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TaKaRa ribonuclease inhibitor
Ribonuclease Inhibitor, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Clone Assay:

Article Title: Three RNA Binding Proteins Form a Complex to Promote Differentiation of Germline Stem Cell Lineage in Drosophila
Article Snippet: .. RNA Immunoprecipitation (RIP) RNase inhibitor (Takara 2313A) was used for all RIP-related experiments. mei-P26 3′UTR (549 bp and 844 bp from stop codon) was cloned into pUAS-GFP vector between GFP stop codon and SV40 3′UTR. .. S2 cells transfected with TAP-Tut and mei-P26 3′UTR or TAP-GFP and mei-P26 3′UTR were lysed in polysome lysis buffer according to .

Recombinant:

Article Title: Developmental heterogeneity of microglia and brain myeloid cells revealed by deep single-cell RNA sequencing
Article Snippet: .. Single cells were index sorted into 96-well plates containing 4uL lysis buffer (4U Recombinant RNase Inhibitor (Takara Bio 2313B), 0.05% Triton X-100, 2.5mM dNTP mix (Thermo Fisher Scientific R0192), 2.5uM Oligo-dT30VN (5′-AAGCAGTGGTATCAACGCAGAGTACT30 VN-3′, ERCC Spike-ins (Thermo Fisher Scientific 4456740) diluted at 1:2.4×107 ). ..

Article Title: Characterization of MazF-Mediated Sequence-Specific RNA Cleavage in Pseudomonas putida Using Massive Parallel Sequencing
Article Snippet: .. This substrate was incubated at 37°C for 15 minutes with two distinct RNA interferases: 2 U of MazF (Takara) in MazF buffer (Takara) containing 4 U of recombinant RNase inhibitor (Takara) or 300 ng of MazFpp in MazFpp buffer (20 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, 0.01% tritonX-100, and 4 U of recombinant RNase inhibitor). .. As the control experiment, the substrate RNA was also treated with 2.5 U of RNase III (New England Biolabs) in NEBNext RNase III Reaction Buffer (New England Biolabs) at 37°C for 15 minutes.

Article Title: Sensitive one-step isothermal detection of pathogen-derived RNAs
Article Snippet: .. Recombinant RNase 340 Inhibitor and pMD20 T-vector were obtained from Takara (Shiga, Japan). .. Malachite green 341 oxalate was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Article Title: Transcriptional Coactivator PC4 Stimulates Promoter Escape and Facilitates Transcriptional Synergy by GAL4-VP16
Article Snippet: .. For 390- and 20-nucleotide (nt) transcripts, in vitro transcription reaction mixtures (25 μl) contained 50 ng of negatively supercoiled pG5HMC2AT or its derivative, 12 mM HEPES-KOH (pH 7.9), 6% glycerol, 60 mM KCl, 0.6 mM EDTA, 8 mM MgCl2 , 5 mM dithiothreitol (DTT), 20 U of RNase inhibitor (TaKaRa), 0.2 mM ATP, 0.2 mM UTP, 0.1 mM 3′- o -methyl GTP, 12.5 μM CTP, 10 μCi of [α-32 P]CTP, 20 ng of TFIIA, 10 ng of TFIIB, 1 μl of FLAG-tagged TFIID (which corresponds to ∼0.1 ng of TATA binding protein [TBP]), 10 ng of TFIIE, 20 ng of TFIIF, 20 ng of recombinant TFIIH, and 100 ng of RNAPII. ..

Article Title: Homodimerization of HYL1 ensures the correct selection of cleavage sites in primary miRNA
Article Snippet: .. Briefly, each 10-μl RNA cleavage assay mixture contained 20 mM Tris HCl (pH 7.0), 50 mM NaCl, 4 mM MgCl2 , 5 mM ATP, 1 mM GTP, 2 units of RNase inhibitor (TaKaRa), RNA substrate, and recombinant DCL1, HYL1 and SE proteins. .. For common reactions, the estimated concentration of proteins and substrate are: DCL1:50 mM; SE:150 mM; HYL1:100 mM; pri-miRNA:200 mM.

In Vitro:

Article Title: Transcriptional Coactivator PC4 Stimulates Promoter Escape and Facilitates Transcriptional Synergy by GAL4-VP16
Article Snippet: .. For 390- and 20-nucleotide (nt) transcripts, in vitro transcription reaction mixtures (25 μl) contained 50 ng of negatively supercoiled pG5HMC2AT or its derivative, 12 mM HEPES-KOH (pH 7.9), 6% glycerol, 60 mM KCl, 0.6 mM EDTA, 8 mM MgCl2 , 5 mM dithiothreitol (DTT), 20 U of RNase inhibitor (TaKaRa), 0.2 mM ATP, 0.2 mM UTP, 0.1 mM 3′- o -methyl GTP, 12.5 μM CTP, 10 μCi of [α-32 P]CTP, 20 ng of TFIIA, 10 ng of TFIIB, 1 μl of FLAG-tagged TFIID (which corresponds to ∼0.1 ng of TATA binding protein [TBP]), 10 ng of TFIIE, 20 ng of TFIIF, 20 ng of recombinant TFIIH, and 100 ng of RNAPII. ..

Immunoprecipitation:

Article Title: Three RNA Binding Proteins Form a Complex to Promote Differentiation of Germline Stem Cell Lineage in Drosophila
Article Snippet: .. RNA Immunoprecipitation (RIP) RNase inhibitor (Takara 2313A) was used for all RIP-related experiments. mei-P26 3′UTR (549 bp and 844 bp from stop codon) was cloned into pUAS-GFP vector between GFP stop codon and SV40 3′UTR. .. S2 cells transfected with TAP-Tut and mei-P26 3′UTR or TAP-GFP and mei-P26 3′UTR were lysed in polysome lysis buffer according to .

Concentration Assay:

Article Title: Vandetanib and ADAM inhibitors synergistically attenuate the pathological migration of EBV-infected retinal pigment epithelial cells by regulating the VEGF-mediated MAPK pathway
Article Snippet: .. Each RNA sample was transcribed into cDNA using oligo (dT) (no. N-7053; Bioneer Corporation, Daejeon, Korea) and AccuPower RT Premix (no. K-2043; 10 mM dNTPs; 2.5 mM, each dNTP; final concentration, 0.5 mM; Bioneer Corporation), oligo (dT)(5 µM), RNase inhibitor (no. 2313A; 10 U/µl; Takara Bio, Inc., Otsu, Japan), and reverse transcriptase (100 U/µl; Bioneer Corporation). .. PCR was performed using Prime Taq Premix with Prime Taq DNA Polymerase, 1 unit/10 µl; 2X reaction buffer; 4 mM MgCl2; enzyme stabilizer; sediment; loading dye (pH 9.0) and 0.5 mM each of dATP, dCTP, dGTP, dTTP (no. G-3002; GeNet Bio, Daejeon, Korea) with the specified primers ( ) and TaKaRa PCR Thermal Cycler Dice (no. TP600; Takara Bio, Inc.).

Incubation:

Article Title: Characterization of MazF-Mediated Sequence-Specific RNA Cleavage in Pseudomonas putida Using Massive Parallel Sequencing
Article Snippet: .. This substrate was incubated at 37°C for 15 minutes with two distinct RNA interferases: 2 U of MazF (Takara) in MazF buffer (Takara) containing 4 U of recombinant RNase inhibitor (Takara) or 300 ng of MazFpp in MazFpp buffer (20 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, 0.01% tritonX-100, and 4 U of recombinant RNase inhibitor). .. As the control experiment, the substrate RNA was also treated with 2.5 U of RNase III (New England Biolabs) in NEBNext RNase III Reaction Buffer (New England Biolabs) at 37°C for 15 minutes.

Lysis:

Article Title: Developmental heterogeneity of microglia and brain myeloid cells revealed by deep single-cell RNA sequencing
Article Snippet: .. Single cells were index sorted into 96-well plates containing 4uL lysis buffer (4U Recombinant RNase Inhibitor (Takara Bio 2313B), 0.05% Triton X-100, 2.5mM dNTP mix (Thermo Fisher Scientific R0192), 2.5uM Oligo-dT30VN (5′-AAGCAGTGGTATCAACGCAGAGTACT30 VN-3′, ERCC Spike-ins (Thermo Fisher Scientific 4456740) diluted at 1:2.4×107 ). ..

Binding Assay:

Article Title: Transcriptional Coactivator PC4 Stimulates Promoter Escape and Facilitates Transcriptional Synergy by GAL4-VP16
Article Snippet: .. For 390- and 20-nucleotide (nt) transcripts, in vitro transcription reaction mixtures (25 μl) contained 50 ng of negatively supercoiled pG5HMC2AT or its derivative, 12 mM HEPES-KOH (pH 7.9), 6% glycerol, 60 mM KCl, 0.6 mM EDTA, 8 mM MgCl2 , 5 mM dithiothreitol (DTT), 20 U of RNase inhibitor (TaKaRa), 0.2 mM ATP, 0.2 mM UTP, 0.1 mM 3′- o -methyl GTP, 12.5 μM CTP, 10 μCi of [α-32 P]CTP, 20 ng of TFIIA, 10 ng of TFIIB, 1 μl of FLAG-tagged TFIID (which corresponds to ∼0.1 ng of TATA binding protein [TBP]), 10 ng of TFIIE, 20 ng of TFIIF, 20 ng of recombinant TFIIH, and 100 ng of RNAPII. ..

Plasmid Preparation:

Article Title: Three RNA Binding Proteins Form a Complex to Promote Differentiation of Germline Stem Cell Lineage in Drosophila
Article Snippet: .. RNA Immunoprecipitation (RIP) RNase inhibitor (Takara 2313A) was used for all RIP-related experiments. mei-P26 3′UTR (549 bp and 844 bp from stop codon) was cloned into pUAS-GFP vector between GFP stop codon and SV40 3′UTR. .. S2 cells transfected with TAP-Tut and mei-P26 3′UTR or TAP-GFP and mei-P26 3′UTR were lysed in polysome lysis buffer according to .

Cleavage Assay:

Article Title: Homodimerization of HYL1 ensures the correct selection of cleavage sites in primary miRNA
Article Snippet: .. Briefly, each 10-μl RNA cleavage assay mixture contained 20 mM Tris HCl (pH 7.0), 50 mM NaCl, 4 mM MgCl2 , 5 mM ATP, 1 mM GTP, 2 units of RNase inhibitor (TaKaRa), RNA substrate, and recombinant DCL1, HYL1 and SE proteins. .. For common reactions, the estimated concentration of proteins and substrate are: DCL1:50 mM; SE:150 mM; HYL1:100 mM; pri-miRNA:200 mM.

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  • 90
    TaKaRa rnase protection
    ( A ) <t>RNase</t> A protection assay (RPA) of rat MC4R in penile tissues. Poly(A) + RNA (6 μg) was hybridized with a rat MC4R <t>cDNA</t> probe (second intracellular loop to mid transmembrane-6) that, upon RNase digestion, would result in a radiolabeled fragment of 313 nucleotides, revealed by denaturing PAGE and autoradiography (as detailed in Materials and Methods ). An internal standard rat MC4R fragment of 240 nucleotides is shown. ( B ) Binding of [ 125 I]MTII to rat penile membranes. Inhibition of [ 125 I]MTII binding by MTII and the THIQ MC4R agonist was performed by using 70 pM radiolabeled MTII for 70 min at 20°C; the results are expressed as percent of [ 125 I]MTII specifically bound. IC 50 values for MTII and MC4R agonist were 1.5 and 11 nM, respectively ( n = 3 experiments).
    Rnase Protection, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase protection/product/TaKaRa
    Average 90 stars, based on 1 article reviews
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    93
    TaKaRa rnase inhibitor
    The inhibition effects of GIDP and RNA on GTPase activity. The GTPase assay was carried out at 37°C for 30 min in a 10-μl reaction mixture (50 mM Tris [pH 7.5], 5 mM Mg 2+ , 5 mM <t>DTT,</t> 0.5 μCi of [α- 32 P]GTP, 40 U of <t>RNase</t> inhibitor, and 30 ng of the helicase-like domain) that contained either 2 mM GIDP (lane 2) or 3 μM 5′ plus-strand RNA of BaMV (lane 3). Lane 1, standard GTPase reaction. The products were then analyzed by TLC.
    Rnase Inhibitor, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase inhibitor/product/TaKaRa
    Average 93 stars, based on 31 article reviews
    Price from $9.99 to $1999.99
    rnase inhibitor - by Bioz Stars, 2020-08
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    85
    TaKaRa wt rnase l
    Expression of <t>RNase</t> L blocks L1 RNP formation. HeLa-M cells were co-transfected with pES2TE1 and either an empty vector (pcDNA 3.0) or a plasmid that encodes an amino-terminal Myc-tagged RNase L expression plasmid. Immunofluorescent confocal microscopy was used to examine L1 ORF2p accumulation in cytoplasmic foci by exploiting the FLAG-HA epitope-tag in pES2TE1 48 h after transfection. The top labels indicate the antibodies used to detect the indicated proteins: anti-HA-ORF2p, red; anti-EBNA-1, green; anti-Myc RNase L, magenta. The labels on the left side of the figure indicate the empty vector or RNase L constructs that were co-transfected into cells. The rightmost column indicates the merged overlay staining. L1 ORF2p formed discrete cytoplasmic punctate localization in co-transfection experiments performed with the empty vector and RNase L catalytically inactive mutant (R667A), but not with WT RNase L. For each condition, either two or three slides were examined per experiment. About 200 cells were examined per slide and representative images were captured. The experiment was conducted three times (biological replicates) with similar results.
    Wt Rnase L, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ( A ) RNase A protection assay (RPA) of rat MC4R in penile tissues. Poly(A) + RNA (6 μg) was hybridized with a rat MC4R cDNA probe (second intracellular loop to mid transmembrane-6) that, upon RNase digestion, would result in a radiolabeled fragment of 313 nucleotides, revealed by denaturing PAGE and autoradiography (as detailed in Materials and Methods ). An internal standard rat MC4R fragment of 240 nucleotides is shown. ( B ) Binding of [ 125 I]MTII to rat penile membranes. Inhibition of [ 125 I]MTII binding by MTII and the THIQ MC4R agonist was performed by using 70 pM radiolabeled MTII for 70 min at 20°C; the results are expressed as percent of [ 125 I]MTII specifically bound. IC 50 values for MTII and MC4R agonist were 1.5 and 11 nM, respectively ( n = 3 experiments).

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

    Article Title: A role for the melanocortin 4 receptor in sexual function

    doi: 10.1073/pnas.172378699

    Figure Lengend Snippet: ( A ) RNase A protection assay (RPA) of rat MC4R in penile tissues. Poly(A) + RNA (6 μg) was hybridized with a rat MC4R cDNA probe (second intracellular loop to mid transmembrane-6) that, upon RNase digestion, would result in a radiolabeled fragment of 313 nucleotides, revealed by denaturing PAGE and autoradiography (as detailed in Materials and Methods ). An internal standard rat MC4R fragment of 240 nucleotides is shown. ( B ) Binding of [ 125 I]MTII to rat penile membranes. Inhibition of [ 125 I]MTII binding by MTII and the THIQ MC4R agonist was performed by using 70 pM radiolabeled MTII for 70 min at 20°C; the results are expressed as percent of [ 125 I]MTII specifically bound. IC 50 values for MTII and MC4R agonist were 1.5 and 11 nM, respectively ( n = 3 experiments).

    Article Snippet: The reverse primers were located in the third intracellular loop. mRNA was isolated from fresh tissues as described under RNase Protection . cDNA was prepared from the 0.5–1 μg mRNA by using the CLONTECH Advantage RT kit per manufacturer's instructions.

    Techniques: Recombinase Polymerase Amplification, Polyacrylamide Gel Electrophoresis, Autoradiography, Binding Assay, Inhibition

    The inhibition effects of GIDP and RNA on GTPase activity. The GTPase assay was carried out at 37°C for 30 min in a 10-μl reaction mixture (50 mM Tris [pH 7.5], 5 mM Mg 2+ , 5 mM DTT, 0.5 μCi of [α- 32 P]GTP, 40 U of RNase inhibitor, and 30 ng of the helicase-like domain) that contained either 2 mM GIDP (lane 2) or 3 μM 5′ plus-strand RNA of BaMV (lane 3). Lane 1, standard GTPase reaction. The products were then analyzed by TLC.

    Journal: Journal of Virology

    Article Title: The Helicase-Like Domain of Plant Potexvirus Replicase Participates in Formation of RNA 5? Cap Structure by Exhibiting RNA 5?-Triphosphatase Activity

    doi: 10.1128/JVI.75.24.12114-12120.2001

    Figure Lengend Snippet: The inhibition effects of GIDP and RNA on GTPase activity. The GTPase assay was carried out at 37°C for 30 min in a 10-μl reaction mixture (50 mM Tris [pH 7.5], 5 mM Mg 2+ , 5 mM DTT, 0.5 μCi of [α- 32 P]GTP, 40 U of RNase inhibitor, and 30 ng of the helicase-like domain) that contained either 2 mM GIDP (lane 2) or 3 μM 5′ plus-strand RNA of BaMV (lane 3). Lane 1, standard GTPase reaction. The products were then analyzed by TLC.

    Article Snippet: The in vitro RNA-capping assay mixture contained 50 mM Tris (pH 7.5), 4 mM MgCl2 , 5 mM DTT, 40 U of RNase inhibitor, 20 μCi of [α-32 P]GTP, 1.2% n -octyl-β- d -glucopyranoside, 800 μM AdoMet, 5 μg of RNA, and 200 ng of the purified capping enzyme domain in a final volume of 20 μl.

    Techniques: Inhibition, Activity Assay, Thin Layer Chromatography

    Restoration of RNase III-dependent regulation of eno - cat expression by putative cis -antisense RNA expression. ( a ) Schematic diagram showing the transcriptional initiation and termination sites (TISs and TTSs, respectively) of putative cis -antisense RNA. The 5′ and 3′ termini of putative cis- antisense RNAs that were inferred from cDNAs were located at positions +451, +272, −36, and −83 (designated as TIS1, TIS2, TIS3, and TIS4, respectively) from the start codon of the eno coding region, and at positions +1,419, +1,265, +736, and +696 from the start codon of the pyrG coding region (designated as TTS1, TTS2, TTS3, and TTS4, respectively). The secondary structure of the TTSs was inferred using the M-fold program. ( b ) Effects of alterations in the eno 5′ UTR on the regulation of eno - cat expression. Top: schematic representation of the region encompassing the eno and pyrG genes in W3110 WT and W3110 PBAD eno strains. Bottom: degree of chloramphenicol resistance of W3110 and W3110 PBAD eno strains harbouring pERS1. The W3110 and W3110 PBAD eno strains harbouring pERS1 were transformed with pPM30, pRNG3 (RNase G), or pRNC3 (RNase III). The transformants were grown to an OD 600 of 0.6 in LB containing 1 mM IPTG and 0.01% arabinose, diluted, and spotted on LB agar containing 0.01% arabinose and 0 (Cm 0) or 100 (Cm 100) μg ml −1 chloramphenicol. (c) Schematic representation of pERS1-derived plasmids that additionally contain a DNA segment encompassing the eno and pyrG genes (from + 875 of the pyrG coding region to + 748, + 320, or + 200 of the eno coding region). (d) Minimal inhibitory concentrations (MICs) of MG1655 harbouring pERS1, pERS-AS748, pERS-AS320, or pERS-AS200 against chloramphenicol. Measurements of the MICs were performed independently, in triplicate, in LB containing various chloramphenicol concentrations; significant differences are indicated with different letters (one-way analysis of variance [ANOVA] followed by the Student–Newman–Keuls test, P

    Journal: Scientific Reports

    Article Title: The coordinated action of RNase III and RNase G controls enolase expression in response to oxygen availability in Escherichia coli

    doi: 10.1038/s41598-019-53883-y

    Figure Lengend Snippet: Restoration of RNase III-dependent regulation of eno - cat expression by putative cis -antisense RNA expression. ( a ) Schematic diagram showing the transcriptional initiation and termination sites (TISs and TTSs, respectively) of putative cis -antisense RNA. The 5′ and 3′ termini of putative cis- antisense RNAs that were inferred from cDNAs were located at positions +451, +272, −36, and −83 (designated as TIS1, TIS2, TIS3, and TIS4, respectively) from the start codon of the eno coding region, and at positions +1,419, +1,265, +736, and +696 from the start codon of the pyrG coding region (designated as TTS1, TTS2, TTS3, and TTS4, respectively). The secondary structure of the TTSs was inferred using the M-fold program. ( b ) Effects of alterations in the eno 5′ UTR on the regulation of eno - cat expression. Top: schematic representation of the region encompassing the eno and pyrG genes in W3110 WT and W3110 PBAD eno strains. Bottom: degree of chloramphenicol resistance of W3110 and W3110 PBAD eno strains harbouring pERS1. The W3110 and W3110 PBAD eno strains harbouring pERS1 were transformed with pPM30, pRNG3 (RNase G), or pRNC3 (RNase III). The transformants were grown to an OD 600 of 0.6 in LB containing 1 mM IPTG and 0.01% arabinose, diluted, and spotted on LB agar containing 0.01% arabinose and 0 (Cm 0) or 100 (Cm 100) μg ml −1 chloramphenicol. (c) Schematic representation of pERS1-derived plasmids that additionally contain a DNA segment encompassing the eno and pyrG genes (from + 875 of the pyrG coding region to + 748, + 320, or + 200 of the eno coding region). (d) Minimal inhibitory concentrations (MICs) of MG1655 harbouring pERS1, pERS-AS748, pERS-AS320, or pERS-AS200 against chloramphenicol. Measurements of the MICs were performed independently, in triplicate, in LB containing various chloramphenicol concentrations; significant differences are indicated with different letters (one-way analysis of variance [ANOVA] followed by the Student–Newman–Keuls test, P

    Article Snippet: An aliquot (4 pmol) of 32 P-5′ end labelled RNA was incubated with 1 ng of purified RNase III with 0.25 mg ml−1 yeast tRNA (Thermo Fisher Scientific), 20 U of RNase inhibitor (Takara), and RNase III cleavage buffer, with or without MgCl2 .

    Techniques: Expressing, RNA Expression, Transformation Assay, Derivative Assay

    Regulation of Eno expression by RNase III and/or RNase G. (a) Effects of rnc and/or rng deletion on the expression level of eno . Escherichia coli MG1655 strains (WT, Δ rng , Δ rnc , and Δ rnc rng ) were grown in LB medium at 37 °C to mid-log phase and harvested for western blot analysis of Eno, Rng, and Rnc using protein-specific polyclonal antibodies. The expression levels of Eno, Rng, and Rnc were compared by setting those of WT to 1. (b) Independent modulation of Eno expression levels by RNase G and RNase III. Western blotting was performed as described for (a) using Δ rnc rng strains harbouring pPM30, pRNG3, or pRNC3. The expression levels of Eno, Rng, and Rnc were compared by setting those of Δ rnc rng harbouring pPM30 to 1. (c) Effects of rnc and/or rng deletion on the eno mRNA abundance. Total cellular RNA was extracted from cultures grown to an OD 600 of 0.6 using an RNeasy mini prep kit. The number of amplicons of enolase and other rnpB mRNA amplified from the cDNAs of the (left) WT, Δ rnc . Δ rng and Δ rnc rng strains (right) harbouring pPM30, pRNC3, or pRNG3. The eno mRNA expression levels were compared by setting those of WT or Δ rnc rng harbouring pPM30 to 1. PCR products were resolved in an 1.5% agarose gel. (d) Identification of the regulatory DNA region that affected the eno expression levels. Top: Schematic diagram of the eno-cat reporter. Bottom: Effects of RNase G and RNase III expression levels on the degree of chloramphenicol resistance of MG1655 cells. MG1655 WT, Δ rng , and Δ rnc cells harbouring pERS1 were transformed with pPM30, pRNG3 (RNase G), or pRNC3 (RNase III). The transformants were grown in LB containing 1 mM IPTG to an OD 600 of 0.6, diluted, and spotted on LB agar containing 0 (Cm 0) or 75 (Cm 75) μg ml −1 chloramphenicol. For (a,b) , the S1 protein was used as an internal standard to evaluate the amount of cell extract in each lane. For (c) , the rnpB mRNA was used as an internal standard to evaluate the amount of cell extract in each lane. For (a–c) , the data are presented as means ± s. e. m. of at least three independent experiments.

    Journal: Scientific Reports

    Article Title: The coordinated action of RNase III and RNase G controls enolase expression in response to oxygen availability in Escherichia coli

    doi: 10.1038/s41598-019-53883-y

    Figure Lengend Snippet: Regulation of Eno expression by RNase III and/or RNase G. (a) Effects of rnc and/or rng deletion on the expression level of eno . Escherichia coli MG1655 strains (WT, Δ rng , Δ rnc , and Δ rnc rng ) were grown in LB medium at 37 °C to mid-log phase and harvested for western blot analysis of Eno, Rng, and Rnc using protein-specific polyclonal antibodies. The expression levels of Eno, Rng, and Rnc were compared by setting those of WT to 1. (b) Independent modulation of Eno expression levels by RNase G and RNase III. Western blotting was performed as described for (a) using Δ rnc rng strains harbouring pPM30, pRNG3, or pRNC3. The expression levels of Eno, Rng, and Rnc were compared by setting those of Δ rnc rng harbouring pPM30 to 1. (c) Effects of rnc and/or rng deletion on the eno mRNA abundance. Total cellular RNA was extracted from cultures grown to an OD 600 of 0.6 using an RNeasy mini prep kit. The number of amplicons of enolase and other rnpB mRNA amplified from the cDNAs of the (left) WT, Δ rnc . Δ rng and Δ rnc rng strains (right) harbouring pPM30, pRNC3, or pRNG3. The eno mRNA expression levels were compared by setting those of WT or Δ rnc rng harbouring pPM30 to 1. PCR products were resolved in an 1.5% agarose gel. (d) Identification of the regulatory DNA region that affected the eno expression levels. Top: Schematic diagram of the eno-cat reporter. Bottom: Effects of RNase G and RNase III expression levels on the degree of chloramphenicol resistance of MG1655 cells. MG1655 WT, Δ rng , and Δ rnc cells harbouring pERS1 were transformed with pPM30, pRNG3 (RNase G), or pRNC3 (RNase III). The transformants were grown in LB containing 1 mM IPTG to an OD 600 of 0.6, diluted, and spotted on LB agar containing 0 (Cm 0) or 75 (Cm 75) μg ml −1 chloramphenicol. For (a,b) , the S1 protein was used as an internal standard to evaluate the amount of cell extract in each lane. For (c) , the rnpB mRNA was used as an internal standard to evaluate the amount of cell extract in each lane. For (a–c) , the data are presented as means ± s. e. m. of at least three independent experiments.

    Article Snippet: An aliquot (4 pmol) of 32 P-5′ end labelled RNA was incubated with 1 ng of purified RNase III with 0.25 mg ml−1 yeast tRNA (Thermo Fisher Scientific), 20 U of RNase inhibitor (Takara), and RNase III cleavage buffer, with or without MgCl2 .

    Techniques: Expressing, Western Blot, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Transformation Assay

    A model for the molecular mechanism involved in RNase III- and RNase G-mediated regulation of eno expression in response to oxygen availability in E. coli . RNase III and RNase G coordinately regulate eno expression in response to oxygen availability in E. coli . Low oxygen activates RNase III activity and promotes the degradation of rng mRNA, leading to decreased expression of RNase G. Enhanced RNase III-mediated cleavage of primary eno mRNA transcripts promotes Eno protein production under anaerobic conditions. This RNase III-mediated processing involves cis -antisense RNA synthesised from the eno coding region to that of pyrG in the opposite direction of mRNA synthesis of these genes.

    Journal: Scientific Reports

    Article Title: The coordinated action of RNase III and RNase G controls enolase expression in response to oxygen availability in Escherichia coli

    doi: 10.1038/s41598-019-53883-y

    Figure Lengend Snippet: A model for the molecular mechanism involved in RNase III- and RNase G-mediated regulation of eno expression in response to oxygen availability in E. coli . RNase III and RNase G coordinately regulate eno expression in response to oxygen availability in E. coli . Low oxygen activates RNase III activity and promotes the degradation of rng mRNA, leading to decreased expression of RNase G. Enhanced RNase III-mediated cleavage of primary eno mRNA transcripts promotes Eno protein production under anaerobic conditions. This RNase III-mediated processing involves cis -antisense RNA synthesised from the eno coding region to that of pyrG in the opposite direction of mRNA synthesis of these genes.

    Article Snippet: An aliquot (4 pmol) of 32 P-5′ end labelled RNA was incubated with 1 ng of purified RNase III with 0.25 mg ml−1 yeast tRNA (Thermo Fisher Scientific), 20 U of RNase inhibitor (Takara), and RNase III cleavage buffer, with or without MgCl2 .

    Techniques: Expressing, Activity Assay

    Identification of RNase cleavage sites in eno mRNA in vivo . (a) Primer extension analysis of the 5′ UTR of eno mRNA in vivo . Total RNA was isolated from MG1655 strains (WT, Δ rng , Δ rnc , and Δ rnc rng ) and hybridised with the 5′ end 32 P-labelled primer (eno + 52 R). Synthesised cDNA products were separated on a 6% polyacrylamide gel containing 8 M of urea. Sequencing ladders were synthesised with the same primers used for cDNA synthesis and PCR DNA encompassing the eno gene was used as a template. (b) S1 nuclease mapping. Total RNA was hybridised with the 5′ end 32 P-labelled DNA probe. The DNA: RNA complex was treated with 1 U of S1 nuclease and separated in denaturing gel as described above. (c) Predicted eno 5′ UTR secondary structure and RNase cleavage sites. The secondary structure was inferred using the M-fold program. RNase III (1, 2, 3, and 4) cleavage sites identified in (a) and (b) are indicated. The putative Shine–Dalgarno sequence and start codon are indicated as blue and red colours, respectively.

    Journal: Scientific Reports

    Article Title: The coordinated action of RNase III and RNase G controls enolase expression in response to oxygen availability in Escherichia coli

    doi: 10.1038/s41598-019-53883-y

    Figure Lengend Snippet: Identification of RNase cleavage sites in eno mRNA in vivo . (a) Primer extension analysis of the 5′ UTR of eno mRNA in vivo . Total RNA was isolated from MG1655 strains (WT, Δ rng , Δ rnc , and Δ rnc rng ) and hybridised with the 5′ end 32 P-labelled primer (eno + 52 R). Synthesised cDNA products were separated on a 6% polyacrylamide gel containing 8 M of urea. Sequencing ladders were synthesised with the same primers used for cDNA synthesis and PCR DNA encompassing the eno gene was used as a template. (b) S1 nuclease mapping. Total RNA was hybridised with the 5′ end 32 P-labelled DNA probe. The DNA: RNA complex was treated with 1 U of S1 nuclease and separated in denaturing gel as described above. (c) Predicted eno 5′ UTR secondary structure and RNase cleavage sites. The secondary structure was inferred using the M-fold program. RNase III (1, 2, 3, and 4) cleavage sites identified in (a) and (b) are indicated. The putative Shine–Dalgarno sequence and start codon are indicated as blue and red colours, respectively.

    Article Snippet: An aliquot (4 pmol) of 32 P-5′ end labelled RNA was incubated with 1 ng of purified RNase III with 0.25 mg ml−1 yeast tRNA (Thermo Fisher Scientific), 20 U of RNase inhibitor (Takara), and RNase III cleavage buffer, with or without MgCl2 .

    Techniques: In Vivo, Isolation, Sequencing, Polymerase Chain Reaction

    Expression of RNase L blocks L1 RNP formation. HeLa-M cells were co-transfected with pES2TE1 and either an empty vector (pcDNA 3.0) or a plasmid that encodes an amino-terminal Myc-tagged RNase L expression plasmid. Immunofluorescent confocal microscopy was used to examine L1 ORF2p accumulation in cytoplasmic foci by exploiting the FLAG-HA epitope-tag in pES2TE1 48 h after transfection. The top labels indicate the antibodies used to detect the indicated proteins: anti-HA-ORF2p, red; anti-EBNA-1, green; anti-Myc RNase L, magenta. The labels on the left side of the figure indicate the empty vector or RNase L constructs that were co-transfected into cells. The rightmost column indicates the merged overlay staining. L1 ORF2p formed discrete cytoplasmic punctate localization in co-transfection experiments performed with the empty vector and RNase L catalytically inactive mutant (R667A), but not with WT RNase L. For each condition, either two or three slides were examined per experiment. About 200 cells were examined per slide and representative images were captured. The experiment was conducted three times (biological replicates) with similar results.

    Journal: Nucleic Acids Research

    Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

    doi: 10.1093/nar/gkt1308

    Figure Lengend Snippet: Expression of RNase L blocks L1 RNP formation. HeLa-M cells were co-transfected with pES2TE1 and either an empty vector (pcDNA 3.0) or a plasmid that encodes an amino-terminal Myc-tagged RNase L expression plasmid. Immunofluorescent confocal microscopy was used to examine L1 ORF2p accumulation in cytoplasmic foci by exploiting the FLAG-HA epitope-tag in pES2TE1 48 h after transfection. The top labels indicate the antibodies used to detect the indicated proteins: anti-HA-ORF2p, red; anti-EBNA-1, green; anti-Myc RNase L, magenta. The labels on the left side of the figure indicate the empty vector or RNase L constructs that were co-transfected into cells. The rightmost column indicates the merged overlay staining. L1 ORF2p formed discrete cytoplasmic punctate localization in co-transfection experiments performed with the empty vector and RNase L catalytically inactive mutant (R667A), but not with WT RNase L. For each condition, either two or three slides were examined per experiment. About 200 cells were examined per slide and representative images were captured. The experiment was conducted three times (biological replicates) with similar results.

    Article Snippet: In a separate control experiment, we demonstrated that co-transfection of WT RNase L did not affect HYG mRNA levels expressed from pIREShyg (Clontech) ( Supplementary Figure S3 ).

    Techniques: Expressing, Transfection, Plasmid Preparation, Confocal Microscopy, Construct, Staining, Cotransfection, Mutagenesis

    Human RNase L alone does not affect G418-resistant foci formation. ( A ) Results from the Assay: HeLa-M cells were co-transfected with pcDNA 3.0 (Gibco/Life Technologies/InVitrogen) and either an empty vector (pFLAG-CMV-2) or an amino-terminal FLAG-tagged RNase L expression plasmid. The cells were subjected to selection for 10 days and G418-resistant foci were fixed and stained with crystal violet for visualization purposes. A representative tissue culture dish for each condition is shown. ( B ) Quantitation of the Assays: The X-axis depicts construct names. The Y-axis depicts the number of G418-resistant foci per cell culture dish. Quantification was performed as outlined in the legend to Figure 2 B. Data are shown as the mean ± standard deviation (SD) from a single experiment with three technical replicates. The experiment was conducted three times (biological replicates) with similar results. No statistically significant difference was found with one-way ANOVA and post hoc tests. ( C ) Protein expression analyses: The WT RNase L, catalytically inactive RNase L mutant (R667A) and constitutively active (NΔ385) RNase L mutant were detected from total cell lysates in western blots with anti-RNase L antibody 2 days after transfection. β-Actin served as loading and transfer control. Size standards are indicated in kDa at the left of the gel.

    Journal: Nucleic Acids Research

    Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

    doi: 10.1093/nar/gkt1308

    Figure Lengend Snippet: Human RNase L alone does not affect G418-resistant foci formation. ( A ) Results from the Assay: HeLa-M cells were co-transfected with pcDNA 3.0 (Gibco/Life Technologies/InVitrogen) and either an empty vector (pFLAG-CMV-2) or an amino-terminal FLAG-tagged RNase L expression plasmid. The cells were subjected to selection for 10 days and G418-resistant foci were fixed and stained with crystal violet for visualization purposes. A representative tissue culture dish for each condition is shown. ( B ) Quantitation of the Assays: The X-axis depicts construct names. The Y-axis depicts the number of G418-resistant foci per cell culture dish. Quantification was performed as outlined in the legend to Figure 2 B. Data are shown as the mean ± standard deviation (SD) from a single experiment with three technical replicates. The experiment was conducted three times (biological replicates) with similar results. No statistically significant difference was found with one-way ANOVA and post hoc tests. ( C ) Protein expression analyses: The WT RNase L, catalytically inactive RNase L mutant (R667A) and constitutively active (NΔ385) RNase L mutant were detected from total cell lysates in western blots with anti-RNase L antibody 2 days after transfection. β-Actin served as loading and transfer control. Size standards are indicated in kDa at the left of the gel.

    Article Snippet: In a separate control experiment, we demonstrated that co-transfection of WT RNase L did not affect HYG mRNA levels expressed from pIREShyg (Clontech) ( Supplementary Figure S3 ).

    Techniques: Transfection, Plasmid Preparation, Expressing, Selection, Staining, Quantitation Assay, Construct, Cell Culture, Standard Deviation, Mutagenesis, Western Blot

    Expression of RNase L reduces L1 protein expression. ( A ) L1 protein expression: HeLa-M cells were co-transfected with pAD2TE1 and either an empty vector (pFLAG-CMV-2) or a plasmid that encodes an amino-terminal FLAG-tagged RNase L expression plasmid. Two days after transfection, cells were selected with hygromycin containing medium for an additional 4 days when total cell lysates and L1 RNPs were prepared. Western blotting, using anti-T7 and anti-TAP antibodies, was used to detect ORF1p and ORF2p, respectively. Shown are two exposures of the ORF2p anti-TAP western blot. Endogenous ribosomal S6 protein was used as the loading/transfer control. β-Actin detection discriminated the total cell lysate (left side of panel) from the L1 RNP fractions (right side of panel). The experiments were repeated twice (biological replicates) with similar results. Shown are data from one representative experiment. ( B ) RNase L does not inhibit exogenous EGFP protein expression: HeLa-M cells were co-transfected with pEGFP-C1 and either an empty vector (pFLAG-CMV-2) or a plasmid that encodes an amino-terminal FLAG-tagged RNase L expression plasmid. Total cell lysates were harvested and the expression of RNase L and GFP was detected in western blot experiments using anti-RNase L and anti-GFP antibodies at 48 h after transfection. GAPDH served as a loading and transfer control.

    Journal: Nucleic Acids Research

    Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

    doi: 10.1093/nar/gkt1308

    Figure Lengend Snippet: Expression of RNase L reduces L1 protein expression. ( A ) L1 protein expression: HeLa-M cells were co-transfected with pAD2TE1 and either an empty vector (pFLAG-CMV-2) or a plasmid that encodes an amino-terminal FLAG-tagged RNase L expression plasmid. Two days after transfection, cells were selected with hygromycin containing medium for an additional 4 days when total cell lysates and L1 RNPs were prepared. Western blotting, using anti-T7 and anti-TAP antibodies, was used to detect ORF1p and ORF2p, respectively. Shown are two exposures of the ORF2p anti-TAP western blot. Endogenous ribosomal S6 protein was used as the loading/transfer control. β-Actin detection discriminated the total cell lysate (left side of panel) from the L1 RNP fractions (right side of panel). The experiments were repeated twice (biological replicates) with similar results. Shown are data from one representative experiment. ( B ) RNase L does not inhibit exogenous EGFP protein expression: HeLa-M cells were co-transfected with pEGFP-C1 and either an empty vector (pFLAG-CMV-2) or a plasmid that encodes an amino-terminal FLAG-tagged RNase L expression plasmid. Total cell lysates were harvested and the expression of RNase L and GFP was detected in western blot experiments using anti-RNase L and anti-GFP antibodies at 48 h after transfection. GAPDH served as a loading and transfer control.

    Article Snippet: In a separate control experiment, we demonstrated that co-transfection of WT RNase L did not affect HYG mRNA levels expressed from pIREShyg (Clontech) ( Supplementary Figure S3 ).

    Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot

    Expression of RNase L inhibits L1 retrotransposition in an EGFP -based retrotransposition assay. ( A ) Results from the assays: HeLa-M cells were co-transfected with an expression construct containing an active human L1 (pLRE3- mEGFPI ) and an empty vector (pFLAG-CMV-2), a plasmid encoding an amino-terminal FLAG-tagged RNase L expression plasmid or an amino-terminal HA-tagged A3A expression plasmid. Experiments with a retrotransposition-defective L1 pJM111-LRE3- mEGFPI served as a negative control. The cells were subjected to puromycin selection for 4 days after transfection. Fluorescence Activated Cell Sorting (FACS) was then used to screen for EGFP-positive cells. The X-axis indicates the construct name. The Y-axis indicates the percentage of EGFP-positive cells. For each sample, 2 × 10 5 cells were analyzed and the percentage of EGFP-positive cells was calculated with using the FlowJo software package. Data were analyzed with one-way ANOVA with post hoc tests and are shown as mean ± SD from a single experiment with three technical replicates. * P

    Journal: Nucleic Acids Research

    Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

    doi: 10.1093/nar/gkt1308

    Figure Lengend Snippet: Expression of RNase L inhibits L1 retrotransposition in an EGFP -based retrotransposition assay. ( A ) Results from the assays: HeLa-M cells were co-transfected with an expression construct containing an active human L1 (pLRE3- mEGFPI ) and an empty vector (pFLAG-CMV-2), a plasmid encoding an amino-terminal FLAG-tagged RNase L expression plasmid or an amino-terminal HA-tagged A3A expression plasmid. Experiments with a retrotransposition-defective L1 pJM111-LRE3- mEGFPI served as a negative control. The cells were subjected to puromycin selection for 4 days after transfection. Fluorescence Activated Cell Sorting (FACS) was then used to screen for EGFP-positive cells. The X-axis indicates the construct name. The Y-axis indicates the percentage of EGFP-positive cells. For each sample, 2 × 10 5 cells were analyzed and the percentage of EGFP-positive cells was calculated with using the FlowJo software package. Data were analyzed with one-way ANOVA with post hoc tests and are shown as mean ± SD from a single experiment with three technical replicates. * P

    Article Snippet: In a separate control experiment, we demonstrated that co-transfection of WT RNase L did not affect HYG mRNA levels expressed from pIREShyg (Clontech) ( Supplementary Figure S3 ).

    Techniques: Expressing, Transfection, Construct, Plasmid Preparation, Negative Control, Selection, Fluorescence, FACS, Software

    Inhibition of L1 retrotransposition by RNase L. ( A ) L1 Retrotransposition Assays: HeLa-M cells were co-transfected with pJM101/L1.3 and either an empty vector (pFLAG-CMV-2) or a plasmid that encodes amino-terminal FLAG-tagged versions of the following proteins: RNase L, A3A or RIG-I. The cells were subjected to selection for 10 days and G418-resistant foci were fixed and stained with crystal violet for visualization purposes. A representative tissue culture dish for each condition is shown. ( B ) Quantitation of the L1 Retrotransposition Assays: The X-axis depicts the co-transfected construct names. The Y-axis depicts the number of G418-resistant foci per cell culture dish. Data are shown as the mean ± standard deviation (SD) from a single experiment with three technical replicates. * P

    Journal: Nucleic Acids Research

    Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

    doi: 10.1093/nar/gkt1308

    Figure Lengend Snippet: Inhibition of L1 retrotransposition by RNase L. ( A ) L1 Retrotransposition Assays: HeLa-M cells were co-transfected with pJM101/L1.3 and either an empty vector (pFLAG-CMV-2) or a plasmid that encodes amino-terminal FLAG-tagged versions of the following proteins: RNase L, A3A or RIG-I. The cells were subjected to selection for 10 days and G418-resistant foci were fixed and stained with crystal violet for visualization purposes. A representative tissue culture dish for each condition is shown. ( B ) Quantitation of the L1 Retrotransposition Assays: The X-axis depicts the co-transfected construct names. The Y-axis depicts the number of G418-resistant foci per cell culture dish. Data are shown as the mean ± standard deviation (SD) from a single experiment with three technical replicates. * P

    Article Snippet: In a separate control experiment, we demonstrated that co-transfection of WT RNase L did not affect HYG mRNA levels expressed from pIREShyg (Clontech) ( Supplementary Figure S3 ).

    Techniques: Inhibition, Transfection, Plasmid Preparation, Selection, Staining, Quantitation Assay, Construct, Cell Culture, Standard Deviation

    An overview of the L1 and IAP retrotransposition assays. ( A ) Schematics of L1 and IAP constructs: The L1 and IAP constructs contain a NEO-based ( mneoI ) or EGFP -based ( mEGFPI ) retrotransposition indicator cassette near their 3′ ends. The indicator cassettes are in an anti-sense (backward) orientation relative to the transcriptional orientation of the L1 or IAP elements. The indicator cassettes also contain an intron that is in the same transcriptional orientation as the retroelement. SD and SA indicate the splice donor and splice acceptor sites of the intron, respectively. Pr′ indicates the promoter driving the expression of the retrotransposition indicator cassette. Closed lollipops indicate the polyadenylation signal on the indicator cassette. A CMV promoter enhances the expression of the pJM101/L1.3, pAD2TE1, pES2TE1 and pAD3TE1 L1 vectors. An SV40 polyadenylation signal is present at the 3′ end of each L1 expression cassette. Notably, the mneoI -based L1 vectors are expressed from a pCEP4 vector that contains a HYG and an EBNA-1 gene. The mEGFPI-based L1 vectors are expressed from a pCEP4 vector that was modified to contain a PURO gene; it also contains the EBNA-1 gene. Flag symbols indicate the names of epitope-tags present in some L1 vectors. The SP and ASP labels indicate the sense and anti-sense promoters located in the L1 5′-UTR. The MS2 24x designation indicates the 24 copies of the MS2-GFP RNA binding motif in the pAD3TE1 construct. The PCR primers for pAD2TE1 are labeled F1, R1, F2 and R2 (see ‘Materials and Methods’ section for details). In the IAP vector [pDJ33/440N1 neo TNF ( 13 )], Pr indicates the viral LTR promoter. The IAP GAG and POL genes also are indicated. ( B ) Rationale of the assay: Transcription from a promoter driving L1 or IAP expression allows splicing of the intron from either the mneoI- or EGFP-based indicator cassettes. Retrotransposition of the resultant RNA leads to activation of the reporter gene, conferring either G418-resistance or EGFP-positivity to host cells. TSD indicates a target site duplication flanking the retrotransposed L1. ( C ) Experimental protocols to detect L1 retrotransposition: Cells were co-transfected with an engineered L1 or IAP retroelement and either an empty vector (pFLAG-CMV-2) or amino-terminal FLAG-tagged RNase L expression plasmid. For the mneoI -based assays, the transfected cells were subjected to G418 selection 2 days after transfection. The numbers of G418-resistant foci serve as a readout of retrotransposition efficiency. For the mEGFPI -based assays, FACS analysis was used to measure the percentage of EGFP-positive cells 4 days after transfection (See ‘Materials and Methods’ section for further details about each assay).

    Journal: Nucleic Acids Research

    Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

    doi: 10.1093/nar/gkt1308

    Figure Lengend Snippet: An overview of the L1 and IAP retrotransposition assays. ( A ) Schematics of L1 and IAP constructs: The L1 and IAP constructs contain a NEO-based ( mneoI ) or EGFP -based ( mEGFPI ) retrotransposition indicator cassette near their 3′ ends. The indicator cassettes are in an anti-sense (backward) orientation relative to the transcriptional orientation of the L1 or IAP elements. The indicator cassettes also contain an intron that is in the same transcriptional orientation as the retroelement. SD and SA indicate the splice donor and splice acceptor sites of the intron, respectively. Pr′ indicates the promoter driving the expression of the retrotransposition indicator cassette. Closed lollipops indicate the polyadenylation signal on the indicator cassette. A CMV promoter enhances the expression of the pJM101/L1.3, pAD2TE1, pES2TE1 and pAD3TE1 L1 vectors. An SV40 polyadenylation signal is present at the 3′ end of each L1 expression cassette. Notably, the mneoI -based L1 vectors are expressed from a pCEP4 vector that contains a HYG and an EBNA-1 gene. The mEGFPI-based L1 vectors are expressed from a pCEP4 vector that was modified to contain a PURO gene; it also contains the EBNA-1 gene. Flag symbols indicate the names of epitope-tags present in some L1 vectors. The SP and ASP labels indicate the sense and anti-sense promoters located in the L1 5′-UTR. The MS2 24x designation indicates the 24 copies of the MS2-GFP RNA binding motif in the pAD3TE1 construct. The PCR primers for pAD2TE1 are labeled F1, R1, F2 and R2 (see ‘Materials and Methods’ section for details). In the IAP vector [pDJ33/440N1 neo TNF ( 13 )], Pr indicates the viral LTR promoter. The IAP GAG and POL genes also are indicated. ( B ) Rationale of the assay: Transcription from a promoter driving L1 or IAP expression allows splicing of the intron from either the mneoI- or EGFP-based indicator cassettes. Retrotransposition of the resultant RNA leads to activation of the reporter gene, conferring either G418-resistance or EGFP-positivity to host cells. TSD indicates a target site duplication flanking the retrotransposed L1. ( C ) Experimental protocols to detect L1 retrotransposition: Cells were co-transfected with an engineered L1 or IAP retroelement and either an empty vector (pFLAG-CMV-2) or amino-terminal FLAG-tagged RNase L expression plasmid. For the mneoI -based assays, the transfected cells were subjected to G418 selection 2 days after transfection. The numbers of G418-resistant foci serve as a readout of retrotransposition efficiency. For the mEGFPI -based assays, FACS analysis was used to measure the percentage of EGFP-positive cells 4 days after transfection (See ‘Materials and Methods’ section for further details about each assay).

    Article Snippet: In a separate control experiment, we demonstrated that co-transfection of WT RNase L did not affect HYG mRNA levels expressed from pIREShyg (Clontech) ( Supplementary Figure S3 ).

    Techniques: Construct, Expressing, Plasmid Preparation, Modification, RNA Binding Assay, Polymerase Chain Reaction, Labeling, Activation Assay, Transfection, Selection, FACS

    RNase L reduces L1 RNA accumulation in cells. ( A ) Results of qRT-PCR experiments: HeLa-M cells were co-transfected with pAD2TE1 and an empty vector (pFLAG-CMV-2) or an amino-terminal FLAG-tagged RNase L expression plasmid. L1 RNA levels were determined 48 h after transfection using the Sybr Green method ( 84 ). The X-axis indicates the RNase L co-transfected samples. The Y-axis indicates the relative expression level of L1 RNA from the transfected construct. The L1 RNA amounts were normalized with hygromycin mRNA levels (see ‘Materials and Methods’ section for detailed PCR strategy). Data are represented as the mean ± SD from three technical replicates of a single representative experiment. * P

    Journal: Nucleic Acids Research

    Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

    doi: 10.1093/nar/gkt1308

    Figure Lengend Snippet: RNase L reduces L1 RNA accumulation in cells. ( A ) Results of qRT-PCR experiments: HeLa-M cells were co-transfected with pAD2TE1 and an empty vector (pFLAG-CMV-2) or an amino-terminal FLAG-tagged RNase L expression plasmid. L1 RNA levels were determined 48 h after transfection using the Sybr Green method ( 84 ). The X-axis indicates the RNase L co-transfected samples. The Y-axis indicates the relative expression level of L1 RNA from the transfected construct. The L1 RNA amounts were normalized with hygromycin mRNA levels (see ‘Materials and Methods’ section for detailed PCR strategy). Data are represented as the mean ± SD from three technical replicates of a single representative experiment. * P

    Article Snippet: In a separate control experiment, we demonstrated that co-transfection of WT RNase L did not affect HYG mRNA levels expressed from pIREShyg (Clontech) ( Supplementary Figure S3 ).

    Techniques: Quantitative RT-PCR, Transfection, Plasmid Preparation, Expressing, SYBR Green Assay, Construct, Polymerase Chain Reaction

    Inhibition of IAP retrotransposition by RNase L. ( A ) IAP Retrotransposition Assays: HeLa-M cells were co-transfected with a mouse IAP expression construct (pDJ33/440N1 neo TNF ) and either an empty vector (pFLAG-CMV-2) or an expression plasmid that encodes an amino-terminal FLAG-tagged version of the following proteins: WT RNase L, a catalytically inactive RNase L mutant (R667A), a constitutively active RNase L mutant (NΔ385), A3A or RIG-I. The cells were subject to selection for 10 days and G418-resistant foci were fixed and stained with crystal violet for visualization purposes. A representative tissue culture dish for each condition is shown. ( B ) Quantitation of the IAP Retrotransposition Assays: The X-axis depicts names of constructs co-transfected into cells with the IAP construct. The Y-axis depicts the number of G418-resistant foci per cell culture dish. Data are represented as the mean ± standard deviation (SD) from a single experiment with three technical replicates. * P

    Journal: Nucleic Acids Research

    Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

    doi: 10.1093/nar/gkt1308

    Figure Lengend Snippet: Inhibition of IAP retrotransposition by RNase L. ( A ) IAP Retrotransposition Assays: HeLa-M cells were co-transfected with a mouse IAP expression construct (pDJ33/440N1 neo TNF ) and either an empty vector (pFLAG-CMV-2) or an expression plasmid that encodes an amino-terminal FLAG-tagged version of the following proteins: WT RNase L, a catalytically inactive RNase L mutant (R667A), a constitutively active RNase L mutant (NΔ385), A3A or RIG-I. The cells were subject to selection for 10 days and G418-resistant foci were fixed and stained with crystal violet for visualization purposes. A representative tissue culture dish for each condition is shown. ( B ) Quantitation of the IAP Retrotransposition Assays: The X-axis depicts names of constructs co-transfected into cells with the IAP construct. The Y-axis depicts the number of G418-resistant foci per cell culture dish. Data are represented as the mean ± standard deviation (SD) from a single experiment with three technical replicates. * P

    Article Snippet: In a separate control experiment, we demonstrated that co-transfection of WT RNase L did not affect HYG mRNA levels expressed from pIREShyg (Clontech) ( Supplementary Figure S3 ).

    Techniques: Inhibition, Transfection, Expressing, Construct, Plasmid Preparation, Mutagenesis, Selection, Staining, Quantitation Assay, Cell Culture, Standard Deviation

    Depletion of endogenous RNase L increases L1 retrotransposition efficiency. ( A ) Knockdown of endogenous RNase L protein: Hey1b cells were transfected with control siRNA pools or RNase L siRNA pools. Western blotting using an anti-RNase L monoclonal antibody confirmed RNase L knockdown 48 h after siRNA transfection. β-Actin served as loading and transfer control. The band intensity was quantified with ImageJ software ( 83 ) and the relative ratio of RNase L to β-actin is shown. ( B ) Representative Retrotransposition Assay Results: Control siRNA (control) and siRNA-mediated RNase L depleted cells (RNase L) were transfected with either pLRE3- mEGFPI or pJM111-LRE3- mEGFPI . L1 retrotransposition was assayed as described in Figure 4 . Representative FACS plots are shown, as is the conservative gating strategy used to detect EGFP-positive cells. ( C ) Quantitation of the Retrotransposition Assays: The X-axis indicates the control siRNA (control) or siRNA-mediated RNase L depleted cells (RNase L). The Y-axis indicates the percentage of EGFP-positive cells. For each sample, 2 × 10 5 cells were analyzed and the percentage of EGFP-positive cells was calculated with using the FlowJo software package. The experiment was conducted four times (biological replicates) with similar results; representative data from one experiment are shown. Data are reported as the mean ± SD from three technical replicates of a single representative experiment. The asterisk indicates a P = 0.0079 and was calculated with two-tailed Student’s t -test.

    Journal: Nucleic Acids Research

    Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

    doi: 10.1093/nar/gkt1308

    Figure Lengend Snippet: Depletion of endogenous RNase L increases L1 retrotransposition efficiency. ( A ) Knockdown of endogenous RNase L protein: Hey1b cells were transfected with control siRNA pools or RNase L siRNA pools. Western blotting using an anti-RNase L monoclonal antibody confirmed RNase L knockdown 48 h after siRNA transfection. β-Actin served as loading and transfer control. The band intensity was quantified with ImageJ software ( 83 ) and the relative ratio of RNase L to β-actin is shown. ( B ) Representative Retrotransposition Assay Results: Control siRNA (control) and siRNA-mediated RNase L depleted cells (RNase L) were transfected with either pLRE3- mEGFPI or pJM111-LRE3- mEGFPI . L1 retrotransposition was assayed as described in Figure 4 . Representative FACS plots are shown, as is the conservative gating strategy used to detect EGFP-positive cells. ( C ) Quantitation of the Retrotransposition Assays: The X-axis indicates the control siRNA (control) or siRNA-mediated RNase L depleted cells (RNase L). The Y-axis indicates the percentage of EGFP-positive cells. For each sample, 2 × 10 5 cells were analyzed and the percentage of EGFP-positive cells was calculated with using the FlowJo software package. The experiment was conducted four times (biological replicates) with similar results; representative data from one experiment are shown. Data are reported as the mean ± SD from three technical replicates of a single representative experiment. The asterisk indicates a P = 0.0079 and was calculated with two-tailed Student’s t -test.

    Article Snippet: In a separate control experiment, we demonstrated that co-transfection of WT RNase L did not affect HYG mRNA levels expressed from pIREShyg (Clontech) ( Supplementary Figure S3 ).

    Techniques: Transfection, Western Blot, Software, FACS, Quantitation Assay, Two Tailed Test