dna template  (New England Biolabs)


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    Name:
    Proteinase K Molecular Biology Grade
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    Proteinase K Molecular Biology Grade 2 ml
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    p8107s
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    Proteases
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    New England Biolabs dna template
    Proteinase K Molecular Biology Grade
    Proteinase K Molecular Biology Grade 2 ml
    https://www.bioz.com/result/dna template/product/New England Biolabs
    Average 93 stars, based on 18016 article reviews
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    dna template - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "Autoregulation of the Streptococcus mutans SloR Metalloregulator Is Constitutive and Driven by an Independent Promoter"

    Article Title: Autoregulation of the Streptococcus mutans SloR Metalloregulator Is Constitutive and Driven by an Independent Promoter

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00214-18

    The S. mutans sloR gene is transcribed even in the absence of a functional sloABC promoter. (a) Map of the sloABCR operon and locations of primer annealing sites. Primer P1 ( sloA .RT_PCR.F) anneals within the sloA coding sequence, and primers P2 and P3 ( sloR .RT_PCR.F and sloR .RT_PCR.R, respectively) anneal within the sloR coding sequence. (b) Products of reverse transcriptase PCR resolved in a 0.8% agarose gel. Amplification of cDNA with the P1/P3 primer pair generated a 2,745-bp amplicon for UA159 but not for GMS611 or GMS611d, consistent with disruption of the sloABC promoter in the mutant strains. In contrast, cDNA amplification with the P2/P3 primer pair gave rise to a 250-bp amplicon even for the sloABC promoter mutants GMS611 and GMS611d, indicating the presence of a sloR -specific promoter in the 184-bp intergenic region that separates the sloABC operon from the downstream sloR gene. gDNA, genomic DNA; P/O, promoter/operator.
    Figure Legend Snippet: The S. mutans sloR gene is transcribed even in the absence of a functional sloABC promoter. (a) Map of the sloABCR operon and locations of primer annealing sites. Primer P1 ( sloA .RT_PCR.F) anneals within the sloA coding sequence, and primers P2 and P3 ( sloR .RT_PCR.F and sloR .RT_PCR.R, respectively) anneal within the sloR coding sequence. (b) Products of reverse transcriptase PCR resolved in a 0.8% agarose gel. Amplification of cDNA with the P1/P3 primer pair generated a 2,745-bp amplicon for UA159 but not for GMS611 or GMS611d, consistent with disruption of the sloABC promoter in the mutant strains. In contrast, cDNA amplification with the P2/P3 primer pair gave rise to a 250-bp amplicon even for the sloABC promoter mutants GMS611 and GMS611d, indicating the presence of a sloR -specific promoter in the 184-bp intergenic region that separates the sloABC operon from the downstream sloR gene. gDNA, genomic DNA; P/O, promoter/operator.

    Techniques Used: Functional Assay, Reverse Transcription Polymerase Chain Reaction, Sequencing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Generated, Mutagenesis

    2) Product Images from "In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation"

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx049

    Schematic representation of yeast  in vivo  RNA–protein Ni 2+ -pull down (RaP-NiP) assay using formaldehyde crosslinking. The basic scheme of the RaP-NiP is described in the form of a flowchart. Green and red balls represent 40S ribosomes and eIF3 complexes, respectively, grey balls stand for the Ni 2+  beads, and purple and blue balls depict some non-specific RNA binding proteins. Exponentially growing yeast cells were crosslinked with 1% formaldehyde. Crosslinking was stopped by adding glycine and the fixed cells were lysed using glass beads by rigorous vortexing. Pre-cleared whole cell extract (WCE) containing RaP-NiP mRNAs in protein-RNA complexes were selectively digested with RNase H using sequence specific custom-made oligos. The resulting specific mRNA segments were purified with the help of the His-tagged a/TIF32 subunit of yeast eIF3 or its mutant variants using the Ni-NTA sepharose beads. Thus isolated protein-RNA complexes were subsequently treated with Proteinase K, and the captured RNAs were further purified by hot phenol extraction, reverse transcribed and their amounts were then quantified by qRT-PCR. The schematic boxed on the right-hand side illustrates typical amounts of RNAse H digested RNA segments of REI-permissive uORF1 and REI-non-permissive uORF4 from the  GCN4  mRNA leader co-purifying with eIF3, the typical ratio of which is ∼4:1.
    Figure Legend Snippet: Schematic representation of yeast in vivo RNA–protein Ni 2+ -pull down (RaP-NiP) assay using formaldehyde crosslinking. The basic scheme of the RaP-NiP is described in the form of a flowchart. Green and red balls represent 40S ribosomes and eIF3 complexes, respectively, grey balls stand for the Ni 2+ beads, and purple and blue balls depict some non-specific RNA binding proteins. Exponentially growing yeast cells were crosslinked with 1% formaldehyde. Crosslinking was stopped by adding glycine and the fixed cells were lysed using glass beads by rigorous vortexing. Pre-cleared whole cell extract (WCE) containing RaP-NiP mRNAs in protein-RNA complexes were selectively digested with RNase H using sequence specific custom-made oligos. The resulting specific mRNA segments were purified with the help of the His-tagged a/TIF32 subunit of yeast eIF3 or its mutant variants using the Ni-NTA sepharose beads. Thus isolated protein-RNA complexes were subsequently treated with Proteinase K, and the captured RNAs were further purified by hot phenol extraction, reverse transcribed and their amounts were then quantified by qRT-PCR. The schematic boxed on the right-hand side illustrates typical amounts of RNAse H digested RNA segments of REI-permissive uORF1 and REI-non-permissive uORF4 from the GCN4 mRNA leader co-purifying with eIF3, the typical ratio of which is ∼4:1.

    Techniques Used: In Vivo, RNA Binding Assay, Sequencing, Purification, Mutagenesis, Isolation, Quantitative RT-PCR

    3) Product Images from "The CRISPR-associated Csx1 protein of Pyrococcus furiosus is an adenosine-specific endoribonuclease"

    Article Title: The CRISPR-associated Csx1 protein of Pyrococcus furiosus is an adenosine-specific endoribonuclease

    Journal: RNA

    doi: 10.1261/rna.039842.113

    Csx1 cleaves ssRNA after adenosines. ( A ) A variety of ssRNAs were treated with no protein (−) or Csx1 for the indicated times, and run alongside 5′-radiolabeled RNA markers (M), RNase T1 ladders (T1), and alkaline hydrolysis ladders (OH).
    Figure Legend Snippet: Csx1 cleaves ssRNA after adenosines. ( A ) A variety of ssRNAs were treated with no protein (−) or Csx1 for the indicated times, and run alongside 5′-radiolabeled RNA markers (M), RNase T1 ladders (T1), and alkaline hydrolysis ladders (OH).

    Techniques Used:

    Csx1 is a temperature-dependent, single-strand-specific ribonuclease. ( A ) Csx1 was tested for nuclease activity (+) on ­ 32 P-labeled single-stranded and double-stranded RNA and DNA (37mer A, 63mer A, 37mer A + B, and 63mer A + B, respectively),
    Figure Legend Snippet: Csx1 is a temperature-dependent, single-strand-specific ribonuclease. ( A ) Csx1 was tested for nuclease activity (+) on ­ 32 P-labeled single-stranded and double-stranded RNA and DNA (37mer A, 63mer A, 37mer A + B, and 63mer A + B, respectively),

    Techniques Used: Activity Assay, Labeling

    4) Product Images from "Allele-Selective Inhibition of Mutant Huntingtin Expression with Antisense Oligonucleotides Targeting the Expanded CAG Repeat"

    Article Title: Allele-Selective Inhibition of Mutant Huntingtin Expression with Antisense Oligonucleotides Targeting the Expanded CAG Repeat

    Journal: Biochemistry

    doi: 10.1021/bi101208k

    Multiple ASOs bind CAG repeat-containing HTT 5′-end transcripts in vitro ( A–B ) Electrophoretic mobility shift assays (EMSA) demonstrate cooperative and near stoichiometric binding of multiple ASOs to CAG repeat sequence in HTT mRNA 5′ end transcripts, as well as a case where no binding is observed. In vitro transcribed and 5′-radiolabeled wild-type and mutant 5′-end HTT mRNA transcripts were incubated with increasing concentrations of ASO. RNA:ASO complexes were resolved on native polyacrylamide gels and visualized by phosphorimager. ASO concentrations are indicated above the gels and shifted bands identified to the right. ( C ) Quantification of LNA(T) binding REP17 and REP69 HTT mRNA transcripts indicates cooperative ASO binding. ASO-bound RNA in gel shifts from panels A and B were quantified and plotted as a function of concentration. Fitting to the Hill equation revealed a sigmoidal binding curve and Hill coefficients (n) near 2, suggesting cooperative ASO binding. ( D ) Putative steric translational blocking mechanism. Translation is repressed on a mutant expanded CAG repeat HTT mRNA due to a proposed steric inhibition of translation by cumulative ASO binding.
    Figure Legend Snippet: Multiple ASOs bind CAG repeat-containing HTT 5′-end transcripts in vitro ( A–B ) Electrophoretic mobility shift assays (EMSA) demonstrate cooperative and near stoichiometric binding of multiple ASOs to CAG repeat sequence in HTT mRNA 5′ end transcripts, as well as a case where no binding is observed. In vitro transcribed and 5′-radiolabeled wild-type and mutant 5′-end HTT mRNA transcripts were incubated with increasing concentrations of ASO. RNA:ASO complexes were resolved on native polyacrylamide gels and visualized by phosphorimager. ASO concentrations are indicated above the gels and shifted bands identified to the right. ( C ) Quantification of LNA(T) binding REP17 and REP69 HTT mRNA transcripts indicates cooperative ASO binding. ASO-bound RNA in gel shifts from panels A and B were quantified and plotted as a function of concentration. Fitting to the Hill equation revealed a sigmoidal binding curve and Hill coefficients (n) near 2, suggesting cooperative ASO binding. ( D ) Putative steric translational blocking mechanism. Translation is repressed on a mutant expanded CAG repeat HTT mRNA due to a proposed steric inhibition of translation by cumulative ASO binding.

    Techniques Used: In Vitro, Electrophoretic Mobility Shift Assay, Binding Assay, Sequencing, Mutagenesis, Incubation, Allele-specific Oligonucleotide, Concentration Assay, Blocking Assay, Inhibition

    5) Product Images from "Allele-Selective Inhibition of Mutant Huntingtin Expression with Antisense Oligonucleotides Targeting the Expanded CAG Repeat"

    Article Title: Allele-Selective Inhibition of Mutant Huntingtin Expression with Antisense Oligonucleotides Targeting the Expanded CAG Repeat

    Journal: Biochemistry

    doi: 10.1021/bi101208k

    Multiple ASOs bind CAG repeat-containing HTT 5′-end transcripts in vitro ( A–B ) Electrophoretic mobility shift assays (EMSA) demonstrate cooperative and near stoichiometric binding of multiple ASOs to CAG repeat sequence in HTT mRNA 5′ end transcripts, as well as a case where no binding is observed. In vitro transcribed and 5′-radiolabeled wild-type and mutant 5′-end HTT mRNA transcripts were incubated with increasing concentrations of ASO. RNA:ASO complexes were resolved on native polyacrylamide gels and visualized by phosphorimager. ASO concentrations are indicated above the gels and shifted bands identified to the right. ( C ) Quantification of LNA(T) binding REP17 and REP69 HTT mRNA transcripts indicates cooperative ASO binding. ASO-bound RNA in gel shifts from panels A and B were quantified and plotted as a function of concentration. Fitting to the Hill equation revealed a sigmoidal binding curve and Hill coefficients (n) near 2, suggesting cooperative ASO binding. ( D ) Putative steric translational blocking mechanism. Translation is repressed on a mutant expanded CAG repeat HTT mRNA due to a proposed steric inhibition of translation by cumulative ASO binding.
    Figure Legend Snippet: Multiple ASOs bind CAG repeat-containing HTT 5′-end transcripts in vitro ( A–B ) Electrophoretic mobility shift assays (EMSA) demonstrate cooperative and near stoichiometric binding of multiple ASOs to CAG repeat sequence in HTT mRNA 5′ end transcripts, as well as a case where no binding is observed. In vitro transcribed and 5′-radiolabeled wild-type and mutant 5′-end HTT mRNA transcripts were incubated with increasing concentrations of ASO. RNA:ASO complexes were resolved on native polyacrylamide gels and visualized by phosphorimager. ASO concentrations are indicated above the gels and shifted bands identified to the right. ( C ) Quantification of LNA(T) binding REP17 and REP69 HTT mRNA transcripts indicates cooperative ASO binding. ASO-bound RNA in gel shifts from panels A and B were quantified and plotted as a function of concentration. Fitting to the Hill equation revealed a sigmoidal binding curve and Hill coefficients (n) near 2, suggesting cooperative ASO binding. ( D ) Putative steric translational blocking mechanism. Translation is repressed on a mutant expanded CAG repeat HTT mRNA due to a proposed steric inhibition of translation by cumulative ASO binding.

    Techniques Used: In Vitro, Electrophoretic Mobility Shift Assay, Binding Assay, Sequencing, Mutagenesis, Incubation, Allele-specific Oligonucleotide, Concentration Assay, Blocking Assay, Inhibition

    6) Product Images from "A stress response that monitors and regulates mRNA structure is central to cold-shock adaptation"

    Article Title: A stress response that monitors and regulates mRNA structure is central to cold-shock adaptation

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2018.02.035

    The 5’UTR of cspA changes in mRNA structure during acclimation (A) The normalized in vivo DMS-seq signal of A/C bases within cspA 5’UTR in WT cells at 37°C (top), 30 min (middle) or 6 hr (bottom) after cold shock. DMS-seq signals were normalized to the maximum signal within cspA message after removing outliers by 98% Winsorisation (see Methods). The red dashed line represents the signal cutoff (0.24), above which the A/C bases are predicted to be unpaired. The region highlighted in red has long-range interactions with the “cold box” element at 10°C. (B-C) The predicted structure of the cspA 5’ UTR at (B) 37°C or (C) 30 min after cold shock. Structure predictions were generated by constraining a minimum free-energy prediction with in vivo DMS-seq data. The start codon of cspA (green), the conserved “cold box” element (blue) and its long-range interaction region at 10°C (red) are highlighted. (D) Scatter plot comparing Gini indices of ORFs (N = 391) and 5’UTR of cspA , cspB , cspG (red dots) at 30 min vs 6 hr after cold shock. Grey dashed line: Y = X. (E) The predicted structure of the cspA .
    Figure Legend Snippet: The 5’UTR of cspA changes in mRNA structure during acclimation (A) The normalized in vivo DMS-seq signal of A/C bases within cspA 5’UTR in WT cells at 37°C (top), 30 min (middle) or 6 hr (bottom) after cold shock. DMS-seq signals were normalized to the maximum signal within cspA message after removing outliers by 98% Winsorisation (see Methods). The red dashed line represents the signal cutoff (0.24), above which the A/C bases are predicted to be unpaired. The region highlighted in red has long-range interactions with the “cold box” element at 10°C. (B-C) The predicted structure of the cspA 5’ UTR at (B) 37°C or (C) 30 min after cold shock. Structure predictions were generated by constraining a minimum free-energy prediction with in vivo DMS-seq data. The start codon of cspA (green), the conserved “cold box” element (blue) and its long-range interaction region at 10°C (red) are highlighted. (D) Scatter plot comparing Gini indices of ORFs (N = 391) and 5’UTR of cspA , cspB , cspG (red dots) at 30 min vs 6 hr after cold shock. Grey dashed line: Y = X. (E) The predicted structure of the cspA .

    Techniques Used: In Vivo, Generated

    7) Product Images from "In vivo probing of nascent RNA structures reveals principles of cotranscriptional folding"

    Article Title: In vivo probing of nascent RNA structures reveals principles of cotranscriptional folding

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx617

    SPET-seq captures RNA folding intermediates in vivo . ( A ) Outline of in vivo SPET-seq applied to Escherichia coli . ( B ) Distribution of read mappings for in vivo SPET-seq on total and ribo-depleted nascent RNA. ( C ) Heatmap of per-base DMS reactivity across transcription intermediates in the 5′-terminal domain of 16S rRNA (rrsB).
    Figure Legend Snippet: SPET-seq captures RNA folding intermediates in vivo . ( A ) Outline of in vivo SPET-seq applied to Escherichia coli . ( B ) Distribution of read mappings for in vivo SPET-seq on total and ribo-depleted nascent RNA. ( C ) Heatmap of per-base DMS reactivity across transcription intermediates in the 5′-terminal domain of 16S rRNA (rrsB).

    Techniques Used: Single Photon Emission Computed Tomography, In Vivo

    8) Product Images from "NDF, a nucleosome-destabilizing factor that facilitates transcription through nucleosomes"

    Article Title: NDF, a nucleosome-destabilizing factor that facilitates transcription through nucleosomes

    Journal: Genes & Development

    doi: 10.1101/gad.313973.118

    NDF facilitates transcription elongation through a nucleosome. ( A ) Schematic diagram of Pol II transcription through a positioned nucleosome. A transcriptionally engaged elongation complex (Pol II EC) was assembled with purified yeast Pol II and a 5′ end-labeled 10-nucleotide (nt) RNA primer. This complex was attached to streptavidin beads and then ligated to a downstream mononucleosome positioned on the 5S rDNA sequence from Xenopus borealis . Transcription elongation was initiated by the addition of ribonucleoside 5′-triphosphates (rNTPs). Where indicated, NDF was added after the ligation step but before the addition of the rNTPs. In parallel experiments, naked DNA (same 5S rDNA sequence) was ligated downstream from the elongation complex instead of a mononucleosome. The distance (∼15 nt) from the leading edge of Pol II to the 3′ end of the transcript is also shown. ( B ) NDF reduces the inhibition of Pol II elongation by a nucleosome. Transcription elongation reactions were performed as described in A in the presence or absence of purified hNDF for the indicated times. Experiments with nucleosomal templates included a 75-fold molar excess (0.3 µM) of free unligated mononucleosomes, which stabilize the low concentration of immobilized nucleosomes. Where indicated, hNDF was included at a concentration of 1.5 µM. The reaction products were resolved by 8% polyacrylamide–urea gel electrophoresis. The diagram shows the locations of the positioned nucleosomes, the sites of Pol II pausing, the runoff product, the ligation junction, and the 10-nt primer RNA. The sizes of the RNA species were estimated by comparison with a 25-nt radiolabeled DNA ladder.
    Figure Legend Snippet: NDF facilitates transcription elongation through a nucleosome. ( A ) Schematic diagram of Pol II transcription through a positioned nucleosome. A transcriptionally engaged elongation complex (Pol II EC) was assembled with purified yeast Pol II and a 5′ end-labeled 10-nucleotide (nt) RNA primer. This complex was attached to streptavidin beads and then ligated to a downstream mononucleosome positioned on the 5S rDNA sequence from Xenopus borealis . Transcription elongation was initiated by the addition of ribonucleoside 5′-triphosphates (rNTPs). Where indicated, NDF was added after the ligation step but before the addition of the rNTPs. In parallel experiments, naked DNA (same 5S rDNA sequence) was ligated downstream from the elongation complex instead of a mononucleosome. The distance (∼15 nt) from the leading edge of Pol II to the 3′ end of the transcript is also shown. ( B ) NDF reduces the inhibition of Pol II elongation by a nucleosome. Transcription elongation reactions were performed as described in A in the presence or absence of purified hNDF for the indicated times. Experiments with nucleosomal templates included a 75-fold molar excess (0.3 µM) of free unligated mononucleosomes, which stabilize the low concentration of immobilized nucleosomes. Where indicated, hNDF was included at a concentration of 1.5 µM. The reaction products were resolved by 8% polyacrylamide–urea gel electrophoresis. The diagram shows the locations of the positioned nucleosomes, the sites of Pol II pausing, the runoff product, the ligation junction, and the 10-nt primer RNA. The sizes of the RNA species were estimated by comparison with a 25-nt radiolabeled DNA ladder.

    Techniques Used: Purification, Labeling, Sequencing, Ligation, Inhibition, Concentration Assay, Nucleic Acid Electrophoresis

    9) Product Images from "DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs"

    Article Title: DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs

    Journal: Genes & Development

    doi: 10.1101/gad.282616.116

    DUSP11 directly dephosphorylates the BLV pre-miRNAs and 5p miRNAs. ( A ) Immunoblot analysis to confirm expression of DUSP11 and DUSP11 catalytic mutant proteins generated using in vitro transcription/translation. The membrane was probed using anti-DUSP11 and anti-tubulin antibodies. ( B ) In vitro phosphatase reactions on the [γ-32P]-BLV-pre-miR-B5 mimic and [γ-32P]-BLV-miR-B5-5p miRNA mimic using CIP (positive control) or the in vitro translated DUSP11, DUSP11 catalytic mutant, or luciferase (negative control) from A . Reactions were fractionated on 15% PAGE/8 M urea, and RNAs were stained with EtBr. RNAs were then transferred to a membrane, exposed to a storage phosphor screen, and imaged on a Typhoon bimolecular imager. ( C ) Northern blot analysis from wild-type and DUSP11 knockout HEK293T cells transfected with a 5′ triphosphorylated BLV-B5 pre-miRNA mimic pretreated with (+) or without (−) RNA 5′ polyphosphatase. The blot was first probed for the 5p miRNA arm (green), stripped, and reprobed for the 3p arm (orange). Note that a lighter exposure for the input RNA is shown as compared with the RNA recovered from cells.
    Figure Legend Snippet: DUSP11 directly dephosphorylates the BLV pre-miRNAs and 5p miRNAs. ( A ) Immunoblot analysis to confirm expression of DUSP11 and DUSP11 catalytic mutant proteins generated using in vitro transcription/translation. The membrane was probed using anti-DUSP11 and anti-tubulin antibodies. ( B ) In vitro phosphatase reactions on the [γ-32P]-BLV-pre-miR-B5 mimic and [γ-32P]-BLV-miR-B5-5p miRNA mimic using CIP (positive control) or the in vitro translated DUSP11, DUSP11 catalytic mutant, or luciferase (negative control) from A . Reactions were fractionated on 15% PAGE/8 M urea, and RNAs were stained with EtBr. RNAs were then transferred to a membrane, exposed to a storage phosphor screen, and imaged on a Typhoon bimolecular imager. ( C ) Northern blot analysis from wild-type and DUSP11 knockout HEK293T cells transfected with a 5′ triphosphorylated BLV-B5 pre-miRNA mimic pretreated with (+) or without (−) RNA 5′ polyphosphatase. The blot was first probed for the 5p miRNA arm (green), stripped, and reprobed for the 3p arm (orange). Note that a lighter exposure for the input RNA is shown as compared with the RNA recovered from cells.

    Techniques Used: Expressing, Mutagenesis, Generated, In Vitro, Positive Control, Luciferase, Negative Control, Polyacrylamide Gel Electrophoresis, Staining, Northern Blot, Knock-Out, Transfection

    10) Product Images from "Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor"

    Article Title: Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor

    Journal: Nature

    doi: 10.1038/nature17978

    Purification of SBP-tagged eIF4A and co-purified RNA from HEK 293 cells (a) Western blot of exogenous SBP-eIF4A and endogenous eIF4A in tetracycline-inducible stable cell line. Expression of physiological levels of the tagged allele attenuated endogenous eIF4A expression but preserved overall eIF4A levels, likely reflecting the same feedback loop previously reported between eIF4AI and eIF4AII 31 . (b) CBB staining of purified SBP-eIF4A and SYBR Gold staining of purified RNA bound to SBP-eIF4A with or without Micrococcal Nuclease (MNase). (c) Correlation of sum of the mRNA fragment reads of each transcript between biological replicates of RIP-seq. r is Pearson’s correlation coefficient. P value is calculated by Student′s t-test. (d) Histogram of the number of transcripts along RNA/eIF4A interaction -fold change by RIP-Seq when cells are treated with 0.03 or 0.3 µM RocA normalized to spiked-in RNA. Data present the same mRNAs analyzed in Figure 1a . Median -fold change is shown. Bin width is 0.1. (e) Correlation of RIP -fold change between different concentration of RocA treatments. ρ: Spearman’s rank correlation coefficient. (f) Correlation of translation -fold change to RIP -fold change with the same concentration of RocA treatment. ρ: Spearman’s rank correlation.
    Figure Legend Snippet: Purification of SBP-tagged eIF4A and co-purified RNA from HEK 293 cells (a) Western blot of exogenous SBP-eIF4A and endogenous eIF4A in tetracycline-inducible stable cell line. Expression of physiological levels of the tagged allele attenuated endogenous eIF4A expression but preserved overall eIF4A levels, likely reflecting the same feedback loop previously reported between eIF4AI and eIF4AII 31 . (b) CBB staining of purified SBP-eIF4A and SYBR Gold staining of purified RNA bound to SBP-eIF4A with or without Micrococcal Nuclease (MNase). (c) Correlation of sum of the mRNA fragment reads of each transcript between biological replicates of RIP-seq. r is Pearson’s correlation coefficient. P value is calculated by Student′s t-test. (d) Histogram of the number of transcripts along RNA/eIF4A interaction -fold change by RIP-Seq when cells are treated with 0.03 or 0.3 µM RocA normalized to spiked-in RNA. Data present the same mRNAs analyzed in Figure 1a . Median -fold change is shown. Bin width is 0.1. (e) Correlation of RIP -fold change between different concentration of RocA treatments. ρ: Spearman’s rank correlation coefficient. (f) Correlation of translation -fold change to RIP -fold change with the same concentration of RocA treatment. ρ: Spearman’s rank correlation.

    Techniques Used: Purification, Western Blot, Stable Transfection, Expressing, Staining, Concentration Assay

    eIF4A/RocA complexes on polypurine motifs block scanning of pre-initiation complex, inducing uORF translation (a) Pre-formation of the complex with RocA and eIF4A on the mRNA bearing seven polypurine motifs represses the translation from the mRNA in RRL. (b) The supplementation of recombinant eIF4A protein to RRL in vitro transaltion reaction with 10 μM Hipp or 3 μM RocA. (c) In vitro translation in RRL with mRNAs with native PV IRES and that with three polypurine motifs ( Extended Data Figure 9a ). (d) Meta-gene analysis of high-sensitivity transcripts to RocA. Reads are normalized to the sum of mitochondrial footprints reads. Histogram of the position of the first polypurine motif (6-mer) after uORF initiation codon (inset). P value is calculated by Fisher’s exact test. Bin width is 12 nt. (e) Western blot of SBP translated from uORF and downstream major ORF in RRL with 0.03 μM RocA treatment. Quantification of bands normalized to long form with DMSO treatment is shown. For gel source data, see Supplementary Fig. 1 . (f) Schematic representation of RocA-mediated translation control. RocA clamps eIF4A onto mRNA by selective affinity enhancement for a polypurine motif in eIF4F-, cap-, and ATP-independent manners, which then blocks scanning of pre-initiation complex, introducing premature translation from uORF and inhibiting downstream ORF translation. In b and c, data represent mean and S.D. (n = 3).
    Figure Legend Snippet: eIF4A/RocA complexes on polypurine motifs block scanning of pre-initiation complex, inducing uORF translation (a) Pre-formation of the complex with RocA and eIF4A on the mRNA bearing seven polypurine motifs represses the translation from the mRNA in RRL. (b) The supplementation of recombinant eIF4A protein to RRL in vitro transaltion reaction with 10 μM Hipp or 3 μM RocA. (c) In vitro translation in RRL with mRNAs with native PV IRES and that with three polypurine motifs ( Extended Data Figure 9a ). (d) Meta-gene analysis of high-sensitivity transcripts to RocA. Reads are normalized to the sum of mitochondrial footprints reads. Histogram of the position of the first polypurine motif (6-mer) after uORF initiation codon (inset). P value is calculated by Fisher’s exact test. Bin width is 12 nt. (e) Western blot of SBP translated from uORF and downstream major ORF in RRL with 0.03 μM RocA treatment. Quantification of bands normalized to long form with DMSO treatment is shown. For gel source data, see Supplementary Fig. 1 . (f) Schematic representation of RocA-mediated translation control. RocA clamps eIF4A onto mRNA by selective affinity enhancement for a polypurine motif in eIF4F-, cap-, and ATP-independent manners, which then blocks scanning of pre-initiation complex, introducing premature translation from uORF and inhibiting downstream ORF translation. In b and c, data represent mean and S.D. (n = 3).

    Techniques Used: Blocking Assay, Recombinant, In Vitro, Western Blot

    Characterization of iCLIP data (a) CBB staining of purified SBP-eIF4A protein in iCLIP procedure. (b) Visualization of RNA-crosslinked with SBP-eIF4A and unknown proteins by 32 P labeling of RNA. We avoided the contamination of RNAs cross-linked to the additional, co-purifying, unknown proteins. (c) Distribution of read length in iCLIP libraries. Avoidance of contaminating RNAs restricted us to short RNAs, which likely correspond to the region of RNA physically protected by eIF4A binding, or footprint (d) Nucleotide bias along the reads in iCLIP libraries. The crosslinking bias for U may underestimate the preference for polypurine motifs. (e) Correlations of iCLIP motif enrichment (4-mer) by different RocA concentrations. (f) Correlations of iCLIP motif enrichment (4-mer) by 3 μM RocA and motif prediction of 0.03 μM RocA effect in RIP-Seq. The motifs shown in Figure 3b are highlighted. ρ: Spearman’s rank correlation.
    Figure Legend Snippet: Characterization of iCLIP data (a) CBB staining of purified SBP-eIF4A protein in iCLIP procedure. (b) Visualization of RNA-crosslinked with SBP-eIF4A and unknown proteins by 32 P labeling of RNA. We avoided the contamination of RNAs cross-linked to the additional, co-purifying, unknown proteins. (c) Distribution of read length in iCLIP libraries. Avoidance of contaminating RNAs restricted us to short RNAs, which likely correspond to the region of RNA physically protected by eIF4A binding, or footprint (d) Nucleotide bias along the reads in iCLIP libraries. The crosslinking bias for U may underestimate the preference for polypurine motifs. (e) Correlations of iCLIP motif enrichment (4-mer) by different RocA concentrations. (f) Correlations of iCLIP motif enrichment (4-mer) by 3 μM RocA and motif prediction of 0.03 μM RocA effect in RIP-Seq. The motifs shown in Figure 3b are highlighted. ρ: Spearman’s rank correlation.

    Techniques Used: Staining, Purification, Labeling, Binding Assay

    11) Product Images from "Isolation and genome-wide characterization of cellular DNA:RNA triplex structures"

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1305

    NEAT1 forms triplexes at numerous genomic sites. ( A ) NEAT1 profiles in TriplexRNA-seq (DNA-IP) (red) and nuclear RNA (blue) from HeLa S3 and U2OS cells with shaded TFR1 and TFR2. Minus (-) and plus (+) strands are shown. The position and sequence of NEAT1-TFR1 and -TFR2 are shown below. ( B ) EMSAs using 10 or 100 pmol of synthetic NEAT1 versions comprising TFR1 (40 or 52 nt) or TFR2 incubated with 0.25 pmol of double–stranded  32 P-labeled oligonucleotides which harbor sequences of NEAT1 target genes predicted from CHART-seq (  Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control, RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( C ) Schematic depiction of the TFR-based capture assay. Biotinylated RNA oligos covering NEAT1-TFR1 and NEAT1-TFR2 were used to capture genomic DNA. ( D ) MEME motif analysis identifying consensus motifs in DNA captured by NEAT1-TFR1 (399 of top 500 peaks) and by NEAT1-TFR2 (500 of top 500 peaks ranked by peak  P -value). Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( E ) TDF analysis of the triplex-forming potential of NEAT1-TFR1 and NEAT1-TFR2 RNAs with top 500 TFR-associated and control DNA peaks (ranked by peak  P -value) compared to 500 randomized regions ( N  = 1000, colored grey).  P -values were obtained from one-tailed Mann–Whitney test. ( F ) Scheme presenting antisense oligo (ASO)-based capture of NEAT1-associated DNA. ( G ) Consensus motif in NEAT1-associated DNA sites (314 of top 500 peaks ranked by peak  P -value). ( H ) TDF analysis predicting the triplex-forming potential of NEAT1 on ASO-captured DNA regions. Significant TFRs along NEAT1 are shown in orange, the number of target sites (DBS) for each TFR in purple. For TFR- and ASO-based capture assays nucleic acids isolated from HeLa S3 chromatin were used.
    Figure Legend Snippet: NEAT1 forms triplexes at numerous genomic sites. ( A ) NEAT1 profiles in TriplexRNA-seq (DNA-IP) (red) and nuclear RNA (blue) from HeLa S3 and U2OS cells with shaded TFR1 and TFR2. Minus (-) and plus (+) strands are shown. The position and sequence of NEAT1-TFR1 and -TFR2 are shown below. ( B ) EMSAs using 10 or 100 pmol of synthetic NEAT1 versions comprising TFR1 (40 or 52 nt) or TFR2 incubated with 0.25 pmol of double–stranded 32 P-labeled oligonucleotides which harbor sequences of NEAT1 target genes predicted from CHART-seq ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control, RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( C ) Schematic depiction of the TFR-based capture assay. Biotinylated RNA oligos covering NEAT1-TFR1 and NEAT1-TFR2 were used to capture genomic DNA. ( D ) MEME motif analysis identifying consensus motifs in DNA captured by NEAT1-TFR1 (399 of top 500 peaks) and by NEAT1-TFR2 (500 of top 500 peaks ranked by peak P -value). Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( E ) TDF analysis of the triplex-forming potential of NEAT1-TFR1 and NEAT1-TFR2 RNAs with top 500 TFR-associated and control DNA peaks (ranked by peak P -value) compared to 500 randomized regions ( N = 1000, colored grey). P -values were obtained from one-tailed Mann–Whitney test. ( F ) Scheme presenting antisense oligo (ASO)-based capture of NEAT1-associated DNA. ( G ) Consensus motif in NEAT1-associated DNA sites (314 of top 500 peaks ranked by peak P -value). ( H ) TDF analysis predicting the triplex-forming potential of NEAT1 on ASO-captured DNA regions. Significant TFRs along NEAT1 are shown in orange, the number of target sites (DBS) for each TFR in purple. For TFR- and ASO-based capture assays nucleic acids isolated from HeLa S3 chromatin were used.

    Techniques Used: Sequencing, Incubation, Labeling, One-tailed Test, MANN-WHITNEY, Allele-specific Oligonucleotide, Isolation

    Validation of triplex-forming RNA and DNAs. ( A ) TDF analysis predicting the potential of top 1000 enriched TriplexRNA (DNA-IP) regions (ranked by peak  P -value) to bind to active promoters defined by ChromHMM. Number of TFRs in RNA (per kilobase of RNA, left) and the number of putative DBSs at promoters (per kilobase of RNA, right) are shown. Boxplot borders are defined by the 1st and 3rd quantiles of the distributions, the middle line corresponds to the median value. The top whisker denotes the maximum value within the third quartile plus 1.5 times the interquartile range (bottom whisker is defined analogously). Dark gray dots represent outliers with values higher or lower than whiskers. Further box plots are based on the same definitions. ( B ) Motif analysis of triplexes formed between TriplexRNA (DNA-IP) and active promoters. The diagram depicts the fraction of antiparallel and parallel triplexes with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( C ) TDF analysis comparing the triplex-forming potential of top 2000 TriplexDNA-seq regions with top 1000 TriplexRNA (DNA-IP) (ranked by peak  P -value). The number of putative DBSs (per kilobase of RNA) is shown. ( D ) Motif analysis of predicted triplexes formed between TriplexRNAs (DNA-IP) and TriplexDNA. The diagram depicts the fraction of antiparallel and parallel triplexes, with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( E ) Box plot classifying triplex interactions between TriplexRNAs (DNA-IP) and TriplexDNA-seq regions as  cis  ( > 10 kb in the same chromosome) and  trans  (at different chromosomes) interactions, excluding underrepresented local interactions (within 10 kb distance). ( F ) EMSAs using 10 or 100 pmol of synthetic TriplexRNAs and 0.25 pmol of double–stranded  32 P-labeled oligonucleotides comprising target regions from TriplexDNA (  Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control (C), RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). TriplexRNA-seq and TriplexDNA-seq data are from HeLa S3 cells. Adjusted  P -values
    Figure Legend Snippet: Validation of triplex-forming RNA and DNAs. ( A ) TDF analysis predicting the potential of top 1000 enriched TriplexRNA (DNA-IP) regions (ranked by peak P -value) to bind to active promoters defined by ChromHMM. Number of TFRs in RNA (per kilobase of RNA, left) and the number of putative DBSs at promoters (per kilobase of RNA, right) are shown. Boxplot borders are defined by the 1st and 3rd quantiles of the distributions, the middle line corresponds to the median value. The top whisker denotes the maximum value within the third quartile plus 1.5 times the interquartile range (bottom whisker is defined analogously). Dark gray dots represent outliers with values higher or lower than whiskers. Further box plots are based on the same definitions. ( B ) Motif analysis of triplexes formed between TriplexRNA (DNA-IP) and active promoters. The diagram depicts the fraction of antiparallel and parallel triplexes with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( C ) TDF analysis comparing the triplex-forming potential of top 2000 TriplexDNA-seq regions with top 1000 TriplexRNA (DNA-IP) (ranked by peak P -value). The number of putative DBSs (per kilobase of RNA) is shown. ( D ) Motif analysis of predicted triplexes formed between TriplexRNAs (DNA-IP) and TriplexDNA. The diagram depicts the fraction of antiparallel and parallel triplexes, with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( E ) Box plot classifying triplex interactions between TriplexRNAs (DNA-IP) and TriplexDNA-seq regions as cis ( > 10 kb in the same chromosome) and trans (at different chromosomes) interactions, excluding underrepresented local interactions (within 10 kb distance). ( F ) EMSAs using 10 or 100 pmol of synthetic TriplexRNAs and 0.25 pmol of double–stranded 32 P-labeled oligonucleotides comprising target regions from TriplexDNA ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control (C), RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). TriplexRNA-seq and TriplexDNA-seq data are from HeLa S3 cells. Adjusted P -values

    Techniques Used: Whisker Assay, Labeling

    12) Product Images from "NDF, a nucleosome-destabilizing factor that facilitates transcription through nucleosomes"

    Article Title: NDF, a nucleosome-destabilizing factor that facilitates transcription through nucleosomes

    Journal: Genes & Development

    doi: 10.1101/gad.313973.118

    NDF facilitates transcription elongation through a nucleosome. ( A ) Schematic diagram of Pol II transcription through a positioned nucleosome. A transcriptionally engaged elongation complex (Pol II EC) was assembled with purified yeast Pol II and a 5′ end-labeled 10-nucleotide (nt) RNA primer. This complex was attached to streptavidin beads and then ligated to a downstream mononucleosome positioned on the 5S rDNA sequence from Xenopus borealis . Transcription elongation was initiated by the addition of ribonucleoside 5′-triphosphates (rNTPs). Where indicated, NDF was added after the ligation step but before the addition of the rNTPs. In parallel experiments, naked DNA (same 5S rDNA sequence) was ligated downstream from the elongation complex instead of a mononucleosome. The distance (∼15 nt) from the leading edge of Pol II to the 3′ end of the transcript is also shown. ( B ) NDF reduces the inhibition of Pol II elongation by a nucleosome. Transcription elongation reactions were performed as described in A in the presence or absence of purified hNDF for the indicated times. Experiments with nucleosomal templates included a 75-fold molar excess (0.3 µM) of free unligated mononucleosomes, which stabilize the low concentration of immobilized nucleosomes. Where indicated, hNDF was included at a concentration of 1.5 µM. The reaction products were resolved by 8% polyacrylamide–urea gel electrophoresis. The diagram shows the locations of the positioned nucleosomes, the sites of Pol II pausing, the runoff product, the ligation junction, and the 10-nt primer RNA. The sizes of the RNA species were estimated by comparison with a 25-nt radiolabeled DNA ladder.
    Figure Legend Snippet: NDF facilitates transcription elongation through a nucleosome. ( A ) Schematic diagram of Pol II transcription through a positioned nucleosome. A transcriptionally engaged elongation complex (Pol II EC) was assembled with purified yeast Pol II and a 5′ end-labeled 10-nucleotide (nt) RNA primer. This complex was attached to streptavidin beads and then ligated to a downstream mononucleosome positioned on the 5S rDNA sequence from Xenopus borealis . Transcription elongation was initiated by the addition of ribonucleoside 5′-triphosphates (rNTPs). Where indicated, NDF was added after the ligation step but before the addition of the rNTPs. In parallel experiments, naked DNA (same 5S rDNA sequence) was ligated downstream from the elongation complex instead of a mononucleosome. The distance (∼15 nt) from the leading edge of Pol II to the 3′ end of the transcript is also shown. ( B ) NDF reduces the inhibition of Pol II elongation by a nucleosome. Transcription elongation reactions were performed as described in A in the presence or absence of purified hNDF for the indicated times. Experiments with nucleosomal templates included a 75-fold molar excess (0.3 µM) of free unligated mononucleosomes, which stabilize the low concentration of immobilized nucleosomes. Where indicated, hNDF was included at a concentration of 1.5 µM. The reaction products were resolved by 8% polyacrylamide–urea gel electrophoresis. The diagram shows the locations of the positioned nucleosomes, the sites of Pol II pausing, the runoff product, the ligation junction, and the 10-nt primer RNA. The sizes of the RNA species were estimated by comparison with a 25-nt radiolabeled DNA ladder.

    Techniques Used: Purification, Labeling, Sequencing, Ligation, Inhibition, Concentration Assay, Nucleic Acid Electrophoresis

    13) Product Images from "Characterizing the structure-function relationship of a naturally-occurring RNA thermometer"

    Article Title: Characterizing the structure-function relationship of a naturally-occurring RNA thermometer

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.7b01170

    Testing agsA thermometer function in a cell-free protein synthesis system using purified pre-transcribed mRNA. (a) Fluorescence trajectories over time for translation of SFGFP from agsA constructs and control mRNA in the PURExpress protein synthesis system at 30 °C and 42 °C. Shading represents standard deviation over three replicates. (b) SFGFP production rates, calculated from the trajectories in (a), during the linear synthesis regime for agsA constructs and control at 30 °C (45–50 minutes) and 42 °C (30–35 minutes), with error bars representing standard deviation.
    Figure Legend Snippet: Testing agsA thermometer function in a cell-free protein synthesis system using purified pre-transcribed mRNA. (a) Fluorescence trajectories over time for translation of SFGFP from agsA constructs and control mRNA in the PURExpress protein synthesis system at 30 °C and 42 °C. Shading represents standard deviation over three replicates. (b) SFGFP production rates, calculated from the trajectories in (a), during the linear synthesis regime for agsA constructs and control at 30 °C (45–50 minutes) and 42 °C (30–35 minutes), with error bars representing standard deviation.

    Techniques Used: Purification, Fluorescence, Construct, Standard Deviation

    14) Product Images from "DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs"

    Article Title: DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs

    Journal: Genes & Development

    doi: 10.1101/gad.282616.116

    DUSP11 catalytic activity is required for BLV 5p miRNA accumulation and RISC activity. ( A ) Immunoblot analysis of parental HEK293T cells (wild-type), parental DUSP11 knockout clone 16 (DUSP11-KO) cells, and DUSP11 knockout clone 16 cells transduced with pLenti-EV (EV), pLenti-DUSP11-3xFlag (D11-3xFlag), or the pLenti-DUSP11-CM-3xFlag catalytic mutant expression vector (D11-CM-3xFlag). ( B ) Northern blot analysis of BLV-B2 and BLV-B5 miRNAs in the indicated cell lines. The membrane was first blotted with probes specific for the 5p miRNAs, stripped, reprobed for the 3p miRNAs, stripped, and reprobed for HSUR4 RNA. ( C ) Luciferase assay to measure the RISC activity of BLV-B5 miRNAs in the DUSP11 knockout cells transduced with the indicated vectors. Bars represent the mean luciferase ratio ( Renilla /firefly) ± SEM from four experiments in which transfections were performed in triplicate.
    Figure Legend Snippet: DUSP11 catalytic activity is required for BLV 5p miRNA accumulation and RISC activity. ( A ) Immunoblot analysis of parental HEK293T cells (wild-type), parental DUSP11 knockout clone 16 (DUSP11-KO) cells, and DUSP11 knockout clone 16 cells transduced with pLenti-EV (EV), pLenti-DUSP11-3xFlag (D11-3xFlag), or the pLenti-DUSP11-CM-3xFlag catalytic mutant expression vector (D11-CM-3xFlag). ( B ) Northern blot analysis of BLV-B2 and BLV-B5 miRNAs in the indicated cell lines. The membrane was first blotted with probes specific for the 5p miRNAs, stripped, reprobed for the 3p miRNAs, stripped, and reprobed for HSUR4 RNA. ( C ) Luciferase assay to measure the RISC activity of BLV-B5 miRNAs in the DUSP11 knockout cells transduced with the indicated vectors. Bars represent the mean luciferase ratio ( Renilla /firefly) ± SEM from four experiments in which transfections were performed in triplicate.

    Techniques Used: Activity Assay, Knock-Out, Transduction, Mutagenesis, Expressing, Plasmid Preparation, Northern Blot, Luciferase, Transfection

    15) Product Images from "The CRISPR-associated Csx1 protein of Pyrococcus furiosus is an adenosine-specific endoribonuclease"

    Article Title: The CRISPR-associated Csx1 protein of Pyrococcus furiosus is an adenosine-specific endoribonuclease

    Journal: RNA

    doi: 10.1261/rna.039842.113

    Csx1 cleaves ssRNA after adenosines. ( A ) A variety of ssRNAs were treated with no protein (−) or Csx1 for the indicated times, and run alongside 5′-radiolabeled RNA markers (M), RNase T1 ladders (T1), and alkaline hydrolysis ladders (OH).
    Figure Legend Snippet: Csx1 cleaves ssRNA after adenosines. ( A ) A variety of ssRNAs were treated with no protein (−) or Csx1 for the indicated times, and run alongside 5′-radiolabeled RNA markers (M), RNase T1 ladders (T1), and alkaline hydrolysis ladders (OH).

    Techniques Used:

    Csx1 is a temperature-dependent, single-strand-specific ribonuclease. ( A ) Csx1 was tested for nuclease activity (+) on ­ 32 P-labeled single-stranded and double-stranded RNA and DNA (37mer A, 63mer A, 37mer A + B, and 63mer A + B, respectively),
    Figure Legend Snippet: Csx1 is a temperature-dependent, single-strand-specific ribonuclease. ( A ) Csx1 was tested for nuclease activity (+) on ­ 32 P-labeled single-stranded and double-stranded RNA and DNA (37mer A, 63mer A, 37mer A + B, and 63mer A + B, respectively),

    Techniques Used: Activity Assay, Labeling

    16) Product Images from "Identification of novel proteins binding the AU-rich element of α-prothymosin mRNA through the selection of open reading frames (RIDome)"

    Article Title: Identification of novel proteins binding the AU-rich element of α-prothymosin mRNA through the selection of open reading frames (RIDome)

    Journal: RNA Biology

    doi: 10.1080/15476286.2015.1107702

    In vitro validation of the selected RNA-binding proteins by ELISA-based assays. ( A) Validation by phage ELISA. The reactivity of 12 top-ranking genes was tested on the ARE PTMA RNA oligonucleotide. To test specificity, a mutated RNA (AREmut PTMA ), an ssDNA oligonucleotide and streptavidin served as controls. Values are indicated as the fold signal vs. the background (uncoated wells). ( B) BLASTP analysis of ORF clones validated by GST ELISA. ELAVL1, RBM38, R3HDM2 and RALY contain at least one conserved RNA-binding domain. ( C) Validation by GST ELISA. Selected ORFs with positive phage ELISA results were subcloned into a compatible pGEX vector and purified as GST fusion proteins. Assays were performed as in A.
    Figure Legend Snippet: In vitro validation of the selected RNA-binding proteins by ELISA-based assays. ( A) Validation by phage ELISA. The reactivity of 12 top-ranking genes was tested on the ARE PTMA RNA oligonucleotide. To test specificity, a mutated RNA (AREmut PTMA ), an ssDNA oligonucleotide and streptavidin served as controls. Values are indicated as the fold signal vs. the background (uncoated wells). ( B) BLASTP analysis of ORF clones validated by GST ELISA. ELAVL1, RBM38, R3HDM2 and RALY contain at least one conserved RNA-binding domain. ( C) Validation by GST ELISA. Selected ORFs with positive phage ELISA results were subcloned into a compatible pGEX vector and purified as GST fusion proteins. Assays were performed as in A.

    Techniques Used: In Vitro, RNA Binding Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Plasmid Preparation, Purification

    17) Product Images from "Nascent SecM Chain Outside the Ribosome Reinforces Translation Arrest"

    Article Title: Nascent SecM Chain Outside the Ribosome Reinforces Translation Arrest

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0122017

    Lifetimes of the translation arrest of HaloTag proteins harbouring the arrest sequence. ( A - D ) Time-course analyses of polypeptidyl-tRNA remaining after the addition of puromycin. Halo-L17-SecM 133–170 ( A ), Halo-L26-SecM 133–170 ( B ), Halo-pD-L8-SecM 133–170 ( C ) and Halo-SecM 1–170 ( D ) were translated in the presence of HaloTag TMR Ligand using the PURExpress ΔRibosome Kit at 37°C for 20 min. Puromycin (1 mg/mL) was added to the reaction mixture at 0 min, and the mixture was incubated at 37°C. Aliquots removed at the indicated time points were subjected to NuPAGE. Polypeptides labelled with HaloTag TMR Ligand were detected using Molecular Imager FX. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. ( E ) Plots of the fraction of polypeptidyl-tRNA remaining in the presence of puromycin as a function of time. Squares, Halo-L17-SecM 133–170 ; diamonds, Halo-L26-SecM 133–170 ; triangles, Halo-pD-L8-SecM 133–170 ; circles, Halo-SecM 1–170 . Data points represent means ± SD of three independent experiments. The solid and dotted lines show the fit to the data obtained using a single exponential function. The lifetimes of the translation arrest of Halo-L17-SecM 133–170 , Halo-L26-SecM 133–170 , Halo-pD-L8-SecM 133–170 and Halo-SecM 1–170 were 5.6 ± 0.066, 11 ± 0.22, 9.4 ± 0.63 and 51 ± 1.6 min, respectively (the errors represent fitting errors). ( F ) Time-course analysis of myc-SecM 1–170 polypeptidyl-tRNA remaining after the addition of puromycin. Myc-SecM 1–170 was translated using the PURExpress ΔRibosome Kit at 37°C for 40 min. Puromycin (1 mg/mL) was added at 0 min, and the mixture was incubated at 37°C. Aliquots were withdrawn at indicated time points and subjected to NuPAGE. Myc-SecM 1–170 was detected by western blotting with anti-c-myc-tag. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. ( G ) The fraction of myc-SecM 1–170 polypeptidyl-tRNA remaining in the presence of puromycin as a function of time. Data points with error bars represent means ± SD for three independent experiments. The solid line shows the fit to the data obtained using a single exponential function. The lifetime of the translation arrest of myc-SecM 1–170 was 48 min ± 4.3 min (the error corresponds to fitting error).
    Figure Legend Snippet: Lifetimes of the translation arrest of HaloTag proteins harbouring the arrest sequence. ( A - D ) Time-course analyses of polypeptidyl-tRNA remaining after the addition of puromycin. Halo-L17-SecM 133–170 ( A ), Halo-L26-SecM 133–170 ( B ), Halo-pD-L8-SecM 133–170 ( C ) and Halo-SecM 1–170 ( D ) were translated in the presence of HaloTag TMR Ligand using the PURExpress ΔRibosome Kit at 37°C for 20 min. Puromycin (1 mg/mL) was added to the reaction mixture at 0 min, and the mixture was incubated at 37°C. Aliquots removed at the indicated time points were subjected to NuPAGE. Polypeptides labelled with HaloTag TMR Ligand were detected using Molecular Imager FX. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. ( E ) Plots of the fraction of polypeptidyl-tRNA remaining in the presence of puromycin as a function of time. Squares, Halo-L17-SecM 133–170 ; diamonds, Halo-L26-SecM 133–170 ; triangles, Halo-pD-L8-SecM 133–170 ; circles, Halo-SecM 1–170 . Data points represent means ± SD of three independent experiments. The solid and dotted lines show the fit to the data obtained using a single exponential function. The lifetimes of the translation arrest of Halo-L17-SecM 133–170 , Halo-L26-SecM 133–170 , Halo-pD-L8-SecM 133–170 and Halo-SecM 1–170 were 5.6 ± 0.066, 11 ± 0.22, 9.4 ± 0.63 and 51 ± 1.6 min, respectively (the errors represent fitting errors). ( F ) Time-course analysis of myc-SecM 1–170 polypeptidyl-tRNA remaining after the addition of puromycin. Myc-SecM 1–170 was translated using the PURExpress ΔRibosome Kit at 37°C for 40 min. Puromycin (1 mg/mL) was added at 0 min, and the mixture was incubated at 37°C. Aliquots were withdrawn at indicated time points and subjected to NuPAGE. Myc-SecM 1–170 was detected by western blotting with anti-c-myc-tag. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. ( G ) The fraction of myc-SecM 1–170 polypeptidyl-tRNA remaining in the presence of puromycin as a function of time. Data points with error bars represent means ± SD for three independent experiments. The solid line shows the fit to the data obtained using a single exponential function. The lifetime of the translation arrest of myc-SecM 1–170 was 48 min ± 4.3 min (the error corresponds to fitting error).

    Techniques Used: Sequencing, Incubation, Western Blot

    In vitro translation of HaloTag proteins with mutated arrest sequence. Each protein construct, with or without a mutation (R163A or P166A) in the arrest sequence, was translated in the presence of HaloTag TMR Ligand using the PURExpress ΔRibosome Kit at 37°C for 20 min. Puromycin (1 mg/mL) was added at 0 min, and the reaction mixture incubated at 37°C for 3 min. Aliquots were withdrawn before and 3 min after the addition of puromycin and subjected to NuPAGE. Polypeptides labelled with HaloTag TMR Ligand were detected using Molecular Imager FX. A , Halo-L8-SecM 133–170 ; B , Halo-L17-SecM 133–170 ; C , Halo-L26-SecM 133–170 ; D , Halo-pD-L8-SecM 133–170 ; E , Halo-SecM 1–170 . Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. The results shown are representative of three independent experiments with similar results.
    Figure Legend Snippet: In vitro translation of HaloTag proteins with mutated arrest sequence. Each protein construct, with or without a mutation (R163A or P166A) in the arrest sequence, was translated in the presence of HaloTag TMR Ligand using the PURExpress ΔRibosome Kit at 37°C for 20 min. Puromycin (1 mg/mL) was added at 0 min, and the reaction mixture incubated at 37°C for 3 min. Aliquots were withdrawn before and 3 min after the addition of puromycin and subjected to NuPAGE. Polypeptides labelled with HaloTag TMR Ligand were detected using Molecular Imager FX. A , Halo-L8-SecM 133–170 ; B , Halo-L17-SecM 133–170 ; C , Halo-L26-SecM 133–170 ; D , Halo-pD-L8-SecM 133–170 ; E , Halo-SecM 1–170 . Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. The results shown are representative of three independent experiments with similar results.

    Techniques Used: In Vitro, Sequencing, Construct, Mutagenesis, Incubation

    In vitro translation of HaloTag proteins harbouring the arrest sequence. ( A ) Halo-L8-SecM 133–170 (lane 1), Halo-L17-SecM 133–170 (lane 2), Halo-L26-SecM 133–170 (lane 3), Halo-pD-L8-SecM 133–170 (lane 4) and Halo-SecM 1–170 (lane 5) were translated in the presence of HaloTag TMR Ligand using the PURExpress ΔRibosome Kit at 37°C for 20 min. Puromycin (1 mg/mL) was added to the reaction mixture at 0 min, and the reaction mixture was incubated at 37°C for 3 min. Aliquots were withdrawn before the addition of puromycin and after 3-min incubation and subjected to NuPAGE. Polypeptides labelled with HaloTag TMR Ligand were detected using Molecular Imager FX. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. The results shown are representative of three independent experiments with similar results. ( B ) Myc-Halo-L8-SecM 133–170 (lane 1), myc-Halo-L17-SecM 133–170 (lane 2), myc-Halo-L26-SecM 133–170 (lane 3), myc-Halo-pD-L8-SecM 133–170 (lane 4) and myc-Halo-SecM 1–170 (lane 5) were translated in the absence of HaloTag TMR Ligand using the PURExpress ΔRibosome Kit at 37°C for 20 min. Puromycin (1 mg/mL) was added at 0 min, and the reaction mixture was incubated at 37°C for 3 min. Aliquots were withdrawn before the addition of puromycin and after a 3-min incubation and subjected to NuPAGE. Myc-tagged polypeptides were detected by western blotting with anti-c-myc-tag. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. The results shown are representative of three independent experiments with similar results. ( C ) Fractions of translation arrest products in the absence (left) and the presence of puromycin (right). Filled bars, fluorescence detection using HaloTag TMR Ligand; open bars, detection by western blotting. Error bars represent the standard deviation (SD) of three independent experiments. The asterisk indicates statistical significance as determined by the Student's t -test ( p
    Figure Legend Snippet: In vitro translation of HaloTag proteins harbouring the arrest sequence. ( A ) Halo-L8-SecM 133–170 (lane 1), Halo-L17-SecM 133–170 (lane 2), Halo-L26-SecM 133–170 (lane 3), Halo-pD-L8-SecM 133–170 (lane 4) and Halo-SecM 1–170 (lane 5) were translated in the presence of HaloTag TMR Ligand using the PURExpress ΔRibosome Kit at 37°C for 20 min. Puromycin (1 mg/mL) was added to the reaction mixture at 0 min, and the reaction mixture was incubated at 37°C for 3 min. Aliquots were withdrawn before the addition of puromycin and after 3-min incubation and subjected to NuPAGE. Polypeptides labelled with HaloTag TMR Ligand were detected using Molecular Imager FX. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. The results shown are representative of three independent experiments with similar results. ( B ) Myc-Halo-L8-SecM 133–170 (lane 1), myc-Halo-L17-SecM 133–170 (lane 2), myc-Halo-L26-SecM 133–170 (lane 3), myc-Halo-pD-L8-SecM 133–170 (lane 4) and myc-Halo-SecM 1–170 (lane 5) were translated in the absence of HaloTag TMR Ligand using the PURExpress ΔRibosome Kit at 37°C for 20 min. Puromycin (1 mg/mL) was added at 0 min, and the reaction mixture was incubated at 37°C for 3 min. Aliquots were withdrawn before the addition of puromycin and after a 3-min incubation and subjected to NuPAGE. Myc-tagged polypeptides were detected by western blotting with anti-c-myc-tag. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. The results shown are representative of three independent experiments with similar results. ( C ) Fractions of translation arrest products in the absence (left) and the presence of puromycin (right). Filled bars, fluorescence detection using HaloTag TMR Ligand; open bars, detection by western blotting. Error bars represent the standard deviation (SD) of three independent experiments. The asterisk indicates statistical significance as determined by the Student's t -test ( p

    Techniques Used: In Vitro, Sequencing, Incubation, Western Blot, Fluorescence, Standard Deviation

    18) Product Images from "DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs"

    Article Title: DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs

    Journal: Genes & Development

    doi: 10.1101/gad.282616.116

    DUSP11 catalytic activity is required for BLV 5p miRNA accumulation and RISC activity. ( A ) Immunoblot analysis of parental HEK293T cells (wild-type), parental DUSP11 knockout clone 16 (DUSP11-KO) cells, and DUSP11 knockout clone 16 cells transduced with pLenti-EV (EV), pLenti-DUSP11-3xFlag (D11-3xFlag), or the pLenti-DUSP11-CM-3xFlag catalytic mutant expression vector (D11-CM-3xFlag). ( B ) Northern blot analysis of BLV-B2 and BLV-B5 miRNAs in the indicated cell lines. The membrane was first blotted with probes specific for the 5p miRNAs, stripped, reprobed for the 3p miRNAs, stripped, and reprobed for HSUR4 RNA. ( C ) Luciferase assay to measure the RISC activity of BLV-B5 miRNAs in the DUSP11 knockout cells transduced with the indicated vectors. Bars represent the mean luciferase ratio ( Renilla /firefly) ± SEM from four experiments in which transfections were performed in triplicate.
    Figure Legend Snippet: DUSP11 catalytic activity is required for BLV 5p miRNA accumulation and RISC activity. ( A ) Immunoblot analysis of parental HEK293T cells (wild-type), parental DUSP11 knockout clone 16 (DUSP11-KO) cells, and DUSP11 knockout clone 16 cells transduced with pLenti-EV (EV), pLenti-DUSP11-3xFlag (D11-3xFlag), or the pLenti-DUSP11-CM-3xFlag catalytic mutant expression vector (D11-CM-3xFlag). ( B ) Northern blot analysis of BLV-B2 and BLV-B5 miRNAs in the indicated cell lines. The membrane was first blotted with probes specific for the 5p miRNAs, stripped, reprobed for the 3p miRNAs, stripped, and reprobed for HSUR4 RNA. ( C ) Luciferase assay to measure the RISC activity of BLV-B5 miRNAs in the DUSP11 knockout cells transduced with the indicated vectors. Bars represent the mean luciferase ratio ( Renilla /firefly) ± SEM from four experiments in which transfections were performed in triplicate.

    Techniques Used: Activity Assay, Knock-Out, Transduction, Mutagenesis, Expressing, Plasmid Preparation, Northern Blot, Luciferase, Transfection

    19) Product Images from "DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs"

    Article Title: DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs

    Journal: Genes & Development

    doi: 10.1101/gad.282616.116

    DUSP11 directly dephosphorylates the BLV pre-miRNAs and 5p miRNAs. ( A ) Immunoblot analysis to confirm expression of DUSP11 and DUSP11 catalytic mutant proteins generated using in vitro transcription/translation. The membrane was probed using anti-DUSP11 and anti-tubulin antibodies. ( B ) In vitro phosphatase reactions on the [γ-32P]-BLV-pre-miR-B5 mimic and [γ-32P]-BLV-miR-B5-5p miRNA mimic using CIP (positive control) or the in vitro translated DUSP11, DUSP11 catalytic mutant, or luciferase (negative control) from A . Reactions were fractionated on 15% PAGE/8 M urea, and RNAs were stained with EtBr. RNAs were then transferred to a membrane, exposed to a storage phosphor screen, and imaged on a Typhoon bimolecular imager. ( C ) Northern blot analysis from wild-type and DUSP11 knockout HEK293T cells transfected with a 5′ triphosphorylated BLV-B5 pre-miRNA mimic pretreated with (+) or without (−) RNA 5′ polyphosphatase. The blot was first probed for the 5p miRNA arm (green), stripped, and reprobed for the 3p arm (orange). Note that a lighter exposure for the input RNA is shown as compared with the RNA recovered from cells.
    Figure Legend Snippet: DUSP11 directly dephosphorylates the BLV pre-miRNAs and 5p miRNAs. ( A ) Immunoblot analysis to confirm expression of DUSP11 and DUSP11 catalytic mutant proteins generated using in vitro transcription/translation. The membrane was probed using anti-DUSP11 and anti-tubulin antibodies. ( B ) In vitro phosphatase reactions on the [γ-32P]-BLV-pre-miR-B5 mimic and [γ-32P]-BLV-miR-B5-5p miRNA mimic using CIP (positive control) or the in vitro translated DUSP11, DUSP11 catalytic mutant, or luciferase (negative control) from A . Reactions were fractionated on 15% PAGE/8 M urea, and RNAs were stained with EtBr. RNAs were then transferred to a membrane, exposed to a storage phosphor screen, and imaged on a Typhoon bimolecular imager. ( C ) Northern blot analysis from wild-type and DUSP11 knockout HEK293T cells transfected with a 5′ triphosphorylated BLV-B5 pre-miRNA mimic pretreated with (+) or without (−) RNA 5′ polyphosphatase. The blot was first probed for the 5p miRNA arm (green), stripped, and reprobed for the 3p arm (orange). Note that a lighter exposure for the input RNA is shown as compared with the RNA recovered from cells.

    Techniques Used: Expressing, Mutagenesis, Generated, In Vitro, Positive Control, Luciferase, Negative Control, Polyacrylamide Gel Electrophoresis, Staining, Northern Blot, Knock-Out, Transfection

    20) Product Images from "Nascent SecM Chain Outside the Ribosome Reinforces Translation Arrest"

    Article Title: Nascent SecM Chain Outside the Ribosome Reinforces Translation Arrest

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0122017

    Lifetimes of the translation arrest of HaloTag proteins harbouring the arrest sequence. ( A - D ) Time-course analyses of polypeptidyl-tRNA remaining after the addition of puromycin. Halo-L17-SecM 133–170 ( A ), Halo-L26-SecM 133–170 ( B ), Halo-pD-L8-SecM 133–170 ( C ) and Halo-SecM 1–170 ( D ) were translated in the presence of HaloTag TMR Ligand using the PURExpress ΔRibosome Kit at 37°C for 20 min. Puromycin (1 mg/mL) was added to the reaction mixture at 0 min, and the mixture was incubated at 37°C. Aliquots removed at the indicated time points were subjected to NuPAGE. Polypeptides labelled with HaloTag TMR Ligand were detected using Molecular Imager FX. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. ( E ) Plots of the fraction of polypeptidyl-tRNA remaining in the presence of puromycin as a function of time. Squares, Halo-L17-SecM 133–170 ; diamonds, Halo-L26-SecM 133–170 ; triangles, Halo-pD-L8-SecM 133–170 ; circles, Halo-SecM 1–170 . Data points represent means ± SD of three independent experiments. The solid and dotted lines show the fit to the data obtained using a single exponential function. The lifetimes of the translation arrest of Halo-L17-SecM 133–170 , Halo-L26-SecM 133–170 , Halo-pD-L8-SecM 133–170 and Halo-SecM 1–170 were 5.6 ± 0.066, 11 ± 0.22, 9.4 ± 0.63 and 51 ± 1.6 min, respectively (the errors represent fitting errors). ( F ) Time-course analysis of myc-SecM 1–170 polypeptidyl-tRNA remaining after the addition of puromycin. Myc-SecM 1–170 was translated using the PURExpress ΔRibosome Kit at 37°C for 40 min. Puromycin (1 mg/mL) was added at 0 min, and the mixture was incubated at 37°C. Aliquots were withdrawn at indicated time points and subjected to NuPAGE. Myc-SecM 1–170 was detected by western blotting with anti-c-myc-tag. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. ( G ) The fraction of myc-SecM 1–170 polypeptidyl-tRNA remaining in the presence of puromycin as a function of time. Data points with error bars represent means ± SD for three independent experiments. The solid line shows the fit to the data obtained using a single exponential function. The lifetime of the translation arrest of myc-SecM 1–170 was 48 min ± 4.3 min (the error corresponds to fitting error).
    Figure Legend Snippet: Lifetimes of the translation arrest of HaloTag proteins harbouring the arrest sequence. ( A - D ) Time-course analyses of polypeptidyl-tRNA remaining after the addition of puromycin. Halo-L17-SecM 133–170 ( A ), Halo-L26-SecM 133–170 ( B ), Halo-pD-L8-SecM 133–170 ( C ) and Halo-SecM 1–170 ( D ) were translated in the presence of HaloTag TMR Ligand using the PURExpress ΔRibosome Kit at 37°C for 20 min. Puromycin (1 mg/mL) was added to the reaction mixture at 0 min, and the mixture was incubated at 37°C. Aliquots removed at the indicated time points were subjected to NuPAGE. Polypeptides labelled with HaloTag TMR Ligand were detected using Molecular Imager FX. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. ( E ) Plots of the fraction of polypeptidyl-tRNA remaining in the presence of puromycin as a function of time. Squares, Halo-L17-SecM 133–170 ; diamonds, Halo-L26-SecM 133–170 ; triangles, Halo-pD-L8-SecM 133–170 ; circles, Halo-SecM 1–170 . Data points represent means ± SD of three independent experiments. The solid and dotted lines show the fit to the data obtained using a single exponential function. The lifetimes of the translation arrest of Halo-L17-SecM 133–170 , Halo-L26-SecM 133–170 , Halo-pD-L8-SecM 133–170 and Halo-SecM 1–170 were 5.6 ± 0.066, 11 ± 0.22, 9.4 ± 0.63 and 51 ± 1.6 min, respectively (the errors represent fitting errors). ( F ) Time-course analysis of myc-SecM 1–170 polypeptidyl-tRNA remaining after the addition of puromycin. Myc-SecM 1–170 was translated using the PURExpress ΔRibosome Kit at 37°C for 40 min. Puromycin (1 mg/mL) was added at 0 min, and the mixture was incubated at 37°C. Aliquots were withdrawn at indicated time points and subjected to NuPAGE. Myc-SecM 1–170 was detected by western blotting with anti-c-myc-tag. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. ( G ) The fraction of myc-SecM 1–170 polypeptidyl-tRNA remaining in the presence of puromycin as a function of time. Data points with error bars represent means ± SD for three independent experiments. The solid line shows the fit to the data obtained using a single exponential function. The lifetime of the translation arrest of myc-SecM 1–170 was 48 min ± 4.3 min (the error corresponds to fitting error).

    Techniques Used: Sequencing, Incubation, Western Blot

    In vitro translation of HaloTag proteins with mutated arrest sequence. Each protein construct, with or without a mutation (R163A or P166A) in the arrest sequence, was translated in the presence of HaloTag TMR Ligand using the PURExpress ΔRibosome Kit at 37°C for 20 min. Puromycin (1 mg/mL) was added at 0 min, and the reaction mixture incubated at 37°C for 3 min. Aliquots were withdrawn before and 3 min after the addition of puromycin and subjected to NuPAGE. Polypeptides labelled with HaloTag TMR Ligand were detected using Molecular Imager FX. A , Halo-L8-SecM 133–170 ; B , Halo-L17-SecM 133–170 ; C , Halo-L26-SecM 133–170 ; D , Halo-pD-L8-SecM 133–170 ; E , Halo-SecM 1–170 . Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. The results shown are representative of three independent experiments with similar results.
    Figure Legend Snippet: In vitro translation of HaloTag proteins with mutated arrest sequence. Each protein construct, with or without a mutation (R163A or P166A) in the arrest sequence, was translated in the presence of HaloTag TMR Ligand using the PURExpress ΔRibosome Kit at 37°C for 20 min. Puromycin (1 mg/mL) was added at 0 min, and the reaction mixture incubated at 37°C for 3 min. Aliquots were withdrawn before and 3 min after the addition of puromycin and subjected to NuPAGE. Polypeptides labelled with HaloTag TMR Ligand were detected using Molecular Imager FX. A , Halo-L8-SecM 133–170 ; B , Halo-L17-SecM 133–170 ; C , Halo-L26-SecM 133–170 ; D , Halo-pD-L8-SecM 133–170 ; E , Halo-SecM 1–170 . Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. The results shown are representative of three independent experiments with similar results.

    Techniques Used: In Vitro, Sequencing, Construct, Mutagenesis, Incubation

    In vitro translation of HaloTag proteins harbouring the arrest sequence. ( A ) Halo-L8-SecM 133–170 (lane 1), Halo-L17-SecM 133–170 (lane 2), Halo-L26-SecM 133–170 (lane 3), Halo-pD-L8-SecM 133–170 (lane 4) and Halo-SecM 1–170 (lane 5) were translated in the presence of HaloTag TMR Ligand using the PURExpress ΔRibosome Kit at 37°C for 20 min. Puromycin (1 mg/mL) was added to the reaction mixture at 0 min, and the reaction mixture was incubated at 37°C for 3 min. Aliquots were withdrawn before the addition of puromycin and after 3-min incubation and subjected to NuPAGE. Polypeptides labelled with HaloTag TMR Ligand were detected using Molecular Imager FX. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. The results shown are representative of three independent experiments with similar results. ( B ) Myc-Halo-L8-SecM 133–170 (lane 1), myc-Halo-L17-SecM 133–170 (lane 2), myc-Halo-L26-SecM 133–170 (lane 3), myc-Halo-pD-L8-SecM 133–170 (lane 4) and myc-Halo-SecM 1–170 (lane 5) were translated in the absence of HaloTag TMR Ligand using the PURExpress ΔRibosome Kit at 37°C for 20 min. Puromycin (1 mg/mL) was added at 0 min, and the reaction mixture was incubated at 37°C for 3 min. Aliquots were withdrawn before the addition of puromycin and after a 3-min incubation and subjected to NuPAGE. Myc-tagged polypeptides were detected by western blotting with anti-c-myc-tag. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. The results shown are representative of three independent experiments with similar results. ( C ) Fractions of translation arrest products in the absence (left) and the presence of puromycin (right). Filled bars, fluorescence detection using HaloTag TMR Ligand; open bars, detection by western blotting. Error bars represent the standard deviation (SD) of three independent experiments. The asterisk indicates statistical significance as determined by the Student's t -test ( p
    Figure Legend Snippet: In vitro translation of HaloTag proteins harbouring the arrest sequence. ( A ) Halo-L8-SecM 133–170 (lane 1), Halo-L17-SecM 133–170 (lane 2), Halo-L26-SecM 133–170 (lane 3), Halo-pD-L8-SecM 133–170 (lane 4) and Halo-SecM 1–170 (lane 5) were translated in the presence of HaloTag TMR Ligand using the PURExpress ΔRibosome Kit at 37°C for 20 min. Puromycin (1 mg/mL) was added to the reaction mixture at 0 min, and the reaction mixture was incubated at 37°C for 3 min. Aliquots were withdrawn before the addition of puromycin and after 3-min incubation and subjected to NuPAGE. Polypeptides labelled with HaloTag TMR Ligand were detected using Molecular Imager FX. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. The results shown are representative of three independent experiments with similar results. ( B ) Myc-Halo-L8-SecM 133–170 (lane 1), myc-Halo-L17-SecM 133–170 (lane 2), myc-Halo-L26-SecM 133–170 (lane 3), myc-Halo-pD-L8-SecM 133–170 (lane 4) and myc-Halo-SecM 1–170 (lane 5) were translated in the absence of HaloTag TMR Ligand using the PURExpress ΔRibosome Kit at 37°C for 20 min. Puromycin (1 mg/mL) was added at 0 min, and the reaction mixture was incubated at 37°C for 3 min. Aliquots were withdrawn before the addition of puromycin and after a 3-min incubation and subjected to NuPAGE. Myc-tagged polypeptides were detected by western blotting with anti-c-myc-tag. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. The results shown are representative of three independent experiments with similar results. ( C ) Fractions of translation arrest products in the absence (left) and the presence of puromycin (right). Filled bars, fluorescence detection using HaloTag TMR Ligand; open bars, detection by western blotting. Error bars represent the standard deviation (SD) of three independent experiments. The asterisk indicates statistical significance as determined by the Student's t -test ( p

    Techniques Used: In Vitro, Sequencing, Incubation, Western Blot, Fluorescence, Standard Deviation

    21) Product Images from "A stress response that monitors and regulates mRNA structure is central to cold-shock adaptation"

    Article Title: A stress response that monitors and regulates mRNA structure is central to cold-shock adaptation

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2018.02.035

    The 5’UTR of cspA changes in mRNA structure during acclimation (A) The normalized in vivo DMS-seq signal of A/C bases within cspA 5’UTR in WT cells at 37°C (top), 30 min (middle) or 6 hr (bottom) after cold shock. DMS-seq signals were normalized to the maximum signal within cspA message after removing outliers by 98% Winsorisation (see Methods). The red dashed line represents the signal cutoff (0.24), above which the A/C bases are predicted to be unpaired. The region highlighted in red has long-range interactions with the “cold box” element at 10°C. (B-C) The predicted structure of the cspA 5’ UTR at (B) 37°C or (C) 30 min after cold shock. Structure predictions were generated by constraining a minimum free-energy prediction with in vivo DMS-seq data. The start codon of cspA (green), the conserved “cold box” element (blue) and its long-range interaction region at 10°C (red) are highlighted. (D) Scatter plot comparing Gini indices of ORFs (N = 391) and 5’UTR of cspA , cspB , cspG (red dots) at 30 min vs 6 hr after cold shock. Grey dashed line: Y = X. (E) The predicted structure of the cspA .
    Figure Legend Snippet: The 5’UTR of cspA changes in mRNA structure during acclimation (A) The normalized in vivo DMS-seq signal of A/C bases within cspA 5’UTR in WT cells at 37°C (top), 30 min (middle) or 6 hr (bottom) after cold shock. DMS-seq signals were normalized to the maximum signal within cspA message after removing outliers by 98% Winsorisation (see Methods). The red dashed line represents the signal cutoff (0.24), above which the A/C bases are predicted to be unpaired. The region highlighted in red has long-range interactions with the “cold box” element at 10°C. (B-C) The predicted structure of the cspA 5’ UTR at (B) 37°C or (C) 30 min after cold shock. Structure predictions were generated by constraining a minimum free-energy prediction with in vivo DMS-seq data. The start codon of cspA (green), the conserved “cold box” element (blue) and its long-range interaction region at 10°C (red) are highlighted. (D) Scatter plot comparing Gini indices of ORFs (N = 391) and 5’UTR of cspA , cspB , cspG (red dots) at 30 min vs 6 hr after cold shock. Grey dashed line: Y = X. (E) The predicted structure of the cspA .

    Techniques Used: In Vivo, Generated

    RNase R facilitates mRNA degradation during acclimation phase (A-C) RNA content of WT and Δrnr cells at 20 min, 4 hr and 8 hr after cold shock for: (A) stable RNA; (B) tmRNA; and (C) mRNA. RNA content was calculated from the fraction of RNA-seq reads mapping to different types of RNA, normalized to total RNA level measured by continuous labeling of 3 H-uridine during 37°C growth and after cold shock (see Methods). (D) mRNA content of WT and Δrnr cells before and after rifampicin (rif) treatment at 10°C. Upper: schematic of experiment. Lower: mRNA / total RNA before and after the 2 hr treatment with rifampicin, calculated from the fraction of RNA-seq reads mapping to mRNA. (E-F) mRNA amount of individual genes (N = 937) in WT (E) or Δrnr cells (F) before and after rif treatment diagrammed in (D). mRNA level was quantified by number of RNA-seq Reads Per Kilobase of transcript per Million mapped reads (RPKM). .
    Figure Legend Snippet: RNase R facilitates mRNA degradation during acclimation phase (A-C) RNA content of WT and Δrnr cells at 20 min, 4 hr and 8 hr after cold shock for: (A) stable RNA; (B) tmRNA; and (C) mRNA. RNA content was calculated from the fraction of RNA-seq reads mapping to different types of RNA, normalized to total RNA level measured by continuous labeling of 3 H-uridine during 37°C growth and after cold shock (see Methods). (D) mRNA content of WT and Δrnr cells before and after rifampicin (rif) treatment at 10°C. Upper: schematic of experiment. Lower: mRNA / total RNA before and after the 2 hr treatment with rifampicin, calculated from the fraction of RNA-seq reads mapping to mRNA. (E-F) mRNA amount of individual genes (N = 937) in WT (E) or Δrnr cells (F) before and after rif treatment diagrammed in (D). mRNA level was quantified by number of RNA-seq Reads Per Kilobase of transcript per Million mapped reads (RPKM). .

    Techniques Used: RNA Sequencing Assay, Labeling

    Global change in translation and mRNA structure during the acclimation phase following cold shock (A) Top: Cell growth after shift to 10°C. Cells gro wing exponentially at 37°C were shifted to 10°C (using dilution to achieve instantaneous tempe rature equilibration indicated by the arrow at t = 0; see Methods), and grown until exponential phase was restored (~25 hours after cold shock). Bottom: Protein synthesis after shift to 10°C. Cultures were simultaneously sampled for OD 420 and protein synthesis ( 35 S-methionine pulse labeling) at different timepoints (see Methods). Protein synthesis rate is calculated as: 35 S-methionine incorporated / minute / OD 420 / 100µL culture. The acclimation phase is shaded gray and marked by a red arrow. (B) Detailed time course of protein synthesis immediately after shift to 10°C, measured as in (A). (C) Meta-gene analysis of ribosome run-off after cold shock. At 5 min and 15 min after cold shock, the median of ribosome density at each position relative to the ORF start codon was calculated across well-expressed genes (N = 492) and normalized to the median ribosome density within the window between 1000 nt and 1180 nt downstream of start codon. Analysis is limited to ORFs ≥ 1200 nt long, and curves are smoothed by calculating the median within rolling windows of 25 nt (see Methods). (D) Histogram of Gini indices of E. coli ORFs calculated from in vivo for determination of read cut-off). P-value
    Figure Legend Snippet: Global change in translation and mRNA structure during the acclimation phase following cold shock (A) Top: Cell growth after shift to 10°C. Cells gro wing exponentially at 37°C were shifted to 10°C (using dilution to achieve instantaneous tempe rature equilibration indicated by the arrow at t = 0; see Methods), and grown until exponential phase was restored (~25 hours after cold shock). Bottom: Protein synthesis after shift to 10°C. Cultures were simultaneously sampled for OD 420 and protein synthesis ( 35 S-methionine pulse labeling) at different timepoints (see Methods). Protein synthesis rate is calculated as: 35 S-methionine incorporated / minute / OD 420 / 100µL culture. The acclimation phase is shaded gray and marked by a red arrow. (B) Detailed time course of protein synthesis immediately after shift to 10°C, measured as in (A). (C) Meta-gene analysis of ribosome run-off after cold shock. At 5 min and 15 min after cold shock, the median of ribosome density at each position relative to the ORF start codon was calculated across well-expressed genes (N = 492) and normalized to the median ribosome density within the window between 1000 nt and 1180 nt downstream of start codon. Analysis is limited to ORFs ≥ 1200 nt long, and curves are smoothed by calculating the median within rolling windows of 25 nt (see Methods). (D) Histogram of Gini indices of E. coli ORFs calculated from in vivo for determination of read cut-off). P-value

    Techniques Used: Labeling, In Vivo

    22) Product Images from "Single-Stranded RNAs Use RNAi to Potently and Allele-Selectively Inhibit Mutant Huntingtin Expression"

    Article Title: Single-Stranded RNAs Use RNAi to Potently and Allele-Selectively Inhibit Mutant Huntingtin Expression

    Journal: Cell

    doi: 10.1016/j.cell.2012.08.002

    Characterization of inhibition by modified ss-siRNAs Western analysis of inhibition of HTT expression by: (A) ss-siRNA 553822 (mismatched base at P9) containing a 5'-phosphate; (B) ss-siRNA 557426 containing three central mismatches; (C) ss-siRNA 556888 containing a mismatch at P6; and (D) ss-siRNA 553822 (mismatched base at P9) in 44-CAG-repeat GM04719 cells. (E) Effect of ss-siRNA 537775 on other genes containing trinucleotide repeats. MM: an RNA containing multiple mismatches. Western analysis for parts A–C is representative data from three or more experiments and error bars are standard error of the mean (SEM).
    Figure Legend Snippet: Characterization of inhibition by modified ss-siRNAs Western analysis of inhibition of HTT expression by: (A) ss-siRNA 553822 (mismatched base at P9) containing a 5'-phosphate; (B) ss-siRNA 557426 containing three central mismatches; (C) ss-siRNA 556888 containing a mismatch at P6; and (D) ss-siRNA 553822 (mismatched base at P9) in 44-CAG-repeat GM04719 cells. (E) Effect of ss-siRNA 537775 on other genes containing trinucleotide repeats. MM: an RNA containing multiple mismatches. Western analysis for parts A–C is representative data from three or more experiments and error bars are standard error of the mean (SEM).

    Techniques Used: Inhibition, Modification, Western Blot, Expressing

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    Article Snippet: .. Proteinase K treatment Mouse sera were mixed with proteinase K (New England Biolabs, Ipswich, MA) at a final concentration of 1 mg/mL, or with the control PBS buffer and were incubated at 55 °C. .. 100 µL of aliquot was removed for qRT-PCR analysis at 0 min, 5 min, 30 min and 60 min.

    Article Title: Functional Investigation of the Plant-Specific Long Coiled-Coil Proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL) in Arabidopsis thaliana
    Article Snippet: .. 75 µl of each sample was added to each of four tubes each containing 1 mM CaCl2 + PK buffer (50 mM Tris-HCl pH 8.0, 1 mM CaCl2 ) with or without proteinase K (200 µg ml−1 , NEB) or 1 mM CaCl2 +1% Triton X-100+ PK buffer (50 mM Tris-HCl pH 8.0, 1 mM CaCl2 ) with or without proteinase K (200 µg ml−1 ) and incubated at 25°C for 30 min. To terminate the reaction, 1 µl of protease inhibitor cocktail (Sigma-Aldrich) was added to each tube and incubated at 25°C for 10 min. 3x SDS protein loading buffer (150 mM Tris-HCl pH 6.8, 6% SDS, 300 mM DTT, 30% glycerol, 0.3% bromophenol blue) was added and samples were boiled for 5 min before subjecting to SDS-PAGE on a 15% SDS-polyacrylamide gel. .. Immunoblot analysis with anti-GFP antibody (Invitrogen) was performed as described below.

    Papanicolaou Stain:

    Article Title: 3?-Phosphoadenosine 5?-Phosphosulfate (PAPS) Synthases, Naturally Fragile Enzymes Specifically Stabilized by Nucleotide Binding *
    Article Snippet: .. Limited Proteolysis To test for protein stability by an orthologous method, 10 μg of PAPS synthase protein was incubated with varying concentrations of proteinase K (New England Biolabs, Frankfurt am Main, Germany) in 20 μl of buffer (20 mm HEPES, pH 7.3, 150 mm NaCl, 0.5 mm EDTA, 1 mm MgCl2 , 1 mm DTT). .. After incubation for 10 min at 20 °C, proteolysis was stopped by addition of 5 mm phenylmethanesulfonyl fluoride (Roth, Karlsruhe, Germany).

    Western Blot:

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation
    Article Snippet: .. The resulting eluates were collected by centrifugation at 500 rcf for 2 min (at this step we always preserved 10 μl of eluate as ‘Elute’ for western blot analysis of our routine check-ups of the Ni2+ -pull down efficiency, as described in ( )) and subsequently all proteins in the samples were digested with 0.8 U of Proteinase K (NE Biolabs) at 37°C for 30 min. .. In the case of the eIF2γ Ni2+ -pull down assay, the YMP34 strain was transformed with the selected RaP-NiP constructs along with the GCD11-His allele-carrying vector (pMP65) ( ) and the resulting transformants were cultured in the SD media and subjected to the RaP-NiP as described above.

    SDS Page:

    Article Title: Functional Investigation of the Plant-Specific Long Coiled-Coil Proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL) in Arabidopsis thaliana
    Article Snippet: .. 75 µl of each sample was added to each of four tubes each containing 1 mM CaCl2 + PK buffer (50 mM Tris-HCl pH 8.0, 1 mM CaCl2 ) with or without proteinase K (200 µg ml−1 , NEB) or 1 mM CaCl2 +1% Triton X-100+ PK buffer (50 mM Tris-HCl pH 8.0, 1 mM CaCl2 ) with or without proteinase K (200 µg ml−1 ) and incubated at 25°C for 30 min. To terminate the reaction, 1 µl of protease inhibitor cocktail (Sigma-Aldrich) was added to each tube and incubated at 25°C for 10 min. 3x SDS protein loading buffer (150 mM Tris-HCl pH 6.8, 6% SDS, 300 mM DTT, 30% glycerol, 0.3% bromophenol blue) was added and samples were boiled for 5 min before subjecting to SDS-PAGE on a 15% SDS-polyacrylamide gel. .. Immunoblot analysis with anti-GFP antibody (Invitrogen) was performed as described below.

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    New England Biolabs superase in rnase inhibitor
    Superase In Rnase Inhibitor, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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