double stranded conformation. (b) Same as (a), but when the RNA sample is treated with RNase S1, which cuts 3′ of single-stranded RNA. Collected reads in this case suggest that the base was unpaired in the original RNA structure. (c) By combining the data extracted from the two complementary experiments (a) and (b), we obtain a nucleotide-resolution score representing the likelihood that the inspected base was in a double- or single-stranded conformation.

Techniques Used: Sequencing, Generated

33) Product Images from "A long-distance RNA–RNA interaction plays an important role in programmed − 1 ribosomal frameshifting in the translation of p88 replicase protein of Red clover necrotic mosaic virus"

Article Title: A long-distance RNA–RNA interaction plays an important role in programmed − 1 ribosomal frameshifting in the translation of p88 replicase protein of Red clover necrotic mosaic virus

Journal: Virology

doi: 10.1016/j.virol.2011.05.012

Enzymatic probing analysis supports the predicted RNA secondary structures of SLCsSL. (A) Enzymatic structure probing of SLCsSL in vitro . RNA transcripts were subjected to enzymatic modifications with RNase A and RNase V1, and the products generated were analyzed by primer extension using a primer 3′SLCsSL-2forSP. The products were separated in a 5% polyacrylamide gel in the presence of 8 M urea along with a sequence ladder generated with the same primer used for reverse transcription. Specific bands of the RNase A treated lane are indicated by white arrowheads and those of the RNase V1 treated lane are indicated by black stars. (B) The results of the structure probing are mapped onto the predicted secondary structure model of the SLCsSL. Different reactivities with residues are indicated by various symbols that are defined in the model.
Figure Legend Snippet: Enzymatic probing analysis supports the predicted RNA secondary structures of SLCsSL. (A) Enzymatic structure probing of SLCsSL in vitro . RNA transcripts were subjected to enzymatic modifications with RNase A and RNase V1, and the products generated were analyzed by primer extension using a primer 3′SLCsSL-2forSP. The products were separated in a 5% polyacrylamide gel in the presence of 8 M urea along with a sequence ladder generated with the same primer used for reverse transcription. Specific bands of the RNase A treated lane are indicated by white arrowheads and those of the RNase V1 treated lane are indicated by black stars. (B) The results of the structure probing are mapped onto the predicted secondary structure model of the SLCsSL. Different reactivities with residues are indicated by various symbols that are defined in the model.

Techniques Used: In Vitro, Generated, Sequencing

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

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

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1002494

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

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

35) Product Images from "Myxoma Virus Protein M029 Is a Dual Function Immunomodulator that Inhibits PKR and Also Conscripts RHA/DHX9 to Promote Expanded Host Tropism and Viral Replication"

Article Title: Myxoma Virus Protein M029 Is a Dual Function Immunomodulator that Inhibits PKR and Also Conscripts RHA/DHX9 to Promote Expanded Host Tropism and Viral Replication

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003465

M029 interacts with human PKR and DHX9/RHA in virus-infected cells. A) HeLa cells were infected with vMyx-GFP (lanes 1, 3, 5, 7, 9 and 11) or vMyx-M029V5N (lanes 2, 4, 6, 8, 10 and 12) for 24 h. Cell lysates were untreated or treated with RNAse A/T1(50 µg/ml RNase A and 125 u/ml RNase T1) at 4°C and co-IP was performed using mouse anti-V5 antibody (lanes 1 to 8), rabbit anti-PKR (lanes 9 and 10) or rabbit anti-DHX9. Proteins associated with the complex were separated on 12% SDS-PAGE, transferred to a PVDF membrane and immunoblotted. The bots were probed with rabbit anti-DHX9 (lanes 1 to 4), rabbit anti-PKR (lanes 5 to 8) or mouse anti-V5 (lanes 9 to 12). B) M029 interaction with PKR is mediated by dsRNA but interaction with DHX9 is dsRNA independent. HeLa cell were infected with vMyxM029V5N for 24 h, cell lysates were treated with RNase V1 (10 u/ml) at 4°C over-night and co-IP was performed using mouse anti-V5 (lanes 1 and 2) or rabbit anti-DHX9 (lanes 3 and 4). After transfer the blots were probed with different antibodies. C) M029 interaction with DHX9 is independent of PKR. HeLa cell lines with constitutive knock down of PKR (HeLa shPKR, described in figure 9 ) were infected with vMyx-M029V5N for 24 h, cell lysates were treated with RNase V1 at 4°C overnight and co-IP was performed using mouse anti-V5 (lanes 1 and 2) or rabbit anti-DHX9 (lanes 3 and 4). After transfer the blots were probed with different antibodies.
Figure Legend Snippet: M029 interacts with human PKR and DHX9/RHA in virus-infected cells. A) HeLa cells were infected with vMyx-GFP (lanes 1, 3, 5, 7, 9 and 11) or vMyx-M029V5N (lanes 2, 4, 6, 8, 10 and 12) for 24 h. Cell lysates were untreated or treated with RNAse A/T1(50 µg/ml RNase A and 125 u/ml RNase T1) at 4°C and co-IP was performed using mouse anti-V5 antibody (lanes 1 to 8), rabbit anti-PKR (lanes 9 and 10) or rabbit anti-DHX9. Proteins associated with the complex were separated on 12% SDS-PAGE, transferred to a PVDF membrane and immunoblotted. The bots were probed with rabbit anti-DHX9 (lanes 1 to 4), rabbit anti-PKR (lanes 5 to 8) or mouse anti-V5 (lanes 9 to 12). B) M029 interaction with PKR is mediated by dsRNA but interaction with DHX9 is dsRNA independent. HeLa cell were infected with vMyxM029V5N for 24 h, cell lysates were treated with RNase V1 (10 u/ml) at 4°C over-night and co-IP was performed using mouse anti-V5 (lanes 1 and 2) or rabbit anti-DHX9 (lanes 3 and 4). After transfer the blots were probed with different antibodies. C) M029 interaction with DHX9 is independent of PKR. HeLa cell lines with constitutive knock down of PKR (HeLa shPKR, described in figure 9 ) were infected with vMyx-M029V5N for 24 h, cell lysates were treated with RNase V1 at 4°C overnight and co-IP was performed using mouse anti-V5 (lanes 1 and 2) or rabbit anti-DHX9 (lanes 3 and 4). After transfer the blots were probed with different antibodies.

Techniques Used: Infection, Co-Immunoprecipitation Assay, SDS Page

36) Product Images from "A T-stem slip in human mitochondrial tRNALeu(CUN) governs its charging capacity"

Article Title: A T-stem slip in human mitochondrial tRNALeu(CUN) governs its charging capacity

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki677

Comparative enzymatic probing of in vitro transcribed human mitochondrial tRNA Leu (CUN) and the U48C mutant. ( A ) Autoradiograms of the various cleavage products of 5′-end-labeled tRNA transcripts separated on denaturing 12% polyacrylamide gels. Lane C, control incubations without probe; lane L, alkaline ladder; lane G, G ladder. The tRNA was 5′-labeled and digested with RNase T1, RNase V1 and nuclease S1. Numbers 1 and 2 refer to increasing concentrations of nuclease. The RNase T1 cleavage product specific for WT transcript was indicated in the solid-line square. The difference can be easily seen in the dashed-line squares. ( B ) Result of the enzymatic probing of WT and U48C mutant hmtRNA Leu (CUN) transcripts. Intensities of cuts are proportional to the darkness of the symbols. The pentacle denotes the specific RNase T1 cleavage at G53 on WT transcript.
Figure Legend Snippet: Comparative enzymatic probing of in vitro transcribed human mitochondrial tRNA Leu (CUN) and the U48C mutant. ( A ) Autoradiograms of the various cleavage products of 5′-end-labeled tRNA transcripts separated on denaturing 12% polyacrylamide gels. Lane C, control incubations without probe; lane L, alkaline ladder; lane G, G ladder. The tRNA was 5′-labeled and digested with RNase T1, RNase V1 and nuclease S1. Numbers 1 and 2 refer to increasing concentrations of nuclease. The RNase T1 cleavage product specific for WT transcript was indicated in the solid-line square. The difference can be easily seen in the dashed-line squares. ( B ) Result of the enzymatic probing of WT and U48C mutant hmtRNA Leu (CUN) transcripts. Intensities of cuts are proportional to the darkness of the symbols. The pentacle denotes the specific RNase T1 cleavage at G53 on WT transcript.

Techniques Used: In Vitro, Mutagenesis, Labeling

Enzymatic probing of the representative tRNA Leu (CUN) variants. ( A ) Cleavage products of 5′-labeled molecules after treatment with nucleases, displayed on autoradiograms of 12% polyacrylamide gels. C stands for H 2 O ladder; G for G ladder; L for alkaline ladder; T1 for RNase T1 digestion ladder; V1 for RNase V1 digestion ladder; S1 for nuclease S1 digestion ladder; numbers 1 and 2 refer to increasing concentrations of nuclease. The RNase T1 cleavage product denoted in the solid-line square indicates the enlarged T-loop. The band difference can be easily seen in the dashed-line squares. ( B ) Location of enzymatic cleavage sites on cloverleaf diagrams of the transcripts. Specifications and intensities of cuts are as indicated in the key. Nucleotides that could not be tested because of technical limitations are marked by a line. Mutated nucleotides are shown in italics. Regions with different cleavage pattern are highlighted by gray background. The pentacles denote the RNase T1 cleavage specific for the enlarged T-loop.
Figure Legend Snippet: Enzymatic probing of the representative tRNA Leu (CUN) variants. ( A ) Cleavage products of 5′-labeled molecules after treatment with nucleases, displayed on autoradiograms of 12% polyacrylamide gels. C stands for H 2 O ladder; G for G ladder; L for alkaline ladder; T1 for RNase T1 digestion ladder; V1 for RNase V1 digestion ladder; S1 for nuclease S1 digestion ladder; numbers 1 and 2 refer to increasing concentrations of nuclease. The RNase T1 cleavage product denoted in the solid-line square indicates the enlarged T-loop. The band difference can be easily seen in the dashed-line squares. ( B ) Location of enzymatic cleavage sites on cloverleaf diagrams of the transcripts. Specifications and intensities of cuts are as indicated in the key. Nucleotides that could not be tested because of technical limitations are marked by a line. Mutated nucleotides are shown in italics. Regions with different cleavage pattern are highlighted by gray background. The pentacles denote the RNase T1 cleavage specific for the enlarged T-loop.

Techniques Used: Labeling

37) Product Images from "New, extended hairpin form of the TAR-2 RNA domain points to the structural polymorphism at the 5? end of the HIV-2 leader RNA"

Article Title: New, extended hairpin form of the TAR-2 RNA domain points to the structural polymorphism at the 5? end of the HIV-2 leader RNA

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl373

Secondary structure probing of HIV-2 TAR RNA. The RNA was treated with selected single-strand specific enzymes (S1, T1, A), DEPC and double-strand specific RNase V1. ( A ) Cleavage patterns obtained for the 5′ end labelled TAR-2 wt transcript. Lanes C represent control sample with untreated RNA; lanes L, formamide ladder; lanes T, limited hydrolysis with RNase T1. ( B ) Cleavage patterns obtained for the 5′ end labelled TAR-2 A21 mutant transcript. ( C ) A summary of the enzymatic cleavages and chemical modifications data for the TAR-2 wt RNA viewed on the secondary structure models (E1, E2 and B). For clarity, only the top part of the E2 conformer that differs from E1 is shown. Sites and intensities of cleavages with the respective reagents are indicated by symbols (see inset); the size of a symbol corresponds to the relative cleavage intensity. The weakest cleavages are not indicated. ( D ) Two different RNase T1 cleavage patterns observed for the particular G-rich region of both TAR-2 wt and A21 mutant along with respective secondary structure motifs. Both patterns point to the mixture of TAR-2 forms; the right pattern is consistent mostly with one of the extended (E1) and with the branched (B) structure models; the most often observed left pattern represents predominantly the second extended conformer E2. ( E ) RNase T1 cleavage pattern obtained for the TAR-2 B4 and ΔC23 mutants stabilized in the branched (B) form.
Figure Legend Snippet: Secondary structure probing of HIV-2 TAR RNA. The RNA was treated with selected single-strand specific enzymes (S1, T1, A), DEPC and double-strand specific RNase V1. ( A ) Cleavage patterns obtained for the 5′ end labelled TAR-2 wt transcript. Lanes C represent control sample with untreated RNA; lanes L, formamide ladder; lanes T, limited hydrolysis with RNase T1. ( B ) Cleavage patterns obtained for the 5′ end labelled TAR-2 A21 mutant transcript. ( C ) A summary of the enzymatic cleavages and chemical modifications data for the TAR-2 wt RNA viewed on the secondary structure models (E1, E2 and B). For clarity, only the top part of the E2 conformer that differs from E1 is shown. Sites and intensities of cleavages with the respective reagents are indicated by symbols (see inset); the size of a symbol corresponds to the relative cleavage intensity. The weakest cleavages are not indicated. ( D ) Two different RNase T1 cleavage patterns observed for the particular G-rich region of both TAR-2 wt and A21 mutant along with respective secondary structure motifs. Both patterns point to the mixture of TAR-2 forms; the right pattern is consistent mostly with one of the extended (E1) and with the branched (B) structure models; the most often observed left pattern represents predominantly the second extended conformer E2. ( E ) RNase T1 cleavage pattern obtained for the TAR-2 B4 and ΔC23 mutants stabilized in the branched (B) form.

Techniques Used: Mutagenesis

38) Product Images from "Molecular analysis of a synthetic tetracycline-binding riboswitch"

Article Title: Molecular analysis of a synthetic tetracycline-binding riboswitch

Journal: RNA

doi: 10.1261/rna.7251305

Enzymatic and chemical probing of the tc-binding aptamer. ( A ) Limited digestion was performed using a 150-nucleotide-long RNA containing the aptamer in its mRNA context. Probing was carried out using RNase T1 (0.5 and 0.25 U), RNase V1 (0.005 and 0.001 U), and S1 nuclease (1 and 0.5 U) in the absence (−) and presence (+) of 10 μM tc. The two left-hand lanes of each nuclease probing correspond to the respective higher enzyme concentration. Alkaline hydrolysis of the RNA is denoted by H. G residues probed with RNase T1 are marked at left , and stem regions proposed by secondary structure prediction are denoted at the right side of the plot with open bars. ( B ) Chemical modifications were performed in the absence and presence of tc and monitored by primer extension reaction. Untreated RNA is marked with an 0. The incubation time (T) and the concentration of tc is given above the figure. (C, U, A, G) Sequencing lanes. (S) DMS modification carried out under semidenaturing conditions in the presence of EDTA. Nucleotide positions with occurring tc-dependent changes in the probing pattern are denoted and marked with arrowheads at the left side of the plot.
Figure Legend Snippet: Enzymatic and chemical probing of the tc-binding aptamer. ( A ) Limited digestion was performed using a 150-nucleotide-long RNA containing the aptamer in its mRNA context. Probing was carried out using RNase T1 (0.5 and 0.25 U), RNase V1 (0.005 and 0.001 U), and S1 nuclease (1 and 0.5 U) in the absence (−) and presence (+) of 10 μM tc. The two left-hand lanes of each nuclease probing correspond to the respective higher enzyme concentration. Alkaline hydrolysis of the RNA is denoted by H. G residues probed with RNase T1 are marked at left , and stem regions proposed by secondary structure prediction are denoted at the right side of the plot with open bars. ( B ) Chemical modifications were performed in the absence and presence of tc and monitored by primer extension reaction. Untreated RNA is marked with an 0. The incubation time (T) and the concentration of tc is given above the figure. (C, U, A, G) Sequencing lanes. (S) DMS modification carried out under semidenaturing conditions in the presence of EDTA. Nucleotide positions with occurring tc-dependent changes in the probing pattern are denoted and marked with arrowheads at the left side of the plot.

Techniques Used: Binding Assay, Concentration Assay, Incubation, Sequencing, Modification

39) Product Images from "6S RNA mimics B-form DNA to regulate Escherichia coli RNA polymerase"

Article Title: 6S RNA mimics B-form DNA to regulate Escherichia coli RNA polymerase

Journal: Molecular cell

doi: 10.1016/j.molcel.2017.09.006

CryoEM structure of the Eco 6S RNA/Eσ 70 complex ( A ) ( top ) Secondary structure of wild-type Eco 6S RNA as observed in the cryoEM structure. Structural elements of the 6S RNA are labeled (CS, closing stem; DB, downstream bulge; DD, downstream duplex; CB, central bubble; UD1-3, upstream duplexes 1-3; UB1-2, upstream bulges 1-2; UTL, upstream terminal loop). The sequence is color-coded according to the RNase footprinting and localized hydroxyl-radical cleavage results comparing 6S RNA with and without Eσ 70 ). RNA positions protected from RNase V1 and/or RNase A cleavage in the presence of Eσ 70 are colored green. RNA positions showing hypersensitivity to RNAse V1 in the presence of Eσ 70 are colored red. RNA positions efficiently cleaved by hydroxyl radicals generated from Fe 2+ in place of the RNAP active site Mg 2+ ). The position of pRNA synthesis initiation (U44) is indicated by a bent arrow. ( below ) Secondary structure of 6S RNA* used in cryoEM structure determination, generated by truncating the CS and shuffling the sequence of the DD to give DD* (shuffled sequences highlighted in the orange box). ( B ) The 3.8 Å resolution cryoEM density map of Eco 6S RNA/Eσ 70 is rendered as a transparent surface colored as shown. Superimposed is the final refined model; the RNAP is shown as a backbone ribbon, the 6S RNA is shown in stick format. ( C ) The 3.8-Å resolution cryoEM density map with the superimposed model of only the 6S RNA. Shown for reference is the RNAP active site Mg 2+ ion (yellow sphere). ( D ) Shown is just the promoter DNA after superimposing the Eσ 70 ) onto the 6S RNA/Eσ 70 structure. The promoter DNA is colored blue but the -35 and -10 elements of the DNA are colored yellow. The -6G of the discriminator and the Core Recognition Element (CRE) are colored violet. Shown for reference is the RNAP active site Mg 2+ ion (yellow sphere). .
Figure Legend Snippet: CryoEM structure of the Eco 6S RNA/Eσ 70 complex ( A ) ( top ) Secondary structure of wild-type Eco 6S RNA as observed in the cryoEM structure. Structural elements of the 6S RNA are labeled (CS, closing stem; DB, downstream bulge; DD, downstream duplex; CB, central bubble; UD1-3, upstream duplexes 1-3; UB1-2, upstream bulges 1-2; UTL, upstream terminal loop). The sequence is color-coded according to the RNase footprinting and localized hydroxyl-radical cleavage results comparing 6S RNA with and without Eσ 70 ). RNA positions protected from RNase V1 and/or RNase A cleavage in the presence of Eσ 70 are colored green. RNA positions showing hypersensitivity to RNAse V1 in the presence of Eσ 70 are colored red. RNA positions efficiently cleaved by hydroxyl radicals generated from Fe 2+ in place of the RNAP active site Mg 2+ ). The position of pRNA synthesis initiation (U44) is indicated by a bent arrow. ( below ) Secondary structure of 6S RNA* used in cryoEM structure determination, generated by truncating the CS and shuffling the sequence of the DD to give DD* (shuffled sequences highlighted in the orange box). ( B ) The 3.8 Å resolution cryoEM density map of Eco 6S RNA/Eσ 70 is rendered as a transparent surface colored as shown. Superimposed is the final refined model; the RNAP is shown as a backbone ribbon, the 6S RNA is shown in stick format. ( C ) The 3.8-Å resolution cryoEM density map with the superimposed model of only the 6S RNA. Shown for reference is the RNAP active site Mg 2+ ion (yellow sphere). ( D ) Shown is just the promoter DNA after superimposing the Eσ 70 ) onto the 6S RNA/Eσ 70 structure. The promoter DNA is colored blue but the -35 and -10 elements of the DNA are colored yellow. The -6G of the discriminator and the Core Recognition Element (CRE) are colored violet. Shown for reference is the RNAP active site Mg 2+ ion (yellow sphere). .

Techniques Used: Labeling, Sequencing, Footprinting, Generated

40) Product Images from "Isolation of specific and high-affinity RNA aptamers against NS3 helicase domain of hepatitis C virus"

Article Title: Isolation of specific and high-affinity RNA aptamers against NS3 helicase domain of hepatitis C virus

Journal: RNA

doi: 10.1261/rna.7100904

Minimal domain and structural determinations of SE RNA that binds to the HCV helicase. ( A ) The 91-nt-long SE RNA (MD1) was truncated at its 3′ end (MD 2–5) or 5′ end (MD 6). The sequences (+25 nt to + 63 nt) selected from the randomized region in the library RNA are presented as shaded box. ( B ) Three hundred and fifty picomolars of radiolabeled pool RNA or MD1–6 RNAs were incubated without (lane b ) or with (lane c , 200 nM) HCV helicase and the RNA–protein complexes were precipitated with Ni–NTA beads. Bound RNAs were extracted and analyzed on a 6% polyacrylamide gel with 7 M urea. Lane a contains 10% of the input-labeled RNAs. Binding % denotes the amount of each bound RNA relative to the input RNA. ( C ) Enzymatic mapping of the RNA secondary structure of the SE RNA. RNA labeled at the 5′ end (2.7 nM) was enzymatically digested with RNases T1 (lane 2 , 1U), nucleases S1 (lane 3 , 2 U), and RNases V1 (lane 4 , 0.0001 U). The partially digested products were then separated on a 12% polyacrylamide gel with urea along with partial alkaline hydrolysis ladder (lane 1 , AH). ( D ) Enzymatic footprinting of HCV helicase-SE RNA complex. The 5′-end-labeled SE RNA was incubated in the absence of (-; lanes 3 , 7 , 11 ) or presence of increasing amounts of HCV helicase (0.105 μM in lanes 4 , 8 , 12 ; 1.05 μM in lanes 5 , 9 , 13 ; 10.5 μM in lanes 6 , 10 , 14 ). Protein–RNA mixtures were digested with RNases T1 (lanes 3 – 6 ), nucleases S1 (lanes 7 – 10 ), and RNases V1 (lanes 11 – 14 ). The cleaved RNA fragments were then resolved on a 12% polyacrylamide gel with urea together with RNA size markers, T1 lad (lane 1 , partially digested SE RNAs with RNases T1) and AH (lane 2 , partially alkaline hydrolyzed SE RNAs). The protected sites from each nuclease by the protein are indicated by bars on the left side. The asterisks on the right side of the autoradiogram indicate the sequences whose accessibility to RNase V1 increased in the presence of the HCV helicase. ( E ) The computer-predicted model of the secondary structure of SE RNA #1 and the first 127 nucleotide of 3′ UTR of the HCV negative strand, HCV(–)3′UTR. The minimal binding domain of the SE RNA was shown in dotted box. Nucleotides 25–63 represented the sequences selected from randomized region of RNA library. Digestion patterns of the RNA to various nucleases are mapped. Squares, triangles, and circles indicate RNases T1, S1 nucleases, and RNases V1 cleavage sites, respectively. The size of each symbol denotes the susceptible intensities on the SE RNA to each nuclease. Protected regions of the RNA in the presence of the target protein are marked as shaded areas. Asterisks indicate the sites at which cleavage by RNases V1 was enhanced following binding of the HCV helicase.
Figure Legend Snippet: Minimal domain and structural determinations of SE RNA that binds to the HCV helicase. ( A ) The 91-nt-long SE RNA (MD1) was truncated at its 3′ end (MD 2–5) or 5′ end (MD 6). The sequences (+25 nt to + 63 nt) selected from the randomized region in the library RNA are presented as shaded box. ( B ) Three hundred and fifty picomolars of radiolabeled pool RNA or MD1–6 RNAs were incubated without (lane b ) or with (lane c , 200 nM) HCV helicase and the RNA–protein complexes were precipitated with Ni–NTA beads. Bound RNAs were extracted and analyzed on a 6% polyacrylamide gel with 7 M urea. Lane a contains 10% of the input-labeled RNAs. Binding % denotes the amount of each bound RNA relative to the input RNA. ( C ) Enzymatic mapping of the RNA secondary structure of the SE RNA. RNA labeled at the 5′ end (2.7 nM) was enzymatically digested with RNases T1 (lane 2 , 1U), nucleases S1 (lane 3 , 2 U), and RNases V1 (lane 4 , 0.0001 U). The partially digested products were then separated on a 12% polyacrylamide gel with urea along with partial alkaline hydrolysis ladder (lane 1 , AH). ( D ) Enzymatic footprinting of HCV helicase-SE RNA complex. The 5′-end-labeled SE RNA was incubated in the absence of (-; lanes 3 , 7 , 11 ) or presence of increasing amounts of HCV helicase (0.105 μM in lanes 4 , 8 , 12 ; 1.05 μM in lanes 5 , 9 , 13 ; 10.5 μM in lanes 6 , 10 , 14 ). Protein–RNA mixtures were digested with RNases T1 (lanes 3 – 6 ), nucleases S1 (lanes 7 – 10 ), and RNases V1 (lanes 11 – 14 ). The cleaved RNA fragments were then resolved on a 12% polyacrylamide gel with urea together with RNA size markers, T1 lad (lane 1 , partially digested SE RNAs with RNases T1) and AH (lane 2 , partially alkaline hydrolyzed SE RNAs). The protected sites from each nuclease by the protein are indicated by bars on the left side. The asterisks on the right side of the autoradiogram indicate the sequences whose accessibility to RNase V1 increased in the presence of the HCV helicase. ( E ) The computer-predicted model of the secondary structure of SE RNA #1 and the first 127 nucleotide of 3′ UTR of the HCV negative strand, HCV(–)3′UTR. The minimal binding domain of the SE RNA was shown in dotted box. Nucleotides 25–63 represented the sequences selected from randomized region of RNA library. Digestion patterns of the RNA to various nucleases are mapped. Squares, triangles, and circles indicate RNases T1, S1 nucleases, and RNases V1 cleavage sites, respectively. The size of each symbol denotes the susceptible intensities on the SE RNA to each nuclease. Protected regions of the RNA in the presence of the target protein are marked as shaded areas. Asterisks indicate the sites at which cleavage by RNases V1 was enhanced following binding of the HCV helicase.

Techniques Used: Incubation, Labeling, Binding Assay, Footprinting

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Article Title: Ifit1 Inhibits Japanese Encephalitis Virus Replication through Binding to 5? Capped 2?-O Unmethylated RNA
Article Snippet: .. The Ifit1 -transfected cells were lysed in RNA-binding buffer (10 mM HEPES, pH 7.3, 500 mM KCl, 1 mM EDTA, 2 mM MgCl2 , 0.1% NP-40, 0.1 μg/μl of yeast tRNA (Ambion), 1 U/ml of RNase inhibitor [Toyobo]), and the lysate (200 μg) was coincubated with 25 pmol of biotin-labeled RNA and streptavidin-agarose (Invitrogen) in RNA-binding buffer for 30 min at room temperature. .. The binding complexes were washed five times with RNA-binding buffer, followed by SDS-PAGE and immunoblotting with an anti-HA probe (F-7) antibody (Santa Cruz Biotechnology).

Positive Control:

Article Title: Physicochemical analysis of rotavirus segment 11 supports a 'modified panhandle' structure and not the predicted alternative tRNA-like structure (TRLS)
Article Snippet: .. boRV RNA11 was not aminoacylated A eukaryotic yeast tRNA mixture (Ambion), used as a positive control, showed effective aminoacylation with 14 C-labelled amino acid (s) in the presence of HeLa S100 (in comparison to reactions in which the cell extract was absent). .. The mean proportion of bound tRNA (i.e., aminoacylated) expressed as a percentage of both bound and unbound tRNAs was 12.2 ± 1.9 % (mean ± standard deviation, calculated from 5 independent experiments).

Synthesized:

Article Title: A 68-Nucleotide Sequence within the 3? Noncoding Region of Simian Hemorrhagic Fever Virus Negative-Strand RNA Binds to Four MA104 Cell Proteins
Article Snippet: .. Nonspecific competitors included a 130-nt RNA (designated plasmid RNA) synthesized from pCR 2.1 DNA, that had been digested with Bam HI, using T7 RNA polymerase as described above; yeast tRNA (Life Technologies); and poly(I)-poly(C) (Sigma). .. RNA-protein binding reactions were performed in a volume of 10 μl as described previously , with some modifications.

Incubation:

Article Title: Hfq-dependent regulation of OmpA synthesis is mediated by an antisense RNA
Article Snippet: .. Binding assays were performed in 1× TMN buffer (20 mM Trisacetate at pH 7.6, 100 mM sodium acetate, 5 mM magnesium acetate) as follows: 5′-end-labeled RNA (0.05 pmol of MicA RNA or ompA leader mRNA) and 1 μg of carrier yeast tRNA (Ambion) were incubated with increasing concentrations of unlabeled RNA (MicA or ompA leader) in 10 μL at 37°C for 30 min (experiment in ). .. The binding reactions were mixed with 2 μL of loading dye (48% glycerol, 0.01% bromophenol blue) and electrophoresed on native 5% polyacrylamide gels in 0.5× TBE buffer at 200 V in a cold room for 3 h. Gels were dried and analyzed using a PhosphorImager and the Image-quant software package (Molecular Dynamics).

Polymerase Chain Reaction:

Article Title: A 68-Nucleotide Sequence within the 3? Noncoding Region of Simian Hemorrhagic Fever Virus Negative-Strand RNA Binds to Four MA104 Cell Proteins
Article Snippet: .. Nonspecific competitors included a 130-nt RNA (designated plasmid RNA) synthesized from pCR 2.1 DNA, that had been digested with Bam HI, using T7 RNA polymerase as described above; yeast tRNA (Life Technologies); and poly(I)-poly(C) (Sigma). .. RNA-protein binding reactions were performed in a volume of 10 μl as described previously , with some modifications.

RNA Binding Assay:

Article Title: Ifit1 Inhibits Japanese Encephalitis Virus Replication through Binding to 5? Capped 2?-O Unmethylated RNA
Article Snippet: .. The Ifit1 -transfected cells were lysed in RNA-binding buffer (10 mM HEPES, pH 7.3, 500 mM KCl, 1 mM EDTA, 2 mM MgCl2 , 0.1% NP-40, 0.1 μg/μl of yeast tRNA (Ambion), 1 U/ml of RNase inhibitor [Toyobo]), and the lysate (200 μg) was coincubated with 25 pmol of biotin-labeled RNA and streptavidin-agarose (Invitrogen) in RNA-binding buffer for 30 min at room temperature. .. The binding complexes were washed five times with RNA-binding buffer, followed by SDS-PAGE and immunoblotting with an anti-HA probe (F-7) antibody (Santa Cruz Biotechnology).

Binding Assay:

Article Title: Hfq-dependent regulation of OmpA synthesis is mediated by an antisense RNA
Article Snippet: .. Binding assays were performed in 1× TMN buffer (20 mM Trisacetate at pH 7.6, 100 mM sodium acetate, 5 mM magnesium acetate) as follows: 5′-end-labeled RNA (0.05 pmol of MicA RNA or ompA leader mRNA) and 1 μg of carrier yeast tRNA (Ambion) were incubated with increasing concentrations of unlabeled RNA (MicA or ompA leader) in 10 μL at 37°C for 30 min (experiment in ). .. The binding reactions were mixed with 2 μL of loading dye (48% glycerol, 0.01% bromophenol blue) and electrophoresed on native 5% polyacrylamide gels in 0.5× TBE buffer at 200 V in a cold room for 3 h. Gels were dried and analyzed using a PhosphorImager and the Image-quant software package (Molecular Dynamics).

Plasmid Preparation:

Article Title: A 68-Nucleotide Sequence within the 3? Noncoding Region of Simian Hemorrhagic Fever Virus Negative-Strand RNA Binds to Four MA104 Cell Proteins
Article Snippet: .. Nonspecific competitors included a 130-nt RNA (designated plasmid RNA) synthesized from pCR 2.1 DNA, that had been digested with Bam HI, using T7 RNA polymerase as described above; yeast tRNA (Life Technologies); and poly(I)-poly(C) (Sigma). .. RNA-protein binding reactions were performed in a volume of 10 μl as described previously , with some modifications.

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    Thermo Fisher rnase v1
    Addition of CA-repeats does not change the secondary structure of the coat RBS stem–loop. ( A ) Enzymatic and chemical structure probing was conducted on control mRNA (00) or mRNAs with tails of 4, 6 or 8 (CA)-repeats, (see Materials and Methods), as indicated. The mRNAs were mock-treated (lanes ‘–’), partially digested with double-strand-specific <t>RNase</t> V1 (V1), or treated with lead(II) acetate (Pb 2+ ). UCGA: sequencing reactions on (CA)8 mRNA. The position of the SD and AUG start codon are indicated by red boxes. Regions of reactivity toward RNase V1 (red solid line) and lead(II) acetate (red dashed line) are indicated on the autoradiogram. ( B ) The localization of RNase V1 (filled triangles) and lead(II) acetate (black dots) cuts are shown on the secondary structure of the 5′-segment of (CA)8 mRNA. Black boxes: SD and AUG.
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    Addition of CA-repeats does not change the secondary structure of the coat RBS stem–loop. ( A ) Enzymatic and chemical structure probing was conducted on control mRNA (00) or mRNAs with tails of 4, 6 or 8 (CA)-repeats, (see Materials and Methods), as indicated. The mRNAs were mock-treated (lanes ‘–’), partially digested with double-strand-specific RNase V1 (V1), or treated with lead(II) acetate (Pb 2+ ). UCGA: sequencing reactions on (CA)8 mRNA. The position of the SD and AUG start codon are indicated by red boxes. Regions of reactivity toward RNase V1 (red solid line) and lead(II) acetate (red dashed line) are indicated on the autoradiogram. ( B ) The localization of RNase V1 (filled triangles) and lead(II) acetate (black dots) cuts are shown on the secondary structure of the 5′-segment of (CA)8 mRNA. Black boxes: SD and AUG.

    Journal: Nucleic Acids Research

    Article Title: Unstructured 5′-tails act through ribosome standby to override inhibitory structure at ribosome binding sites

    doi: 10.1093/nar/gky073

    Figure Lengend Snippet: Addition of CA-repeats does not change the secondary structure of the coat RBS stem–loop. ( A ) Enzymatic and chemical structure probing was conducted on control mRNA (00) or mRNAs with tails of 4, 6 or 8 (CA)-repeats, (see Materials and Methods), as indicated. The mRNAs were mock-treated (lanes ‘–’), partially digested with double-strand-specific RNase V1 (V1), or treated with lead(II) acetate (Pb 2+ ). UCGA: sequencing reactions on (CA)8 mRNA. The position of the SD and AUG start codon are indicated by red boxes. Regions of reactivity toward RNase V1 (red solid line) and lead(II) acetate (red dashed line) are indicated on the autoradiogram. ( B ) The localization of RNase V1 (filled triangles) and lead(II) acetate (black dots) cuts are shown on the secondary structure of the 5′-segment of (CA)8 mRNA. Black boxes: SD and AUG.

    Article Snippet: RNase V1 probing used a final concentration of 0.01 U/μl (ThermoFisher Scientific, #AM2275) for 5 min at 37°C.

    Techniques: Sequencing

    Addition of CA-repeats does not change the secondary structure of the coat RBS stem–loop. ( A ) Enzymatic and chemical structure probing was conducted on control mRNA (00) or mRNAs with tails of 4, 6 or 8 (CA)-repeats, (see Materials and Methods), as indicated. The mRNAs were mock-treated (lanes ‘–’), partially digested with double-strand-specific RNase V1 (V1), or treated with lead(II) acetate (Pb 2+ ). UCGA: sequencing reactions on (CA)8 mRNA. The position of the SD and AUG start codon are indicated by red boxes. Regions of reactivity toward RNase V1 (red solid line) and lead(II) acetate (red dashed line) are indicated on the autoradiogram. ( B ) The localization of RNase V1 (filled triangles) and lead(II) acetate (black dots) cuts are shown on the secondary structure of the 5′-segment of (CA)8 mRNA. Black boxes: SD and AUG.

    Journal: Nucleic Acids Research

    Article Title: Unstructured 5′-tails act through ribosome standby to override inhibitory structure at ribosome binding sites

    doi: 10.1093/nar/gky073

    Figure Lengend Snippet: Addition of CA-repeats does not change the secondary structure of the coat RBS stem–loop. ( A ) Enzymatic and chemical structure probing was conducted on control mRNA (00) or mRNAs with tails of 4, 6 or 8 (CA)-repeats, (see Materials and Methods), as indicated. The mRNAs were mock-treated (lanes ‘–’), partially digested with double-strand-specific RNase V1 (V1), or treated with lead(II) acetate (Pb 2+ ). UCGA: sequencing reactions on (CA)8 mRNA. The position of the SD and AUG start codon are indicated by red boxes. Regions of reactivity toward RNase V1 (red solid line) and lead(II) acetate (red dashed line) are indicated on the autoradiogram. ( B ) The localization of RNase V1 (filled triangles) and lead(II) acetate (black dots) cuts are shown on the secondary structure of the 5′-segment of (CA)8 mRNA. Black boxes: SD and AUG.

    Article Snippet: RNase V1 probing used a final concentration of 0.01 U/μl (ThermoFisher Scientific, #AM2275) for 5 min at 37°C.

    Techniques: Sequencing

    Enzymatic degradation of RNA causes protein aggregation. a Diagram showing the experimental design. b SDS-PAGE analysis of soluble (Supernatant) and insoluble proteins (Pellet) from human neurons after treatment with a mixture of RNase A and RNase T1 (A/T1), or vehicle (Ve-). c Protein aggregation (Pellet) after incubation with different ribonucleases or DNase I in the presence of either EDTA or Mg 2+ . Ribonucleases used were RNase A (A), RNase T1 (T1), a mixture of RNase A and RNase T1 (A/T1), RNase 1f (1f), and RNase V1 (V1),

    Journal: bioRxiv

    Article Title: Enzymatic degradation of RNA causes widespread protein aggregation in cell and tissue lysates

    doi: 10.1101/841577

    Figure Lengend Snippet: Enzymatic degradation of RNA causes protein aggregation. a Diagram showing the experimental design. b SDS-PAGE analysis of soluble (Supernatant) and insoluble proteins (Pellet) from human neurons after treatment with a mixture of RNase A and RNase T1 (A/T1), or vehicle (Ve-). c Protein aggregation (Pellet) after incubation with different ribonucleases or DNase I in the presence of either EDTA or Mg 2+ . Ribonucleases used were RNase A (A), RNase T1 (T1), a mixture of RNase A and RNase T1 (A/T1), RNase 1f (1f), and RNase V1 (V1),

    Article Snippet: Enzymes and reagents RNase T1 (AM2280), RNase V1 (AM2275), RNase A/T1 cocktail (EN0551), DNase I (2222), and Yeast t-RNA (15401-011) were from Thermo Fisher Scientific.

    Techniques: SDS Page, Incubation

    Representative probing experiments using 5′-endlabeled aptamer A011 RNA and analyzed by ( A ) 20% or ( B ) 10% denaturing PAGE. Lanes 1 and 13, undigested RNA control (con.); lanes 2 and 14, limited alkaline hydrolysis; lanes 3, 12, 15 and 24, partial digestion with RNase T1 under denaturing (denat.) conditions; lanes 4, 5, 16 and 17, with two concentrations of RNase T1 under native conditions; lanes 6, 7, 18 and 19, with two concentrations of RNase V1; lanes 8, 9, 20 and 21, with two concentrations of nuclease P1; lane 10 and 22, with nuclease S1; lanes 11 and 23, Pb2+-induced hydrolysis. For experimental details, see Materials and Methods. Alkaline hydrolysis bands and the corresponding structure elements are indicated at the left and right margins, respectively, according to the numbering system presented in Figure 2 (A011, center). ( C , D ) Illustration of prominent cleavage sites in the context of the secondary structure of A011, derived from probing experiments such as those shown in panels A and B . Symbol sizes suggests the relative intensity of cleavage bands based on visual inspection.

    Journal: International Journal of Molecular Sciences

    Article Title: 2′-Fluoro-Pyrimidine-Modified RNA Aptamers Specific for Lipopolysaccharide Binding Protein (LBP)

    doi: 10.3390/ijms19123883

    Figure Lengend Snippet: Representative probing experiments using 5′-endlabeled aptamer A011 RNA and analyzed by ( A ) 20% or ( B ) 10% denaturing PAGE. Lanes 1 and 13, undigested RNA control (con.); lanes 2 and 14, limited alkaline hydrolysis; lanes 3, 12, 15 and 24, partial digestion with RNase T1 under denaturing (denat.) conditions; lanes 4, 5, 16 and 17, with two concentrations of RNase T1 under native conditions; lanes 6, 7, 18 and 19, with two concentrations of RNase V1; lanes 8, 9, 20 and 21, with two concentrations of nuclease P1; lane 10 and 22, with nuclease S1; lanes 11 and 23, Pb2+-induced hydrolysis. For experimental details, see Materials and Methods. Alkaline hydrolysis bands and the corresponding structure elements are indicated at the left and right margins, respectively, according to the numbering system presented in Figure 2 (A011, center). ( C , D ) Illustration of prominent cleavage sites in the context of the secondary structure of A011, derived from probing experiments such as those shown in panels A and B . Symbol sizes suggests the relative intensity of cleavage bands based on visual inspection.

    Article Snippet: Enzymatic digestions were performed as follows: RNase T1 (Thermo Fisher Scientific, Waltham, MA, USA) either in 20 mM sodium citrat (pH 5.0), 7 mM urea, 1 mM EDTA and 1 unit RNase T1 for 30 min at 55 °C (denaturing conditions) or in binding buffer (see above) with 0.5 or 0.05 units for 20 min at room temperature (RT); 0.01 or 0.05 units RNase V1 (Thermo Fisher Scientific Pierce) in binding buffer for 20 min at RT; 1 unit RNase S1 (Thermo Fisher Scientific, Waltham, MA, USA) in binding buffer containing 4.5 mM ZnSO4 for 20 min at RT; 0.01 or 0.05 units nuclease P1 (Sigma-Aldrich, St. Louis, MO, USA) in binding buffer containing 0.4 mM ZnSO4 for 20 min at RT.

    Techniques: Polyacrylamide Gel Electrophoresis, Derivative Assay