rnase inhibitor murine  (New England Biolabs)


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    RNase Inhibitor Murine
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    RNase Inhibitor Murine 15 000 units
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    m0314l
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    Enzyme Inhibitors
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    New England Biolabs rnase inhibitor murine
    RNase Inhibitor Murine
    RNase Inhibitor Murine 15 000 units
    https://www.bioz.com/result/rnase inhibitor murine/product/New England Biolabs
    Average 99 stars, based on 35 article reviews
    Price from $9.99 to $1999.99
    rnase inhibitor murine - by Bioz Stars, 2020-09
    99/100 stars

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    1) Product Images from "Transforming activity of an oncoprotein-encoding circular RNA from human papillomavirus"

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

    Journal: Nature Communications

    doi: 10.1038/s41467-019-10246-5

    Biological functions of circE7 in tumors and episomal HPV. CaSki cells (4×10 6 ), which had been stably transduced with the indicated construct, were xenografted onto the flanks of NSG mice ( n = 8 per construct). Mice were given water with or without doxycycline (1 mg/mL) as indicated. a Image of representative CaSki tumor xenografts dissected from the indicated mice after 21 days (top). Weights of CaSki tumors with or without dox-induced circE7 sh1/2 expression (bottom). b Representative images of tumors formed by CaSki xenografts without (top) or with (bottom) doxycycline. Arrowhead indicates an area of invasive tumor. Arrows indicate mitotic figures and Ki-67-positive cells. Dashed box indicates area of detail. Scale bars, 200 μm. c TCGA RNA-Seq data (CESC, HNSC) was analyzed with vircircRNA and backsplices with ≥2 reads were tabulated. d RT-PCR from CaSki or HPV BP cells that possess integrated or episomal HPV16 genomes with or without RNase R reveals the presence of circE7 in both samples. e Human foreskin keratinocyte (HFK), keratinocytes infected with religated HPV31 (HFK + HPV31), or a HPV31 infected cell line derived from a grade II cervical biopsy (CIN612) were induced to differentiate with high calcium. Levels of HPV31 circE7 were assessed by RT-PCR (left) or RT-qPCR (right). Calcium-induced differentiation significantly decreased levels of HPV31 circE7. RT-PCR is representative of 4 independent experiments. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with two-tailed t test ( a , e )
    Figure Legend Snippet: Biological functions of circE7 in tumors and episomal HPV. CaSki cells (4×10 6 ), which had been stably transduced with the indicated construct, were xenografted onto the flanks of NSG mice ( n = 8 per construct). Mice were given water with or without doxycycline (1 mg/mL) as indicated. a Image of representative CaSki tumor xenografts dissected from the indicated mice after 21 days (top). Weights of CaSki tumors with or without dox-induced circE7 sh1/2 expression (bottom). b Representative images of tumors formed by CaSki xenografts without (top) or with (bottom) doxycycline. Arrowhead indicates an area of invasive tumor. Arrows indicate mitotic figures and Ki-67-positive cells. Dashed box indicates area of detail. Scale bars, 200 μm. c TCGA RNA-Seq data (CESC, HNSC) was analyzed with vircircRNA and backsplices with ≥2 reads were tabulated. d RT-PCR from CaSki or HPV BP cells that possess integrated or episomal HPV16 genomes with or without RNase R reveals the presence of circE7 in both samples. e Human foreskin keratinocyte (HFK), keratinocytes infected with religated HPV31 (HFK + HPV31), or a HPV31 infected cell line derived from a grade II cervical biopsy (CIN612) were induced to differentiate with high calcium. Levels of HPV31 circE7 were assessed by RT-PCR (left) or RT-qPCR (right). Calcium-induced differentiation significantly decreased levels of HPV31 circE7. RT-PCR is representative of 4 independent experiments. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with two-tailed t test ( a , e )

    Techniques Used: Stable Transfection, Transduction, Construct, Mouse Assay, Expressing, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Infection, Derivative Assay, Quantitative RT-PCR, Two Tailed Test

    Identification of HPV circRNAs. a A transcript map generated by vircircRNA summarizing the splicing events identified for HPV16 from the combined SRA datasets (Supplementary Fig. 1e ). Lines (top) indicate forward splicing events; arcs (bottom) indicate backsplicing; thickness = log 2 (read count); red arc highlights circE7. The lower panel represents a partial HPV16 genome with promoters (P, green arrowheads) and the early polyadenylation (A E , red line) indicated. Numbering from the NC_001526 reference sequence. b Alignment of sequencing reads spanning the circE7 backsplice junction from SRS2410540. Red indicates E7-E1 sequences, and blue indicates E6 sequence. c Predicted formation and size of HPV16 circE7. Arrows indicate primers used to detect linear E6/E7 and circE7. d RT-PCR of random hexamer primed total RNA from HPV16+ cancer cell lines. 2 μg of total RNA were treated with 5U of RNase R (or water for mock) in the presence of RNase inhibitor for 40 min prior to RT reaction. Results are representative of 4 independent experiments. e Sanger sequencing of PCR products from d confirmed the presence of the expected circE7 backsplice junction without the insertion of additional nucleotides. Sequencing traces were identical for 3 independent reactions from each cell line. f Northern blot of total RNA after mock (8 μg) or with RNase R treatment (20 μg) from the indicated HPV16+ cell line probed with HPV16 E7. Arrows indicates RNase resistant band with E7 sequence. Ethidium Bromide staining (bottom), RNase R treatment control. Results representative of 5 independent northerns
    Figure Legend Snippet: Identification of HPV circRNAs. a A transcript map generated by vircircRNA summarizing the splicing events identified for HPV16 from the combined SRA datasets (Supplementary Fig. 1e ). Lines (top) indicate forward splicing events; arcs (bottom) indicate backsplicing; thickness = log 2 (read count); red arc highlights circE7. The lower panel represents a partial HPV16 genome with promoters (P, green arrowheads) and the early polyadenylation (A E , red line) indicated. Numbering from the NC_001526 reference sequence. b Alignment of sequencing reads spanning the circE7 backsplice junction from SRS2410540. Red indicates E7-E1 sequences, and blue indicates E6 sequence. c Predicted formation and size of HPV16 circE7. Arrows indicate primers used to detect linear E6/E7 and circE7. d RT-PCR of random hexamer primed total RNA from HPV16+ cancer cell lines. 2 μg of total RNA were treated with 5U of RNase R (or water for mock) in the presence of RNase inhibitor for 40 min prior to RT reaction. Results are representative of 4 independent experiments. e Sanger sequencing of PCR products from d confirmed the presence of the expected circE7 backsplice junction without the insertion of additional nucleotides. Sequencing traces were identical for 3 independent reactions from each cell line. f Northern blot of total RNA after mock (8 μg) or with RNase R treatment (20 μg) from the indicated HPV16+ cell line probed with HPV16 E7. Arrows indicates RNase resistant band with E7 sequence. Ethidium Bromide staining (bottom), RNase R treatment control. Results representative of 5 independent northerns

    Techniques Used: Generated, Sequencing, Reverse Transcription Polymerase Chain Reaction, Random Hexamer Labeling, Polymerase Chain Reaction, Northern Blot, Staining

    Protein encoding circE7 is essential for CaSki cell growth. a CaSki cells were lentivirally transduced with doxycycline (dox)-inducible hairpins specific for the circE7 backsplice junction (circE7 sh1/2). RT-qPCR for levels of circE7 revealed that circE7 sh1/2 resulted in significant decreases of circE7 levels. ( n = 3 independent experiments, run in duplicate). b Northern blot of RNase R treated total RNA (30 μg) from CaSki cells with or without circE7 sh1/2 induction (2 days). Band density (bottom number) was quantitated and normalized to the uninduced control. c Western blots for E7 and E6 after circE7 sh1/2 induction (3 days). Western blots representative of 3 independent experiments. GAPDH, loading control. d A total of 6.0 × 10 4 CaSki cells were seeded in triplicate in six-well plates at day 0 and absolute cell number quantitated daily after day 2. CircE7 sh1/2 induction resulted in significantly slower growth of CaSki cells after day 4. Similar results were obtained in 3 independent experiments. e CaSki cells with or without circE7 sh1/2 induction (1 day) were plated in chamber slides and labeled with BrdU (10 μM for 1.5 h). Cells were stained with αBrdU and DAPI and scored as % of DAPI + cells. f 1.0 × 10 4 CaSki circE7 sh1/2 cells with or without induction (1 day) were seeded in triplicate in soft agar with or without dox (14 days). Average colonies per 35 mm. n = 4 independent transfections. g CaSki were doubly transduced with a shRNA resistant WT circE7 (circResist_WT) and circE7 sh1/2. MTT assay of circResist_WT cells with and without Dox induction. MTT values normalized to the uninduced (-Dox) condition. h CaSki were doubly transduced with a shRNA resistant circE7 with no start codons (circResist_noATG) and circE7 sh1/2. MTT assay of circResist_noATG cells with and without Dox induction. MTT values normalized to the uninduced (-Dox) condition. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with two-tailed t test ( d , g , h ) and one-way analysis of variance (ANOVA) with Holm–Sidak tests ( a , e , f ). Source data for b , c provided in Source Data file
    Figure Legend Snippet: Protein encoding circE7 is essential for CaSki cell growth. a CaSki cells were lentivirally transduced with doxycycline (dox)-inducible hairpins specific for the circE7 backsplice junction (circE7 sh1/2). RT-qPCR for levels of circE7 revealed that circE7 sh1/2 resulted in significant decreases of circE7 levels. ( n = 3 independent experiments, run in duplicate). b Northern blot of RNase R treated total RNA (30 μg) from CaSki cells with or without circE7 sh1/2 induction (2 days). Band density (bottom number) was quantitated and normalized to the uninduced control. c Western blots for E7 and E6 after circE7 sh1/2 induction (3 days). Western blots representative of 3 independent experiments. GAPDH, loading control. d A total of 6.0 × 10 4 CaSki cells were seeded in triplicate in six-well plates at day 0 and absolute cell number quantitated daily after day 2. CircE7 sh1/2 induction resulted in significantly slower growth of CaSki cells after day 4. Similar results were obtained in 3 independent experiments. e CaSki cells with or without circE7 sh1/2 induction (1 day) were plated in chamber slides and labeled with BrdU (10 μM for 1.5 h). Cells were stained with αBrdU and DAPI and scored as % of DAPI + cells. f 1.0 × 10 4 CaSki circE7 sh1/2 cells with or without induction (1 day) were seeded in triplicate in soft agar with or without dox (14 days). Average colonies per 35 mm. n = 4 independent transfections. g CaSki were doubly transduced with a shRNA resistant WT circE7 (circResist_WT) and circE7 sh1/2. MTT assay of circResist_WT cells with and without Dox induction. MTT values normalized to the uninduced (-Dox) condition. h CaSki were doubly transduced with a shRNA resistant circE7 with no start codons (circResist_noATG) and circE7 sh1/2. MTT assay of circResist_noATG cells with and without Dox induction. MTT values normalized to the uninduced (-Dox) condition. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with two-tailed t test ( d , g , h ) and one-way analysis of variance (ANOVA) with Holm–Sidak tests ( a , e , f ). Source data for b , c provided in Source Data file

    Techniques Used: Transduction, Quantitative RT-PCR, Northern Blot, Western Blot, Labeling, Staining, Transfection, shRNA, MTT Assay, Two Tailed Test

    Characterization of circE7. a CircE7-transfected cells were fractionated and indicated fractions analyzed by northern blot. Total RNA (4 μg) with mock or RNase R treatment of fractions from 293 T cells confirms that circE7 is enriched in the cytoplasm and is RNase R-resistant. MALAT1 and β-actin, fractionation controls. Band density (bottom) quantitated after normalization to the enriched fraction. Results are representative of 3 independent blots. b CircE7-transfected 293T (left) or untransfected CaSki (right) were fractionated and analyzed by RT-qPCR. MALAT1 and 18 S (top), fractionation controls. Values normalized to the enriched fraction. Results are representative of 3 independent fractionation experiments. c RT-qPCR of RNA IP (m 6 A or IgG control) after transfection with the indicated plasmid (24 h) ( n = 8 biological replicates from 4 transfections). SON, m 6 A RNA IP control. d Western blot for METTL3 from 293T co-transfected with control or METTL3 siRNA and circE7 construct (top). GAPDH, loading control. RT-qPCR of RNA IP (m 6 A or IgG control) from 293 T cells co-transfected with indicated siRNA and circE7 construct. RT-PCR is representative of 4 independent experiments. e Schematic of the DRACH consensus motifs for METTL3/14 and the sites mutated in the circE7_noDRACH construct (top). RT-qPCR for circE7 in cells transfected with the indicated construct. Loss of UTR DRACH motifs in circE7 results in a significant decrease in the abundance of circE7, but not linear E6/E7. ( n = 4 independent experiments). f Western blot for E7 from 293 T transfected with indicated circE7 construct. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with one-way analysis of variance (ANOVA) with Holm–Sidak tests. g Representative tracing of circE7-transfected cells after polysome enrichment assay with the monosome (M), light polysome (L), and heavy polysome (H) fractions indicated (left). Dashed lines indicate collected fraction. Detection of circE7 in polysome fraction by RT-PCR after transfection with circE7 or circE7_noATG (right). β-actin, control. Source data for a provided in Source Data file
    Figure Legend Snippet: Characterization of circE7. a CircE7-transfected cells were fractionated and indicated fractions analyzed by northern blot. Total RNA (4 μg) with mock or RNase R treatment of fractions from 293 T cells confirms that circE7 is enriched in the cytoplasm and is RNase R-resistant. MALAT1 and β-actin, fractionation controls. Band density (bottom) quantitated after normalization to the enriched fraction. Results are representative of 3 independent blots. b CircE7-transfected 293T (left) or untransfected CaSki (right) were fractionated and analyzed by RT-qPCR. MALAT1 and 18 S (top), fractionation controls. Values normalized to the enriched fraction. Results are representative of 3 independent fractionation experiments. c RT-qPCR of RNA IP (m 6 A or IgG control) after transfection with the indicated plasmid (24 h) ( n = 8 biological replicates from 4 transfections). SON, m 6 A RNA IP control. d Western blot for METTL3 from 293T co-transfected with control or METTL3 siRNA and circE7 construct (top). GAPDH, loading control. RT-qPCR of RNA IP (m 6 A or IgG control) from 293 T cells co-transfected with indicated siRNA and circE7 construct. RT-PCR is representative of 4 independent experiments. e Schematic of the DRACH consensus motifs for METTL3/14 and the sites mutated in the circE7_noDRACH construct (top). RT-qPCR for circE7 in cells transfected with the indicated construct. Loss of UTR DRACH motifs in circE7 results in a significant decrease in the abundance of circE7, but not linear E6/E7. ( n = 4 independent experiments). f Western blot for E7 from 293 T transfected with indicated circE7 construct. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with one-way analysis of variance (ANOVA) with Holm–Sidak tests. g Representative tracing of circE7-transfected cells after polysome enrichment assay with the monosome (M), light polysome (L), and heavy polysome (H) fractions indicated (left). Dashed lines indicate collected fraction. Detection of circE7 in polysome fraction by RT-PCR after transfection with circE7 or circE7_noATG (right). β-actin, control. Source data for a provided in Source Data file

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

    Related Articles

    Concentration Assay:

    Article Title: Transforming activity of an oncoprotein-encoding circular RNA from human papillomavirus
    Article Snippet: .. A concentration of 2 µg of total RNA was incubated with 5U RNase R (Lucigen, RNR07250), 10U murine ribonuclease inhibitor (New England Biolabs, M0314S), 0.5U DNase (Qiagen, 79254), and 1X RNase R buffer for 40 min at 37 °C and then placed on ice. ..

    Incubation:

    Article Title: Transforming activity of an oncoprotein-encoding circular RNA from human papillomavirus
    Article Snippet: .. A concentration of 2 µg of total RNA was incubated with 5U RNase R (Lucigen, RNR07250), 10U murine ribonuclease inhibitor (New England Biolabs, M0314S), 0.5U DNase (Qiagen, 79254), and 1X RNase R buffer for 40 min at 37 °C and then placed on ice. ..

    Ligation:

    Article Title: nextPARS: parallel probing of RNA structures in Illumina
    Article Snippet: .. Samples were then put on ice, and 2 µL of 5× HM Ligation Buffer, 1 µL of RNase inhibitor and 1 µL of T4 RNA Ligase 2, truncated (New England BioLabs) were added. .. After 1 h incubation at 28°C, we added 1 µL of stop solution, gently pipetted up and down, incubated for 15 min more at 28°C and placed the samples on ice.

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    New England Biolabs murine rnase inhibitor
    Biochemical characterization of LwaCas13a <t>RNA</t> cleavage activity a, LwaCas13a has more active <t>RNAse</t> activity than LshCas13a. b, Gel electrophoresis of ssRNA1 after incubation with LwaCas13a and with and without crRNA 1 for varying amounts of times. c, Gel electrophoresis of ssRNA1 after incubation with varying amounts of LwaCas13a-crRNA complex. d, Sequence and structure of ssRNA 4 and ssRNA 5. crRNA spacer sequence is highlighted in blue. e, Gel electrophoresis of ssRNA 4 and ssRNA 5 after incubation with LwaCas13a and crRNA 1. f, Sequence and structure of ssRNA 4 with sites of poly-x modifications highlighted in red. crRNA spacer sequence is highlighted in blue. g, Gel electrophoresis of ssRNA 4 with each of 4 possible poly-x modifications incubated with LwaCas13a and crRNA 1. h, LwaCas13a can process pre-crRNA from the L. wadei CRISPR-Cas locus. i, Cleavage efficiency of ssRNA 1 for crRNA spacer truncations after incubation with LwaCas13a.
    Murine Rnase Inhibitor, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs rnase a inhibitor
    Effect of the growth parameters on RiCF. (A) Schematics of a hypothetical scenario when RNA inhibits NAPs that could potentially cleave DNA. During lysis, quick RNA degradation removes the inhibition resulting in breakage of chromosomes. (B) Growth phase dependence of RiCF. AB1157 was grown at 37°C with periodic OD measurements, and samples for plugs were withdrawn at various times. The cells were made into plugs using lysis agarose and RNase (50 μg/plug) and the plugs were lysed and electrophoresed under standard conditions. Data points are means of at least three independent assays ± SEM. (C) Effect of translation and transcription inhibition on RiCF. Cells were grown till OD 0.5–0.6, split into three parts and chloramphenicol (40 μg/ml) or rifampicin (150 μg/ml) were added to two samples. All sample were shaken for another 2–3 hours at 37°C before making plugs as described in (B). Data points are means of four independent assays ± SEM. (D) Growth in minimal medium reduces RiCF. Cells were grown in LB or MOPS till the OD reached 0.6 and made into plugs using standard conditions. The values presented are means of six independent assays ± SEM. (E) Effect of growth temperature on RNase-induced chromosomal fragmentation. Cultures of AB1157 were grown at 20°C, 30°C, 37°C, 42°C or 45°C to same cell densities (A 600 = 0.6), and plugs were made in lysis agarose with <t>RNAse</t> A (50 μg/plug), as described in (A). Data are means of three to six independent measurements ± SEM.
    Rnase A Inhibitor, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biochemical characterization of LwaCas13a RNA cleavage activity a, LwaCas13a has more active RNAse activity than LshCas13a. b, Gel electrophoresis of ssRNA1 after incubation with LwaCas13a and with and without crRNA 1 for varying amounts of times. c, Gel electrophoresis of ssRNA1 after incubation with varying amounts of LwaCas13a-crRNA complex. d, Sequence and structure of ssRNA 4 and ssRNA 5. crRNA spacer sequence is highlighted in blue. e, Gel electrophoresis of ssRNA 4 and ssRNA 5 after incubation with LwaCas13a and crRNA 1. f, Sequence and structure of ssRNA 4 with sites of poly-x modifications highlighted in red. crRNA spacer sequence is highlighted in blue. g, Gel electrophoresis of ssRNA 4 with each of 4 possible poly-x modifications incubated with LwaCas13a and crRNA 1. h, LwaCas13a can process pre-crRNA from the L. wadei CRISPR-Cas locus. i, Cleavage efficiency of ssRNA 1 for crRNA spacer truncations after incubation with LwaCas13a.

    Journal: Nature

    Article Title: RNA targeting with CRISPR-Cas13a

    doi: 10.1038/nature24049

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

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

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

    eIF3d cap-binding activity is required for efficient 48S initiation complex formation on specific mRNAs a , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to eIF3 in the presence of competitor ligands. b , Electrostatic surface view of the eIF3d cap-binding domain colored by charge, with a zoomed view of single stranded RNA (ssRNA) and cap analog modeled according to their positions bound to DXO 15 . Positive charge is colored blue and negative charge is in red, and the RNA gate is removed for clarity. c , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to wild type or helix α5 or helix α11-mutant eIF3. eIF3d-helix α5 mutant (D249Q/V262I/Y263A), helix α11 mutant (T317E/N320E/H321A). WT, wild type. d , Incorporation of c-Jun and ACTB mRNA into initiation complexes by wild type, helix α5, or helix α11-mutant eIF3d as measured by quantitative RT-PCR. mRNA-ribosome association is expressed as the ratio between the quantity of mRNA transcripts to 18S rRNA and normalized to the wild type sample. The results are representative of three independent experiments and given as the mean ± s.d. from a representative quantitative RT-PCR experiment performed in duplicate.

    Journal: Nature

    Article Title: eIF3d is an mRNA cap-binding protein required for specialized translation initiation

    doi: 10.1038/nature18954

    Figure Lengend Snippet: eIF3d cap-binding activity is required for efficient 48S initiation complex formation on specific mRNAs a , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to eIF3 in the presence of competitor ligands. b , Electrostatic surface view of the eIF3d cap-binding domain colored by charge, with a zoomed view of single stranded RNA (ssRNA) and cap analog modeled according to their positions bound to DXO 15 . Positive charge is colored blue and negative charge is in red, and the RNA gate is removed for clarity. c , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to wild type or helix α5 or helix α11-mutant eIF3. eIF3d-helix α5 mutant (D249Q/V262I/Y263A), helix α11 mutant (T317E/N320E/H321A). WT, wild type. d , Incorporation of c-Jun and ACTB mRNA into initiation complexes by wild type, helix α5, or helix α11-mutant eIF3d as measured by quantitative RT-PCR. mRNA-ribosome association is expressed as the ratio between the quantity of mRNA transcripts to 18S rRNA and normalized to the wild type sample. The results are representative of three independent experiments and given as the mean ± s.d. from a representative quantitative RT-PCR experiment performed in duplicate.

    Article Snippet: Each translation reaction contained 50% in vitro translation lysate and buffer to make the final reaction with 0.84 mM ATP, 0.21 mM GTP, 21 mM creatine phosphate (Roche), 45 U ml-1 creatine phosphokinase (Roche), 10 mM HEPES-KOH pH 7.6, 2 mM DTT, 8 mM amino acids (Promega), 255 mM spermidine, 1 U ml-1 murine RNase inhibitor (NEB), and mRNA-specific concentrations of Mg(OAc)2 and KOAc.

    Techniques: Binding Assay, Activity Assay, SDS-Gel, Labeling, Mutagenesis, Quantitative RT-PCR

    eIF4E recognizes the 5′ end of the c-Jun mRNA less efficiently than ACTB mRNA a , Coomassie blue stained SDS gel of recombinant human eIF4E expressed in E. coli. b , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled ACTB or c-Jun 5′ UTR RNA crosslinked to eIF4E. The result is representative of three independent experiments. For gel source data, see Supplementary Figure 1 .

    Journal: Nature

    Article Title: eIF3d is an mRNA cap-binding protein required for specialized translation initiation

    doi: 10.1038/nature18954

    Figure Lengend Snippet: eIF4E recognizes the 5′ end of the c-Jun mRNA less efficiently than ACTB mRNA a , Coomassie blue stained SDS gel of recombinant human eIF4E expressed in E. coli. b , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled ACTB or c-Jun 5′ UTR RNA crosslinked to eIF4E. The result is representative of three independent experiments. For gel source data, see Supplementary Figure 1 .

    Article Snippet: Each translation reaction contained 50% in vitro translation lysate and buffer to make the final reaction with 0.84 mM ATP, 0.21 mM GTP, 21 mM creatine phosphate (Roche), 45 U ml-1 creatine phosphokinase (Roche), 10 mM HEPES-KOH pH 7.6, 2 mM DTT, 8 mM amino acids (Promega), 255 mM spermidine, 1 U ml-1 murine RNase inhibitor (NEB), and mRNA-specific concentrations of Mg(OAc)2 and KOAc.

    Techniques: Staining, SDS-Gel, Recombinant, Labeling

    5' end recognition of c-Jun mRNA is eIF4F-independent a , Distribution of c-Jun or ACTB mRNA-containing initiation complexes in programmed 293T cell in vitro translation extracts. The mRNA abundance (black line) is expressed as the fraction of total recovered transcripts. The results are given as the mean ± standard deviation (s.d.) of a representative quantitative RT-PCR experiment performed in duplicate. The polysome profile (gray line) is plotted as relative absorbance at 254 nm versus elution fractions. b , Western blot analysis of initiation factors in 48S translation complexes formed on c-Jun and ACTB mRNAs. 293T, total protein from 293T in vitro translation extracts. For gel source data, see Supplementary Figure 1 . c , Phosphorimage of SDS gel resolving RNase-protected 32 P-internal or 32 P-cap-labeled c-Jun 5' UTR RNA crosslinked to eIF3 subunits. Recombinant eIF3a migrates at ~100 kDa due to a C-terminal truncation 26 . The results of a - c are representative of three independent experiments.

    Journal: Nature

    Article Title: eIF3d is an mRNA cap-binding protein required for specialized translation initiation

    doi: 10.1038/nature18954

    Figure Lengend Snippet: 5' end recognition of c-Jun mRNA is eIF4F-independent a , Distribution of c-Jun or ACTB mRNA-containing initiation complexes in programmed 293T cell in vitro translation extracts. The mRNA abundance (black line) is expressed as the fraction of total recovered transcripts. The results are given as the mean ± standard deviation (s.d.) of a representative quantitative RT-PCR experiment performed in duplicate. The polysome profile (gray line) is plotted as relative absorbance at 254 nm versus elution fractions. b , Western blot analysis of initiation factors in 48S translation complexes formed on c-Jun and ACTB mRNAs. 293T, total protein from 293T in vitro translation extracts. For gel source data, see Supplementary Figure 1 . c , Phosphorimage of SDS gel resolving RNase-protected 32 P-internal or 32 P-cap-labeled c-Jun 5' UTR RNA crosslinked to eIF3 subunits. Recombinant eIF3a migrates at ~100 kDa due to a C-terminal truncation 26 . The results of a - c are representative of three independent experiments.

    Article Snippet: Each translation reaction contained 50% in vitro translation lysate and buffer to make the final reaction with 0.84 mM ATP, 0.21 mM GTP, 21 mM creatine phosphate (Roche), 45 U ml-1 creatine phosphokinase (Roche), 10 mM HEPES-KOH pH 7.6, 2 mM DTT, 8 mM amino acids (Promega), 255 mM spermidine, 1 U ml-1 murine RNase inhibitor (NEB), and mRNA-specific concentrations of Mg(OAc)2 and KOAc.

    Techniques: In Vitro, Standard Deviation, Quantitative RT-PCR, Western Blot, SDS-Gel, Labeling, Recombinant

    CBP interacts with RNA in vivo A) Native RNA-IP of CBP. Top, RNA immunprecipitated with CBP. Bottom, CBP western blot. B) PAR-CLIP protocol. 4-Thiouridine (4-SU). C) CBP PAR-CLIP required 4-SU: top, autoradiography; bottom, CBP western blot.. D) Quantification of CBP PAR-CLIP. Error bars represent mean +/− s.e.m; n=4. E) CBP PAR-CLIP signal was sensitive to RNAse. 1× RNAse cocktail contained: RNAse A (0.01mU/ul) + RNase T1 (0.4mU/ul). F) Quantification of RNase titration. Error bars represent mean +/− s.e.m; n=4; P -values from two-tailed Student’s t-test: *P

    Journal: Cell

    Article Title: RNA binding to CBP stimulates histone acetylation and transcription

    doi: 10.1016/j.cell.2016.12.020

    Figure Lengend Snippet: CBP interacts with RNA in vivo A) Native RNA-IP of CBP. Top, RNA immunprecipitated with CBP. Bottom, CBP western blot. B) PAR-CLIP protocol. 4-Thiouridine (4-SU). C) CBP PAR-CLIP required 4-SU: top, autoradiography; bottom, CBP western blot.. D) Quantification of CBP PAR-CLIP. Error bars represent mean +/− s.e.m; n=4. E) CBP PAR-CLIP signal was sensitive to RNAse. 1× RNAse cocktail contained: RNAse A (0.01mU/ul) + RNase T1 (0.4mU/ul). F) Quantification of RNase titration. Error bars represent mean +/− s.e.m; n=4; P -values from two-tailed Student’s t-test: *P

    Article Snippet: Reactions contained 1× HAT assay buffer (50mM Tris-HCl pH 7.5 (RT), 5% glycerol, 0.1mM EDTA, 50mM KCl), 1mM DTT, 10mM Na-Butyrate, 1× Complete EDTA protease inhibitor cocktail (Roche), 0.4U/ul murine RNAse inhibitor (NEB), 0.1mg/ml BSA (NEB), 80nM H31–21 peptide (Anaspec) and the required dilution of RNA probe.

    Techniques: In Vivo, Western Blot, Cross-linking Immunoprecipitation, Autoradiography, Titration, Two Tailed Test

    Effect of the growth parameters on RiCF. (A) Schematics of a hypothetical scenario when RNA inhibits NAPs that could potentially cleave DNA. During lysis, quick RNA degradation removes the inhibition resulting in breakage of chromosomes. (B) Growth phase dependence of RiCF. AB1157 was grown at 37°C with periodic OD measurements, and samples for plugs were withdrawn at various times. The cells were made into plugs using lysis agarose and RNase (50 μg/plug) and the plugs were lysed and electrophoresed under standard conditions. Data points are means of at least three independent assays ± SEM. (C) Effect of translation and transcription inhibition on RiCF. Cells were grown till OD 0.5–0.6, split into three parts and chloramphenicol (40 μg/ml) or rifampicin (150 μg/ml) were added to two samples. All sample were shaken for another 2–3 hours at 37°C before making plugs as described in (B). Data points are means of four independent assays ± SEM. (D) Growth in minimal medium reduces RiCF. Cells were grown in LB or MOPS till the OD reached 0.6 and made into plugs using standard conditions. The values presented are means of six independent assays ± SEM. (E) Effect of growth temperature on RNase-induced chromosomal fragmentation. Cultures of AB1157 were grown at 20°C, 30°C, 37°C, 42°C or 45°C to same cell densities (A 600 = 0.6), and plugs were made in lysis agarose with RNAse A (50 μg/plug), as described in (A). Data are means of three to six independent measurements ± SEM.

    Journal: PLoS ONE

    Article Title: Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome

    doi: 10.1371/journal.pone.0190177

    Figure Lengend Snippet: Effect of the growth parameters on RiCF. (A) Schematics of a hypothetical scenario when RNA inhibits NAPs that could potentially cleave DNA. During lysis, quick RNA degradation removes the inhibition resulting in breakage of chromosomes. (B) Growth phase dependence of RiCF. AB1157 was grown at 37°C with periodic OD measurements, and samples for plugs were withdrawn at various times. The cells were made into plugs using lysis agarose and RNase (50 μg/plug) and the plugs were lysed and electrophoresed under standard conditions. Data points are means of at least three independent assays ± SEM. (C) Effect of translation and transcription inhibition on RiCF. Cells were grown till OD 0.5–0.6, split into three parts and chloramphenicol (40 μg/ml) or rifampicin (150 μg/ml) were added to two samples. All sample were shaken for another 2–3 hours at 37°C before making plugs as described in (B). Data points are means of four independent assays ± SEM. (D) Growth in minimal medium reduces RiCF. Cells were grown in LB or MOPS till the OD reached 0.6 and made into plugs using standard conditions. The values presented are means of six independent assays ± SEM. (E) Effect of growth temperature on RNase-induced chromosomal fragmentation. Cultures of AB1157 were grown at 20°C, 30°C, 37°C, 42°C or 45°C to same cell densities (A 600 = 0.6), and plugs were made in lysis agarose with RNAse A (50 μg/plug), as described in (A). Data are means of three to six independent measurements ± SEM.

    Article Snippet: XRN-I, RNase If , Exonuclease T (Exo T), EcoRI and RNase A inhibitor were all from New England Biolabs.

    Techniques: Lysis, Inhibition

    RNA degradation causes chromosomal fragmentation. (A)  Schematics of a hypothetical scenario when RNA makes the central core of nucleoids, and its degradation results in collapse of the nucleoid structure, causing chromosomal fragmentation.  (B)  Radiogram of a pulsed field gel showing chromosomal fragmentation in AB1157 when cells were embedded in agarose plugs in the presence and absence of proteinase K (25 μg/plug) and/or RNase (50 μg/plug) and lysed overnight at 62°C.  (C)  Radiogram showing DNase I sensitivity of the signal entering the gel. Plugs were lysed at 62°C, washed extensively to remove traces of lysis buffer and then treated with DNase I at 37°C before PFGE.  (D)  A representative gel showing that RNA degradation by different enzymes causes chromosomal fragmentation. Plugs were made in the absence of proteinase K in 1x restriction enzyme buffer (NEBuffer 3 for RNase A, XRN-1 and RNAse I f  and NEBuffer 4 for Exo T). The concentrations of the enzymes used were, RNase, 50 μg/plug; XRN-1, 5 U/plug; RNAse I f , 100 U/plug and Exo T, 20 U/plug.  (E)  Quantification of the chromosomal fragmentation when plugs were made in the presence of various RNA degrading enzymes. The values presented are means of four independent assays ± SEM. CZ, compression zone.

    Journal: PLoS ONE

    Article Title: Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome

    doi: 10.1371/journal.pone.0190177

    Figure Lengend Snippet: RNA degradation causes chromosomal fragmentation. (A) Schematics of a hypothetical scenario when RNA makes the central core of nucleoids, and its degradation results in collapse of the nucleoid structure, causing chromosomal fragmentation. (B) Radiogram of a pulsed field gel showing chromosomal fragmentation in AB1157 when cells were embedded in agarose plugs in the presence and absence of proteinase K (25 μg/plug) and/or RNase (50 μg/plug) and lysed overnight at 62°C. (C) Radiogram showing DNase I sensitivity of the signal entering the gel. Plugs were lysed at 62°C, washed extensively to remove traces of lysis buffer and then treated with DNase I at 37°C before PFGE. (D) A representative gel showing that RNA degradation by different enzymes causes chromosomal fragmentation. Plugs were made in the absence of proteinase K in 1x restriction enzyme buffer (NEBuffer 3 for RNase A, XRN-1 and RNAse I f and NEBuffer 4 for Exo T). The concentrations of the enzymes used were, RNase, 50 μg/plug; XRN-1, 5 U/plug; RNAse I f , 100 U/plug and Exo T, 20 U/plug. (E) Quantification of the chromosomal fragmentation when plugs were made in the presence of various RNA degrading enzymes. The values presented are means of four independent assays ± SEM. CZ, compression zone.

    Article Snippet: XRN-I, RNase If , Exonuclease T (Exo T), EcoRI and RNase A inhibitor were all from New England Biolabs.

    Techniques: Pulsed-Field Gel, Lysis