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  • 99
    Millipore ribonucleic acid rna
    Transcriptional analysis of <t>RNA</t> recovered from wild type and allelic exchange mutant E . chaffeensis organisms assessed by <t>RT-PCR.</t> ( A ) RT-PCR products from wild type (W) and Ech_0230 mutant (M) organisms were resolved (L, 1 kb plus molecular weight DNA markers resolved; +, genomic DNA from wild type E . chaffeensis was used as the template; -, negative control reaction with no template added). ( B ) As in panel A, except that the analysis was performed using RNA recovered from Ech_0379 disruption (M) and restoration (R) mutant organisms. Positive controls for this experiments included genomic DNAs as the templates from W, M and R. (0.38 kb amplicons are expected for DNA templates in PCRs of W and R and 1.6 kb product is expected for M DNA as the template.) ( C ) Mutations to inactivate and restore the gene activity in Ech_0379 did not alter the gene expression from its neighboring genes. Semi-quantitative RT-PCR assays were performed at 30, 35 and 40 PCR cycles for Ech_0378, Ech_0379 and Ech_0380 for wild type, gene inactivation and gene rescue mutant organisms and the data for 35 cycles were presented. W, M and R had similar quantities of amplicons for Ech_0378 and Ech_0380; Ech_0379 amplicons were also similar for W and R, while absent for M. (Full-length gels and blots were included in the Supplementary Figure file, as parts of the Figure had cropped images).
    Ribonucleic Acid Rna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2801 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Worthington Biochemical rna
    Mechanism of FXN activation by repeat-targeted duplex RNAs. ( a ) RIP examining the association of Ago2 with FXN pre-mRNA after treatment with 50 nM duplex <t>RNA</t> and analysis by real-time <t>PCR.</t> An arrow marks the PCR product of FXN pre-mRNA, which was confirmed by sequencing ( Supplementary Fig. 7 ) ( b ) Anti-GAA duplex RNA with central mismatches (siGAA 9,10 mm with mismatches on both strands; 25 nM) activates FXN expression at a level similar to the analogous fully complementary duplex RNA ( n =3). siExon3 is a positive control for transfection efficiency targeting exon 3 of FXN . ( c ) ChIP for RNAP2 using four different primer sets ( n =4). ( d ) ChIP for transcription-associated histone modification markers H3K4me3, H3K9me2, H3K9me3, H3K9Ac, H3K27me3 and H4Ac ( n =4–8). ( e ) FXN mRNA stability assay. Cells were transfected with duplex RNAs siGAA or CM at 25 nM ( n =3). Actinomycin D (5 μg ml −1 ) was added with fresh media 3 days after transfection and cells were collected at the indicated time points. HPRT expression was measured for normalization. All experiments were performed in GM03816 patient-derived cells. All data are presented as mean±STDEV. * P
    Rna, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 99/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore rna
    Validation by RIP-qPCR of a set of ARA-lincRNAs, which were detected in the RIP-seq assay as associated to AR. <t>LNCaP</t> cells treated with 0.1 nM androgen (red lines) or with vehicle control (blue lines) were assayed as indicated on the x -axis. The amount of the indicated lincRNA that was co-immunoprecipitated with antiAR antibody or with IgG from non-immunized rabbit (negative control) was measured by RT-qPCR in three different biological replicates (each represented with a different symbol), and the corresponding points for antiAR and IgG for each replicate are connected with a straight line. The results are shown as % input <t>RNA</t> (mean ± SEM) of three technical replicates for each individual biological replicate. For the four lincRNA genes in the experiment with androgen whose experimental points are connected with red dotted lines, the amount of RIP material was only enough for two technical replicates each, and the enrichment t -test was not applied. Red and blue solid lines = significant difference between antiAR and IgG ( p
    Rna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7260 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore transfer rna
    Validation by RIP-qPCR of a set of ARA-lincRNAs, which were detected in the RIP-seq assay as associated to AR. <t>LNCaP</t> cells treated with 0.1 nM androgen (red lines) or with vehicle control (blue lines) were assayed as indicated on the x -axis. The amount of the indicated lincRNA that was co-immunoprecipitated with antiAR antibody or with IgG from non-immunized rabbit (negative control) was measured by RT-qPCR in three different biological replicates (each represented with a different symbol), and the corresponding points for antiAR and IgG for each replicate are connected with a straight line. The results are shown as % input <t>RNA</t> (mean ± SEM) of three technical replicates for each individual biological replicate. For the four lincRNA genes in the experiment with androgen whose experimental points are connected with red dotted lines, the amount of RIP material was only enough for two technical replicates each, and the enrichment t -test was not applied. Red and blue solid lines = significant difference between antiAR and IgG ( p
    Transfer Rna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Transcriptional analysis of RNA recovered from wild type and allelic exchange mutant E . chaffeensis organisms assessed by RT-PCR. ( A ) RT-PCR products from wild type (W) and Ech_0230 mutant (M) organisms were resolved (L, 1 kb plus molecular weight DNA markers resolved; +, genomic DNA from wild type E . chaffeensis was used as the template; -, negative control reaction with no template added). ( B ) As in panel A, except that the analysis was performed using RNA recovered from Ech_0379 disruption (M) and restoration (R) mutant organisms. Positive controls for this experiments included genomic DNAs as the templates from W, M and R. (0.38 kb amplicons are expected for DNA templates in PCRs of W and R and 1.6 kb product is expected for M DNA as the template.) ( C ) Mutations to inactivate and restore the gene activity in Ech_0379 did not alter the gene expression from its neighboring genes. Semi-quantitative RT-PCR assays were performed at 30, 35 and 40 PCR cycles for Ech_0378, Ech_0379 and Ech_0380 for wild type, gene inactivation and gene rescue mutant organisms and the data for 35 cycles were presented. W, M and R had similar quantities of amplicons for Ech_0378 and Ech_0380; Ech_0379 amplicons were also similar for W and R, while absent for M. (Full-length gels and blots were included in the Supplementary Figure file, as parts of the Figure had cropped images).

    Journal: Scientific Reports

    Article Title: A genetic system for targeted mutations to disrupt and restore genes in the obligate bacterium, Ehrlichia chaffeensis

    doi: 10.1038/s41598-017-16023-y

    Figure Lengend Snippet: Transcriptional analysis of RNA recovered from wild type and allelic exchange mutant E . chaffeensis organisms assessed by RT-PCR. ( A ) RT-PCR products from wild type (W) and Ech_0230 mutant (M) organisms were resolved (L, 1 kb plus molecular weight DNA markers resolved; +, genomic DNA from wild type E . chaffeensis was used as the template; -, negative control reaction with no template added). ( B ) As in panel A, except that the analysis was performed using RNA recovered from Ech_0379 disruption (M) and restoration (R) mutant organisms. Positive controls for this experiments included genomic DNAs as the templates from W, M and R. (0.38 kb amplicons are expected for DNA templates in PCRs of W and R and 1.6 kb product is expected for M DNA as the template.) ( C ) Mutations to inactivate and restore the gene activity in Ech_0379 did not alter the gene expression from its neighboring genes. Semi-quantitative RT-PCR assays were performed at 30, 35 and 40 PCR cycles for Ech_0378, Ech_0379 and Ech_0380 for wild type, gene inactivation and gene rescue mutant organisms and the data for 35 cycles were presented. W, M and R had similar quantities of amplicons for Ech_0378 and Ech_0380; Ech_0379 amplicons were also similar for W and R, while absent for M. (Full-length gels and blots were included in the Supplementary Figure file, as parts of the Figure had cropped images).

    Article Snippet: RNA analysis by RT-PCR to verify the loss and restoration of transcription Total RNAs from wild type and mutant E . chaffeensis organisms grown in ISE6 or DH82 cell cultures were isolated by following the Tri-reagent total RNA isolation method (Sigma-Aldrich, St. Louis, MO).

    Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Negative Control, Activity Assay, Expressing, Quantitative RT-PCR, Polymerase Chain Reaction

    Discovery of two hydrophobic small molecule-RNA conjugates with [M-H] − m/z = 824.200 and 868.189. (a) Scheme of the general method for discovering biological small molecule-RNA conjugates applied in this work. Total cellular RNA samples are treated with active nuclease P1 or with heat-inactivated nuclease P1 under otherwise identical conditions, then subjected to comparative LC/MS analysis. Nucleotide ions more abundant in the active nuclease sample than in the heat-inactivated nuclease (control) sample represent candidate cellular small molecule-RNA conjugates. (b) Extracted ion chromatogram of AMP-tryptophan and the two unknown nucleotides elucidated in this work.

    Journal: Nature chemical biology

    Article Title: Discovery and biological characterization of geranylated RNA in bacteria

    doi: 10.1038/nchembio.1070

    Figure Lengend Snippet: Discovery of two hydrophobic small molecule-RNA conjugates with [M-H] − m/z = 824.200 and 868.189. (a) Scheme of the general method for discovering biological small molecule-RNA conjugates applied in this work. Total cellular RNA samples are treated with active nuclease P1 or with heat-inactivated nuclease P1 under otherwise identical conditions, then subjected to comparative LC/MS analysis. Nucleotide ions more abundant in the active nuclease sample than in the heat-inactivated nuclease (control) sample represent candidate cellular small molecule-RNA conjugates. (b) Extracted ion chromatogram of AMP-tryptophan and the two unknown nucleotides elucidated in this work.

    Article Snippet: RNA digestion with nuclease P1 and alkaline phosphatase For positive ion mode mass spectrometric analysis, 500 μg of isolated total RNA or commercial RNA were digested with 10 U nuclease P1 and 100 U alkaline phophatase (Sigma) in 500 μL of 50 mM NH4 OAc, pH 6 at 37 °C for 4 hours.

    Techniques: Liquid Chromatography with Mass Spectroscopy

    Comparison of growth, gene transcription, translation and isobutanol production in engineered strains Syn-pEEK2, pEEK2- kivd , Syn-IB-7, -8, -9 and -6. A) RT-PCR, SDS-PAGE and Strep-tag Western-immunoblot (top to bottom). Each lane represents the results from a single engineered strain. –RT is a negative RT-PCR control using RNA as template without addition of RT enzyme to control for possible DNA contamination. The positive control is an RT-PCR carried out using the corresponding plasmid as template. Red arrows indicate the location of kivd , blue arrows indicate the expected location for different AHDs. The first row of the Strep-tag Western-immunoblot shows the expression of kivd and the second row shows the expression of all the ADHs. The negative control for protein gel and Western-immunoblot is the empty vector strain Syn-pEEK2. B) Growth curve during 7 days of cultivation. C) Isobutanol production at day 3, day 5 and day 6 from all the engineered strains. Results are the mean of 4 biological replicates and 3 technical replicates, error bars represent standard deviation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Journal: Metabolic Engineering Communications

    Article Title: Isobutanol production in Synechocystis PCC 6803 using heterologous and endogenous alcohol dehydrogenases

    doi: 10.1016/j.meteno.2017.07.003

    Figure Lengend Snippet: Comparison of growth, gene transcription, translation and isobutanol production in engineered strains Syn-pEEK2, pEEK2- kivd , Syn-IB-7, -8, -9 and -6. A) RT-PCR, SDS-PAGE and Strep-tag Western-immunoblot (top to bottom). Each lane represents the results from a single engineered strain. –RT is a negative RT-PCR control using RNA as template without addition of RT enzyme to control for possible DNA contamination. The positive control is an RT-PCR carried out using the corresponding plasmid as template. Red arrows indicate the location of kivd , blue arrows indicate the expected location for different AHDs. The first row of the Strep-tag Western-immunoblot shows the expression of kivd and the second row shows the expression of all the ADHs. The negative control for protein gel and Western-immunoblot is the empty vector strain Syn-pEEK2. B) Growth curve during 7 days of cultivation. C) Isobutanol production at day 3, day 5 and day 6 from all the engineered strains. Results are the mean of 4 biological replicates and 3 technical replicates, error bars represent standard deviation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Article Snippet: 2.6 RNA isolation and semi-quantitative reverse transcript PCR (RT-PCR) Total RNA was isolated from cultures (OD750 = 0.5) using RTI Reagent (Sigma-Aldrich) according to the manufacturer's instructions.

    Techniques: Reverse Transcription Polymerase Chain Reaction, SDS Page, Strep-tag, Western Blot, Positive Control, Plasmid Preparation, Expressing, Negative Control, Standard Deviation

    Comparison of isobutanol production in engineered Synechocystis PCC 6803 strains Syn-IB-1, -2, -3, -4, -5, and -6. A) RT-PCR, SDS-PAGE and Strep-tag Western-immunoblot (top to bottom). Every dashed line separated each lane represents the results from a single engineered strain. –RT is a negative RT-PCR control using RNA as template without addition of RT enzyme to control for possible DNA contamination. The positive control is an RT-PCR carried out using the corresponding plasmid as template. Red arrows in the SDS-PAGE indicate the location of kivd , blue arrows indicate the expected location for slr1192 . The Strep-tag Western-immunoblot examines the expression of all the ADHs. The negative control for SDS-PAGE and Western-immunoblot is an extract from strain Syn-pDDH. B) Growth curve during 7 days of cultivation. C) Isobutanol production at day 3, day 5 and day 6 from the different engineered strains. Results are the mean of 4 biological replicates and 3 technical replicates, error bars represent standard deviation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Journal: Metabolic Engineering Communications

    Article Title: Isobutanol production in Synechocystis PCC 6803 using heterologous and endogenous alcohol dehydrogenases

    doi: 10.1016/j.meteno.2017.07.003

    Figure Lengend Snippet: Comparison of isobutanol production in engineered Synechocystis PCC 6803 strains Syn-IB-1, -2, -3, -4, -5, and -6. A) RT-PCR, SDS-PAGE and Strep-tag Western-immunoblot (top to bottom). Every dashed line separated each lane represents the results from a single engineered strain. –RT is a negative RT-PCR control using RNA as template without addition of RT enzyme to control for possible DNA contamination. The positive control is an RT-PCR carried out using the corresponding plasmid as template. Red arrows in the SDS-PAGE indicate the location of kivd , blue arrows indicate the expected location for slr1192 . The Strep-tag Western-immunoblot examines the expression of all the ADHs. The negative control for SDS-PAGE and Western-immunoblot is an extract from strain Syn-pDDH. B) Growth curve during 7 days of cultivation. C) Isobutanol production at day 3, day 5 and day 6 from the different engineered strains. Results are the mean of 4 biological replicates and 3 technical replicates, error bars represent standard deviation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Article Snippet: 2.6 RNA isolation and semi-quantitative reverse transcript PCR (RT-PCR) Total RNA was isolated from cultures (OD750 = 0.5) using RTI Reagent (Sigma-Aldrich) according to the manufacturer's instructions.

    Techniques: Periodic Counter-current Chromatography, Reverse Transcription Polymerase Chain Reaction, SDS Page, Strep-tag, Western Blot, Positive Control, Plasmid Preparation, Expressing, Negative Control, Standard Deviation

    Model for the type III secretion system (T3SS) regulatory network in D. dadantii 3937. The D. dadantii 3937 T3SS is regulated by the HrpX/HrpY-HrpS-HrpL and the GacS/GacA- rsmB -RsmA-HrpL pathways. In this study, the flagellar master regulator FlhDC was observed to hierarchically regulate the expression of T3SS encoding genes. (i) FlhDC positively regulates the PilZ domain protein encoding gene ycgR 3937 at transcriptional level through a sigma factor FliA. Under high c-di-GMP levels (Δ egcpB ), YcgR 3937 binds c-di-GMP, which negatively regulates the T3SS. (ii) FlhDC controls the expression of phosphodiesterase encoding gene ecpC . EcpC degrades intracellular c-di-GMP, which counteracts the negative impact of c-di-GMP on the RpoN, which is required for the transcription of hrpL . (iii) FlhDC and FliA divergently regulate the regulatory small RNA RsmB at the post-transcriptional level. ⊥represents negative control; → represents positive control. The dotted lines indicate regulatory mechanisms identified in this study.

    Journal: Environmental microbiology

    Article Title: Cross-talk between a regulatory small RNA, cyclic-di-GMP signalling and flagellar regulator FlhDC for virulence and bacterial behaviours

    doi: 10.1111/1462-2920.13029

    Figure Lengend Snippet: Model for the type III secretion system (T3SS) regulatory network in D. dadantii 3937. The D. dadantii 3937 T3SS is regulated by the HrpX/HrpY-HrpS-HrpL and the GacS/GacA- rsmB -RsmA-HrpL pathways. In this study, the flagellar master regulator FlhDC was observed to hierarchically regulate the expression of T3SS encoding genes. (i) FlhDC positively regulates the PilZ domain protein encoding gene ycgR 3937 at transcriptional level through a sigma factor FliA. Under high c-di-GMP levels (Δ egcpB ), YcgR 3937 binds c-di-GMP, which negatively regulates the T3SS. (ii) FlhDC controls the expression of phosphodiesterase encoding gene ecpC . EcpC degrades intracellular c-di-GMP, which counteracts the negative impact of c-di-GMP on the RpoN, which is required for the transcription of hrpL . (iii) FlhDC and FliA divergently regulate the regulatory small RNA RsmB at the post-transcriptional level. ⊥represents negative control; → represents positive control. The dotted lines indicate regulatory mechanisms identified in this study.

    Article Snippet: To measure the RNA levels of rsmB in wild type, Δ flhDC , Δ fliA and complemented strains, bacterial cells grown in MM for 12 h were harvested and total RNA was isolated using TRI reagent (Sigma-Aldrich, St Louis, MO).

    Techniques: Expressing, Negative Control, Positive Control

    FlhDC and FliA inversely regulate RsmB at a post-transcriptional level. A. Promoter activity of rsmB was measured in the wild-type D. dadantii , the flhDC mutant and the fliA mutant strains. B. Northern blot analysis of rsmB mRNA in the wild-type D. dadantii harbouring empty vector pCL1920, Δ fliA harbouring empty vector pCL1920, Δ fliA harbouring plasmid pCL1920- fliA , Δ flhDC harbouring empty pCL1920 and Δ flhDC harbouring pCL1920- flhDC . 16S rRNA was used as RNA loading control. C. Pectate lyase production assay was performed in the wild-type D. dadantii harbouring empty vector pCL1920, Δ fliA harbouring empty vector pCL1920, Δ fliA harbouring plasmid pCL1920- fliA , Δ flhDC harbouring empty pCL1920 and Δ flhDC harbouring pCL1920- flhDC . Values are a representative of three independent experiments. Three replicates were used in each experiment. Error bars indicate standard errors of the means. Asterisks indicate statistically significant differences of the means ( P

    Journal: Environmental microbiology

    Article Title: Cross-talk between a regulatory small RNA, cyclic-di-GMP signalling and flagellar regulator FlhDC for virulence and bacterial behaviours

    doi: 10.1111/1462-2920.13029

    Figure Lengend Snippet: FlhDC and FliA inversely regulate RsmB at a post-transcriptional level. A. Promoter activity of rsmB was measured in the wild-type D. dadantii , the flhDC mutant and the fliA mutant strains. B. Northern blot analysis of rsmB mRNA in the wild-type D. dadantii harbouring empty vector pCL1920, Δ fliA harbouring empty vector pCL1920, Δ fliA harbouring plasmid pCL1920- fliA , Δ flhDC harbouring empty pCL1920 and Δ flhDC harbouring pCL1920- flhDC . 16S rRNA was used as RNA loading control. C. Pectate lyase production assay was performed in the wild-type D. dadantii harbouring empty vector pCL1920, Δ fliA harbouring empty vector pCL1920, Δ fliA harbouring plasmid pCL1920- fliA , Δ flhDC harbouring empty pCL1920 and Δ flhDC harbouring pCL1920- flhDC . Values are a representative of three independent experiments. Three replicates were used in each experiment. Error bars indicate standard errors of the means. Asterisks indicate statistically significant differences of the means ( P

    Article Snippet: To measure the RNA levels of rsmB in wild type, Δ flhDC , Δ fliA and complemented strains, bacterial cells grown in MM for 12 h were harvested and total RNA was isolated using TRI reagent (Sigma-Aldrich, St Louis, MO).

    Techniques: Activity Assay, Mutagenesis, Northern Blot, Plasmid Preparation

    Effect of ZFP57 loss on gene expression and genomic imprinting in inbred and hybrid ESCs, determined by global and allele-specific RNA-seq analyses. ( A ) Empirical cumulative distribution of the distances (bp) of deregulated genes (all genes, black; imprinted genes, green; non imprinted genes, orange) from ZFP57 binding sites in both Zfp57 −/− E14 and JB1 ESCs. ( B ) Scatter plot showing deregulated genes in both Zfp57 −/− E14 and JB1 ESCs. Deregulated genes are indicated by red dots if distant

    Journal: Nucleic Acids Research

    Article Title: ZFP57 maintains the parent-of-origin-specific expression of the imprinted genes and differentially affects non-imprinted targets in mouse embryonic stem cells

    doi: 10.1093/nar/gkw505

    Figure Lengend Snippet: Effect of ZFP57 loss on gene expression and genomic imprinting in inbred and hybrid ESCs, determined by global and allele-specific RNA-seq analyses. ( A ) Empirical cumulative distribution of the distances (bp) of deregulated genes (all genes, black; imprinted genes, green; non imprinted genes, orange) from ZFP57 binding sites in both Zfp57 −/− E14 and JB1 ESCs. ( B ) Scatter plot showing deregulated genes in both Zfp57 −/− E14 and JB1 ESCs. Deregulated genes are indicated by red dots if distant

    Article Snippet: RNA analysis RNA was extracted from cultured ESCs by lysing the cells on the culture dish with the addition of the TRI reagent (Sigma-Aldrich) and following the protocol of the manufacturer.

    Techniques: Expressing, RNA Sequencing Assay, Binding Assay

    Time courses for the formation of SAH (blue triangles), 5’-dA (red circles), ms 2 i 6 A (black triangles) and d 3 -ms 2 i 6 A (green triangles) upon ( A ) initial incubation of 150 μM Tm MiaB with a stoichiometric concentration of SAM in the absence of dithionite followed by ( B ) introduction of excess (500 μM) [ methyl - d 3 ]-SAM, 130 μM i 6 A ACSL RNA, and 1 mM dithionite in 50 mM Tris-HCl pH 7.5. The lines are fits to a first-order single-exponential equation, with the following obtained kinetic parameters: ( A ) SAH formation: A = 110 ± 4 μM, ν = 8.5 ± 1.3 min −1 ( B ) 5’-dA formation: A = 105 ± 3 μM, ν = 5.7 ± 0.7 μM min −1 ; SAH formation: A = 230 ± 3 μM, ν = 9.7 ± 0.8 μM min −1 ; ms 2 i 6 A formation: A = 40 ± 1 μM, ν = 11.6 ± 2.6 μM min −1 ; d 3 -ms 2 i 6 A formation: A = 48 ± 2 μM, ν = 1.3 ± 0.2 μM min −1 .

    Journal: Journal of the American Chemical Society

    Article Title: Identification of an Intermediate Methyl Carrier in the Radical SAM Methylthiotransferases, RimO and MiaB

    doi: 10.1021/ja4048448

    Figure Lengend Snippet: Time courses for the formation of SAH (blue triangles), 5’-dA (red circles), ms 2 i 6 A (black triangles) and d 3 -ms 2 i 6 A (green triangles) upon ( A ) initial incubation of 150 μM Tm MiaB with a stoichiometric concentration of SAM in the absence of dithionite followed by ( B ) introduction of excess (500 μM) [ methyl - d 3 ]-SAM, 130 μM i 6 A ACSL RNA, and 1 mM dithionite in 50 mM Tris-HCl pH 7.5. The lines are fits to a first-order single-exponential equation, with the following obtained kinetic parameters: ( A ) SAH formation: A = 110 ± 4 μM, ν = 8.5 ± 1.3 min −1 ( B ) 5’-dA formation: A = 105 ± 3 μM, ν = 5.7 ± 0.7 μM min −1 ; SAH formation: A = 230 ± 3 μM, ν = 9.7 ± 0.8 μM min −1 ; ms 2 i 6 A formation: A = 40 ± 1 μM, ν = 11.6 ± 2.6 μM min −1 ; d 3 -ms 2 i 6 A formation: A = 48 ± 2 μM, ν = 1.3 ± 0.2 μM min −1 .

    Article Snippet: The ACSL RNA was determined to be completely modified by MiaA, with each mol of RNA containing one mol of i6 A. Quantification was conducted by LC/MS using a standard curve generated from commercially available i6 A (Sigma).

    Techniques: Mass Spectrometry, Incubation, Concentration Assay

    Isotopic distribution of ms 2 i 6 A in assays containing 20 μM Tm MiaB, 100 μM i 6 A ACSL RNA substrate, and 1 mM dithionite in the presence of: ( A ) 500 μM SAM; ( B ) 500 μM d 3 -SAM and 500 μM NaSCH 3 ; and ( C ) 500 μM d 3 -SAM. After 2 h at 37 °C, ( A ) 19.4 μM ms 2 i 6 A was generated in the presence of SAM only ( B ) 11.7 μM ms 2 i 6 A and 9.8 μM d 3 -ms 2 i 6 A were generated in the presence both of SAM and NaSCH 3 , and ( C ) 21.2 μM d 3 -ms 2 i 6 A was generated in the presence of d 3 -SAM only.

    Journal: Journal of the American Chemical Society

    Article Title: Identification of an Intermediate Methyl Carrier in the Radical SAM Methylthiotransferases, RimO and MiaB

    doi: 10.1021/ja4048448

    Figure Lengend Snippet: Isotopic distribution of ms 2 i 6 A in assays containing 20 μM Tm MiaB, 100 μM i 6 A ACSL RNA substrate, and 1 mM dithionite in the presence of: ( A ) 500 μM SAM; ( B ) 500 μM d 3 -SAM and 500 μM NaSCH 3 ; and ( C ) 500 μM d 3 -SAM. After 2 h at 37 °C, ( A ) 19.4 μM ms 2 i 6 A was generated in the presence of SAM only ( B ) 11.7 μM ms 2 i 6 A and 9.8 μM d 3 -ms 2 i 6 A were generated in the presence both of SAM and NaSCH 3 , and ( C ) 21.2 μM d 3 -ms 2 i 6 A was generated in the presence of d 3 -SAM only.

    Article Snippet: The ACSL RNA was determined to be completely modified by MiaA, with each mol of RNA containing one mol of i6 A. Quantification was conducted by LC/MS using a standard curve generated from commercially available i6 A (Sigma).

    Techniques: Mass Spectrometry, Generated

    DOX-induced DNA damage induces TP53-dependent differential binding of specific miRNAs to AGO2. ( A ) Fold changes in let-7 family levels determined by a combined small RNA-seq and AGO2-RIP-seq approach (miRNAs demonstrating a positive or negative fold change

    Journal: Genome Research

    Article Title: TP53 regulates miRNA association with AGO2 to remodel the miRNA–mRNA interaction network

    doi: 10.1101/gr.191759.115

    Figure Lengend Snippet: DOX-induced DNA damage induces TP53-dependent differential binding of specific miRNAs to AGO2. ( A ) Fold changes in let-7 family levels determined by a combined small RNA-seq and AGO2-RIP-seq approach (miRNAs demonstrating a positive or negative fold change

    Article Snippet: To check for RNA dependence in TP53–AGO2 interaction, matrix-bound complexes were incubated with RNase A (Sigma) for 30 min at 37°C.

    Techniques: Binding Assay, RNA Sequencing Assay

    AGO2 interacts with TP53 in an RNA-dependent manner. HCT116 TP53 +/+ were treated with vehicle or DOX for 12 h, and coimmunoprecipitation assays were then performed to determine whether an interaction exists between TP53 and AGO2 proteins. ( A ) Reciprocal

    Journal: Genome Research

    Article Title: TP53 regulates miRNA association with AGO2 to remodel the miRNA–mRNA interaction network

    doi: 10.1101/gr.191759.115

    Figure Lengend Snippet: AGO2 interacts with TP53 in an RNA-dependent manner. HCT116 TP53 +/+ were treated with vehicle or DOX for 12 h, and coimmunoprecipitation assays were then performed to determine whether an interaction exists between TP53 and AGO2 proteins. ( A ) Reciprocal

    Article Snippet: To check for RNA dependence in TP53–AGO2 interaction, matrix-bound complexes were incubated with RNase A (Sigma) for 30 min at 37°C.

    Techniques:

    HPIV3 viral RNA triggers SG formation. (A and B) HeLa cells were transfected with the indicated RNA samples from mock-infected or HPIV3-infected MK2 cells, treated with pIC for 12 h or AS for 1 h. (A) Cells were immunostained for TIA-1 (green) and G3BP (red). Nuclei were stained with DAPI (blue). The white scale bar corresponds to 10 μm. (B) Cell lysates were analyzed via western blot using anti-phosphorylated PKR, anti-PKR, anti-phosphorylated eIF2α, anti-eIF2α, and anti-GAPDH antibodies. (C and D) HeLa cells were transfected with the RNA from HPIV3-infected MK2 cells, the respective PolyA + RNA fraction, or the respective PolyA - RNA fraction for 12 h. (C) Cells were immunostained for TIA-1 (green) and G3BP (red). Nuclei were stained with DAPI (blue). The white scale bar corresponds to 10 μm. (D) The percentage of cells containing SGs was quantified in three independent experiments. (E and F) HeLa cells were transfected with the respective PolyA + RNA fraction from HPIV3-infected MK2 cells for 12 h and subsequently mock-treated or treated with CHX for another 1 h, 2 h, or 3 h. (E) Cells were immunostained for TIA-1 (green) and G3BP (red). Nuclei were stained with DAPI (blue). The white scale bar corresponds to 10 μm. (F) The percentage of cells containing SGs was quantified in three independent experiments. Data are represented as means ±SD. Student’s t test: * P

    Journal: PLoS Pathogens

    Article Title: Inclusion bodies of human parainfluenza virus type 3 inhibit antiviral stress granule formation by shielding viral RNAs

    doi: 10.1371/journal.ppat.1006948

    Figure Lengend Snippet: HPIV3 viral RNA triggers SG formation. (A and B) HeLa cells were transfected with the indicated RNA samples from mock-infected or HPIV3-infected MK2 cells, treated with pIC for 12 h or AS for 1 h. (A) Cells were immunostained for TIA-1 (green) and G3BP (red). Nuclei were stained with DAPI (blue). The white scale bar corresponds to 10 μm. (B) Cell lysates were analyzed via western blot using anti-phosphorylated PKR, anti-PKR, anti-phosphorylated eIF2α, anti-eIF2α, and anti-GAPDH antibodies. (C and D) HeLa cells were transfected with the RNA from HPIV3-infected MK2 cells, the respective PolyA + RNA fraction, or the respective PolyA - RNA fraction for 12 h. (C) Cells were immunostained for TIA-1 (green) and G3BP (red). Nuclei were stained with DAPI (blue). The white scale bar corresponds to 10 μm. (D) The percentage of cells containing SGs was quantified in three independent experiments. (E and F) HeLa cells were transfected with the respective PolyA + RNA fraction from HPIV3-infected MK2 cells for 12 h and subsequently mock-treated or treated with CHX for another 1 h, 2 h, or 3 h. (E) Cells were immunostained for TIA-1 (green) and G3BP (red). Nuclei were stained with DAPI (blue). The white scale bar corresponds to 10 μm. (F) The percentage of cells containing SGs was quantified in three independent experiments. Data are represented as means ±SD. Student’s t test: * P

    Article Snippet: PolyA+ RNA was subsequently isolated from the total RNA according to the manufacturer’s instructions for the GenElute mRNA Miniprep Kit (Sigma).

    Techniques: Transfection, Infection, Staining, Western Blot

    GRK2 plays a role in YFV-17D entry independently of β-arrestins. (A) HuH-7 cells treated with siNSC or siGRK2_2 were incubated with YFV-17D at a M.O.I. of 5 at 37°C for 2 hr. Samples were treated for 3 min with high-salt alkaline solution to remove the membrane-bound virus. Total RNA was then collected and YFV-17D genome was detected by quantitative RT-PCR. Each sample was normalized to the amount of beta-actin detected by qRT-PCR. The amount of YFV-17D genome detected is expressed in fold change over the control siNSC. Statistical significance was tested using Mann-Whitney non-parametric t-test and the p-value is indicated in the figure. (B and C) Percentage of YFV-17D (B) and DEN2-NGC (C) infected wild type (WT) and β-arrestin 1/2-KO MEFs. The error bars represent the standard deviation of at least 4 wells. Statistical significance was tested using Welch's t-test, conditions with an inhibitory effect with a p-value

    Journal: PLoS Neglected Tropical Diseases

    Article Title: G Protein-Coupled Receptor Kinase 2 Promotes Flaviviridae Entry and Replication

    doi: 10.1371/journal.pntd.0001820

    Figure Lengend Snippet: GRK2 plays a role in YFV-17D entry independently of β-arrestins. (A) HuH-7 cells treated with siNSC or siGRK2_2 were incubated with YFV-17D at a M.O.I. of 5 at 37°C for 2 hr. Samples were treated for 3 min with high-salt alkaline solution to remove the membrane-bound virus. Total RNA was then collected and YFV-17D genome was detected by quantitative RT-PCR. Each sample was normalized to the amount of beta-actin detected by qRT-PCR. The amount of YFV-17D genome detected is expressed in fold change over the control siNSC. Statistical significance was tested using Mann-Whitney non-parametric t-test and the p-value is indicated in the figure. (B and C) Percentage of YFV-17D (B) and DEN2-NGC (C) infected wild type (WT) and β-arrestin 1/2-KO MEFs. The error bars represent the standard deviation of at least 4 wells. Statistical significance was tested using Welch's t-test, conditions with an inhibitory effect with a p-value

    Article Snippet: Quantitative PCR analysis of viral RNA Total RNA was isolated from infected HuH-7 cells using TRI Reagent (Sigma). cDNA was generated using High-Capacity cDNA Reverse Transcription kit (Applied Biosystems).

    Techniques: Incubation, Quantitative RT-PCR, MANN-WHITNEY, Infection, Standard Deviation

    RNA Binding Characteristics of HCF152. (A) Scheme of the Arabidopsis psbB - petB region. The RNA probes for the binding assays are indicated by arrows and letters (BDb to BDf). The length of each arrow indicates the length of the probe, and a scale bar for 400 nucleotides is indicated. Stars indicate the high-affinity binding sites for HCF152. (B) RNA binding of HCF152-F to several RNAs derived from the psbB - petD operon was analyzed with the UV cross-linking assay. (C) Competition UV cross-linking assay. The indicated RNAs competed with the BDd RNA binding to the recombinant HCF152-F. The experiments were performed with radiolabeled BDd RNA and 25-, 50-, or 100-fold molar excess of the nonradioactive RNAs BDb to BDf. The IC 50 values calculated from the graphs shown in (D) of at least three independent experiments are shown. (D) Intensities of the bands in (C) were quantified and plotted. The intensity without a competitor RNA was defined as 100%. Closed circle, BDb; closed triangle, BDc; closed square, BDd; open circle, BDe; open square, BDf.

    Journal: The Plant Cell

    Article Title: HCF152, an Arabidopsis RNA Binding Pentatricopeptide Repeat Protein Involved in the Processing of Chloroplast psbB-psbT-psbH-petB-petD RNAs

    doi: 10.1105/tpc.010397

    Figure Lengend Snippet: RNA Binding Characteristics of HCF152. (A) Scheme of the Arabidopsis psbB - petB region. The RNA probes for the binding assays are indicated by arrows and letters (BDb to BDf). The length of each arrow indicates the length of the probe, and a scale bar for 400 nucleotides is indicated. Stars indicate the high-affinity binding sites for HCF152. (B) RNA binding of HCF152-F to several RNAs derived from the psbB - petD operon was analyzed with the UV cross-linking assay. (C) Competition UV cross-linking assay. The indicated RNAs competed with the BDd RNA binding to the recombinant HCF152-F. The experiments were performed with radiolabeled BDd RNA and 25-, 50-, or 100-fold molar excess of the nonradioactive RNAs BDb to BDf. The IC 50 values calculated from the graphs shown in (D) of at least three independent experiments are shown. (D) Intensities of the bands in (C) were quantified and plotted. The intensity without a competitor RNA was defined as 100%. Closed circle, BDb; closed triangle, BDc; closed square, BDd; open circle, BDe; open square, BDf.

    Article Snippet: For UV cross-linking of the HCF152 protein to radiolabeled RNA, the protein was incubated with 32 P-RNA in buffer containing 10 mM Hepes-NaOH, pH 7.9, 30 mM KCl, 6 mM MgCl2 , 0.05 mM EDTA, 2 mM DTT, 8% glycerol, 0.0067% Triton X-100, and 67 μg/mL yeast tRNA (Sigma) for 15 min.

    Techniques: RNA Binding Assay, Binding Assay, Derivative Assay, Recombinant

    Transcript Pattern of the psbB-psbT-psbH-petB-petD Operon in the Mutants hcf152-1 and hcf152-2 . Eight micrograms of total leaf RNA from 3-week-old mutant and wild-type (WT) seedlings was analyzed by RNA gel blot hybridization. The nylon filters were hybridized with the gene-specific probes indicated with arrows and letters (A to H). Introns of the petB and petD genes are designated I B and I D , respectively. The sizes (in nucleotides) and identities of the transcripts are indicated.

    Journal: The Plant Cell

    Article Title: HCF152, an Arabidopsis RNA Binding Pentatricopeptide Repeat Protein Involved in the Processing of Chloroplast psbB-psbT-psbH-petB-petD RNAs

    doi: 10.1105/tpc.010397

    Figure Lengend Snippet: Transcript Pattern of the psbB-psbT-psbH-petB-petD Operon in the Mutants hcf152-1 and hcf152-2 . Eight micrograms of total leaf RNA from 3-week-old mutant and wild-type (WT) seedlings was analyzed by RNA gel blot hybridization. The nylon filters were hybridized with the gene-specific probes indicated with arrows and letters (A to H). Introns of the petB and petD genes are designated I B and I D , respectively. The sizes (in nucleotides) and identities of the transcripts are indicated.

    Article Snippet: For UV cross-linking of the HCF152 protein to radiolabeled RNA, the protein was incubated with 32 P-RNA in buffer containing 10 mM Hepes-NaOH, pH 7.9, 30 mM KCl, 6 mM MgCl2 , 0.05 mM EDTA, 2 mM DTT, 8% glycerol, 0.0067% Triton X-100, and 67 μg/mL yeast tRNA (Sigma) for 15 min.

    Techniques: Mutagenesis, Western Blot, Hybridization

    RNase Protection Assay for Processed and Spliced RNAs of the psbB-psbT-psbH-petB-petD Operon in hcf152-1 , hcf152-2 , and the Wild Type. RNase protection assay for spliced and processed petB , petD (A) , and psbH (B) RNAs. Total leaf RNA isolated from the wild type (WT) or the mutants ( hcf152-1 and hcf152-2 ) was annealed with uniformly labeled probes spanning the petB , petD , and psbH splice junction and processing sites. The transcribed probes included a small amount of vector sequence on both sides to be separated from the protected fragment of the unspliced RNA. A parallel control reaction contained no leaf RNA but an equivalent amount of yeast tRNA (tRNA). The RNA was digested with RNase T1, and the protected fragments were analyzed on sequencing gels and by autoradiography. The locations of the probes are indicated in the schemes (thick lines). The sizes of the protected fragments (thin lines and numbers in boldface to the left of the lanes) were determined by coelectrophoresis with a DNA sequence ladder. For subsequent identification of protected bands, a 100-bp DNA sequence ladder was used that was radioactively labeled (M) (sizes of the marker fragments are indicated with lightface numbers). Protected probe fragments correspond to unspliced (u), spliced (s), spliced intron (si), unspliced processed (u,p), or processed (p) RNA and are indicated at right. A protected band of the petD probe of ∼220 nucleotides of unknown origin is indicated with an asterisk. S1 protection experiments revealed a single protected fragment of 430 nucleotides for the unspliced petD RNAs, indicating that the triplet detected here reflects heterogeneous cutting by RNase T1.

    Journal: The Plant Cell

    Article Title: HCF152, an Arabidopsis RNA Binding Pentatricopeptide Repeat Protein Involved in the Processing of Chloroplast psbB-psbT-psbH-petB-petD RNAs

    doi: 10.1105/tpc.010397

    Figure Lengend Snippet: RNase Protection Assay for Processed and Spliced RNAs of the psbB-psbT-psbH-petB-petD Operon in hcf152-1 , hcf152-2 , and the Wild Type. RNase protection assay for spliced and processed petB , petD (A) , and psbH (B) RNAs. Total leaf RNA isolated from the wild type (WT) or the mutants ( hcf152-1 and hcf152-2 ) was annealed with uniformly labeled probes spanning the petB , petD , and psbH splice junction and processing sites. The transcribed probes included a small amount of vector sequence on both sides to be separated from the protected fragment of the unspliced RNA. A parallel control reaction contained no leaf RNA but an equivalent amount of yeast tRNA (tRNA). The RNA was digested with RNase T1, and the protected fragments were analyzed on sequencing gels and by autoradiography. The locations of the probes are indicated in the schemes (thick lines). The sizes of the protected fragments (thin lines and numbers in boldface to the left of the lanes) were determined by coelectrophoresis with a DNA sequence ladder. For subsequent identification of protected bands, a 100-bp DNA sequence ladder was used that was radioactively labeled (M) (sizes of the marker fragments are indicated with lightface numbers). Protected probe fragments correspond to unspliced (u), spliced (s), spliced intron (si), unspliced processed (u,p), or processed (p) RNA and are indicated at right. A protected band of the petD probe of ∼220 nucleotides of unknown origin is indicated with an asterisk. S1 protection experiments revealed a single protected fragment of 430 nucleotides for the unspliced petD RNAs, indicating that the triplet detected here reflects heterogeneous cutting by RNase T1.

    Article Snippet: For UV cross-linking of the HCF152 protein to radiolabeled RNA, the protein was incubated with 32 P-RNA in buffer containing 10 mM Hepes-NaOH, pH 7.9, 30 mM KCl, 6 mM MgCl2 , 0.05 mM EDTA, 2 mM DTT, 8% glycerol, 0.0067% Triton X-100, and 67 μg/mL yeast tRNA (Sigma) for 15 min.

    Techniques: Rnase Protection Assay, Isolation, Labeling, Plasmid Preparation, Sequencing, Autoradiography, Marker

    Characterization of Complemented hcf152 Transformants. (A) Chlorophyll fluorescence induction kinetics of the wild type, hcf152-1 mutants, and hcf152-1 transformants complemented with the open reading frame of the At3g09650 gene. A representative curve for each plant is shown (for details, see Methods). Fm, maximal fluorescence; Fo, initial fluorescence. (B) Transcript pattern of the psbB-psbT-psbH-petB-petD operon in the T1 generation of complemented hcf152-1 . The RNA gel blot was hybridized with the probe against the petD gene. Arrows indicate significant differences between the mutants and the transformants.

    Journal: The Plant Cell

    Article Title: HCF152, an Arabidopsis RNA Binding Pentatricopeptide Repeat Protein Involved in the Processing of Chloroplast psbB-psbT-psbH-petB-petD RNAs

    doi: 10.1105/tpc.010397

    Figure Lengend Snippet: Characterization of Complemented hcf152 Transformants. (A) Chlorophyll fluorescence induction kinetics of the wild type, hcf152-1 mutants, and hcf152-1 transformants complemented with the open reading frame of the At3g09650 gene. A representative curve for each plant is shown (for details, see Methods). Fm, maximal fluorescence; Fo, initial fluorescence. (B) Transcript pattern of the psbB-psbT-psbH-petB-petD operon in the T1 generation of complemented hcf152-1 . The RNA gel blot was hybridized with the probe against the petD gene. Arrows indicate significant differences between the mutants and the transformants.

    Article Snippet: For UV cross-linking of the HCF152 protein to radiolabeled RNA, the protein was incubated with 32 P-RNA in buffer containing 10 mM Hepes-NaOH, pH 7.9, 30 mM KCl, 6 mM MgCl2 , 0.05 mM EDTA, 2 mM DTT, 8% glycerol, 0.0067% Triton X-100, and 67 μg/mL yeast tRNA (Sigma) for 15 min.

    Techniques: Fluorescence, Western Blot

    Map of the Genomic Region Around the T-DNA Insertion in hcf152-1 and Analysis of Transcript Levels of the Corresponding Genomic Region. In the scheme at top, the T-DNA insertion is located in a genomic segment of BAC F11F8. Genes adjacent to the insertion point are shown and designated according to the MIPS database code. Black bars represent exon sequences, and white bars represent intron sequences. Arrows indicate gene orientation. The probes used in RNA gel blot hybridization experiments are depicted below the scheme. (A) to (C) Transcript levels of the At3g09650 (A) , At3g09660 (B) , and At3g09670 (C) genes in wild-type (WT) and hcf152-1/2 detected with double-stranded probes. Five micrograms of poly(A) RNA was loaded in each lane. (D) Wild-type and mutant RNA hybridized with an antisense probe of the At3g09650 gene. The transcript of HCF152 is indicated by the arrow. Two micrograms of poly(A) RNA was loaded in each lane.

    Journal: The Plant Cell

    Article Title: HCF152, an Arabidopsis RNA Binding Pentatricopeptide Repeat Protein Involved in the Processing of Chloroplast psbB-psbT-psbH-petB-petD RNAs

    doi: 10.1105/tpc.010397

    Figure Lengend Snippet: Map of the Genomic Region Around the T-DNA Insertion in hcf152-1 and Analysis of Transcript Levels of the Corresponding Genomic Region. In the scheme at top, the T-DNA insertion is located in a genomic segment of BAC F11F8. Genes adjacent to the insertion point are shown and designated according to the MIPS database code. Black bars represent exon sequences, and white bars represent intron sequences. Arrows indicate gene orientation. The probes used in RNA gel blot hybridization experiments are depicted below the scheme. (A) to (C) Transcript levels of the At3g09650 (A) , At3g09660 (B) , and At3g09670 (C) genes in wild-type (WT) and hcf152-1/2 detected with double-stranded probes. Five micrograms of poly(A) RNA was loaded in each lane. (D) Wild-type and mutant RNA hybridized with an antisense probe of the At3g09650 gene. The transcript of HCF152 is indicated by the arrow. Two micrograms of poly(A) RNA was loaded in each lane.

    Article Snippet: For UV cross-linking of the HCF152 protein to radiolabeled RNA, the protein was incubated with 32 P-RNA in buffer containing 10 mM Hepes-NaOH, pH 7.9, 30 mM KCl, 6 mM MgCl2 , 0.05 mM EDTA, 2 mM DTT, 8% glycerol, 0.0067% Triton X-100, and 67 μg/mL yeast tRNA (Sigma) for 15 min.

    Techniques: BAC Assay, Western Blot, Hybridization, Mutagenesis

    OND depletes intracellular ATP levels, inhibits transcription, induces relocation of the cellular polyamine pool to the nucleus and reduces the staining density of histone H3 with antibody. a The intracellular concentration of ATP in untreated, OND-exposed and recovering cells was determined using a luciferase dependent assay. b Global rates of transcription, determined by the incorporation of bromouridine into RNA, in untreated cells, cells under OND and cells recovering from OND are presented. HL-1 cells, either untreated or subjected to 1 hour of OND, were fixed, permeabilized and stained either with anti-polyamine antibody ( c , d ) or with anti-total H3 antibody ( e , f ) and counterstained with the fluorescent DNA binding dye Hoechst 33342. Cells were then examined using confocal microscopy. The content of immunostained histone H3 was evaluated by SMLM in untreated ( e ) and OND-treated ( f ) HL-1 cells. BrU bromouridine. The error bars represent the standard deviation of three independent samples

    Journal: Genome Biology

    Article Title: A transient ischemic environment induces reversible compaction of chromatin

    doi: 10.1186/s13059-015-0802-2

    Figure Lengend Snippet: OND depletes intracellular ATP levels, inhibits transcription, induces relocation of the cellular polyamine pool to the nucleus and reduces the staining density of histone H3 with antibody. a The intracellular concentration of ATP in untreated, OND-exposed and recovering cells was determined using a luciferase dependent assay. b Global rates of transcription, determined by the incorporation of bromouridine into RNA, in untreated cells, cells under OND and cells recovering from OND are presented. HL-1 cells, either untreated or subjected to 1 hour of OND, were fixed, permeabilized and stained either with anti-polyamine antibody ( c , d ) or with anti-total H3 antibody ( e , f ) and counterstained with the fluorescent DNA binding dye Hoechst 33342. Cells were then examined using confocal microscopy. The content of immunostained histone H3 was evaluated by SMLM in untreated ( e ) and OND-treated ( f ) HL-1 cells. BrU bromouridine. The error bars represent the standard deviation of three independent samples

    Article Snippet: Bromouridine labeling of nascent RNA Newly synthesized RNA was pulse labeled by incubating confluent 10-cm culture dishes with 2 mM bromouridine (BrU; Sigma Aldrich) for 1 hour, either under normal culture conditions, under OND, or after 1 hour of recovery from OND.

    Techniques: Staining, Concentration Assay, Luciferase, Binding Assay, Confocal Microscopy, Standard Deviation

    Estimating the effect of the tissue heterogenity. Normalized gene expressions levels comparing diapausing and non-diapausing D . montana females A) from the RNAseq analysis (for comparison), and B) from the qPCR using the females whose ovaries had been removed before RNA extractions. Both data sets present the combined data for all the three isofemales strains (see text for details). CPM = counts per million. Significance levels for qPCR: • 0.1 > P > 0.05, **0.01 > P > 0.001, ***P

    Journal: PLoS ONE

    Article Title: Transcriptional Differences between Diapausing and Non-Diapausing D. montana Females Reared under the Same Photoperiod and Temperature

    doi: 10.1371/journal.pone.0161852

    Figure Lengend Snippet: Estimating the effect of the tissue heterogenity. Normalized gene expressions levels comparing diapausing and non-diapausing D . montana females A) from the RNAseq analysis (for comparison), and B) from the qPCR using the females whose ovaries had been removed before RNA extractions. Both data sets present the combined data for all the three isofemales strains (see text for details). CPM = counts per million. Significance levels for qPCR: • 0.1 > P > 0.05, **0.01 > P > 0.001, ***P

    Article Snippet: RNA extraction and RNAseq RNA extraction was performed using a Tri Reagent (Sigma-Aldrich) extraction kit followed by a RNeasy Mini (Qiagen) kit with DNase treatment.

    Techniques: Real-time Polymerase Chain Reaction

    Sensitivity of RT-RPA assay for purified GII.4 New Orleans RNA as predicted using probit regression analyses. Serial dilutions of purified RNA from three selected patient stool samples were used as templates in RT-RPA reactions for 8 replicates each and the number of positive samples at each dilution was used for separate probit regressions. A representative regression analysis (for sample 29) is pictured. The points mark the proportion of samples positive at each log 10 genomic copy level; the solid line marks the predicted frequency of samples positive as a function of log 10 genomic copy input, and the dotted line is a visual marker at the 95% level of sample positivity.

    Journal: Scientific Reports

    Article Title: Development of a Recombinase Polymerase Amplification Assay for Detection of Epidemic Human Noroviruses

    doi: 10.1038/srep40244

    Figure Lengend Snippet: Sensitivity of RT-RPA assay for purified GII.4 New Orleans RNA as predicted using probit regression analyses. Serial dilutions of purified RNA from three selected patient stool samples were used as templates in RT-RPA reactions for 8 replicates each and the number of positive samples at each dilution was used for separate probit regressions. A representative regression analysis (for sample 29) is pictured. The points mark the proportion of samples positive at each log 10 genomic copy level; the solid line marks the predicted frequency of samples positive as a function of log 10 genomic copy input, and the dotted line is a visual marker at the 95% level of sample positivity.

    Article Snippet: These earlier studies used an RNA standard to determine analytical sensitivity and showed RT-RPA detection limits < 1 LGC for bovine coronavirus , and Rift Valley fever, Ebola, Marburg, Sudan and Sigma viruses .

    Techniques: RT RPA Assay, Purification, Recombinase Polymerase Amplification, Marker

    Time to signal detection of different RT-RPA primer pairs. The RT-RPA assay was performed as described using the NOP1 probe. The template used was 7.0 LGC of purified GII.4 New Orleans RNA per reaction.

    Journal: Scientific Reports

    Article Title: Development of a Recombinase Polymerase Amplification Assay for Detection of Epidemic Human Noroviruses

    doi: 10.1038/srep40244

    Figure Lengend Snippet: Time to signal detection of different RT-RPA primer pairs. The RT-RPA assay was performed as described using the NOP1 probe. The template used was 7.0 LGC of purified GII.4 New Orleans RNA per reaction.

    Article Snippet: These earlier studies used an RNA standard to determine analytical sensitivity and showed RT-RPA detection limits < 1 LGC for bovine coronavirus , and Rift Valley fever, Ebola, Marburg, Sudan and Sigma viruses .

    Techniques: Recombinase Polymerase Amplification, RT RPA Assay, Purification

    Characterization of PKI-NL4.3 and H89-NL4.3 viral particles. (A) 293T cells expressing HIV-1 NL4.3 were maintained in the presence of medium alone or supplemented with Myr-PKI (PKI) or H89 inhibitors. Normalized amounts of cells lysates (left panel) or sucrose cushion purified viruses (right panel) were sequentially probed with rabbit anti-RT or anti-gp41 sera or anti-p24 mAbs. (B) Genomic RNA in viral particles was quantified by qRT-PCR. Values are expressed as percentages of NL4.3 values ± SD. (C) NL4.3, PKI-NL4.3 and H89-NL4.3 viral particles were imaged by electron microscopy. Bar = 100 nm. (D) The number of fully mature and aberrant viruses was counted in each sample and expressed as a percentage of total observation (NL4.3 n = 57; PKI-NL4.3 n = 105; H89-NL4.3 n = 18). Error bars represent 95% confidence intervals.

    Journal: Retrovirology

    Article Title: HIV-1-associated PKA acts as a cofactor for genome reverse transcription

    doi: 10.1186/1742-4690-10-157

    Figure Lengend Snippet: Characterization of PKI-NL4.3 and H89-NL4.3 viral particles. (A) 293T cells expressing HIV-1 NL4.3 were maintained in the presence of medium alone or supplemented with Myr-PKI (PKI) or H89 inhibitors. Normalized amounts of cells lysates (left panel) or sucrose cushion purified viruses (right panel) were sequentially probed with rabbit anti-RT or anti-gp41 sera or anti-p24 mAbs. (B) Genomic RNA in viral particles was quantified by qRT-PCR. Values are expressed as percentages of NL4.3 values ± SD. (C) NL4.3, PKI-NL4.3 and H89-NL4.3 viral particles were imaged by electron microscopy. Bar = 100 nm. (D) The number of fully mature and aberrant viruses was counted in each sample and expressed as a percentage of total observation (NL4.3 n = 57; PKI-NL4.3 n = 105; H89-NL4.3 n = 18). Error bars represent 95% confidence intervals.

    Article Snippet: qPCR analysis of genomic RNA and proviral DNA Total RNA isolated from cells challenged with HIV for 1 h or genomic RNA contained in HIV-1 particles (100 ng p24) was extracted with Tri-Reagent (Sigma, France) and subjected to qRT-PCR analysis as previously described [ ].

    Techniques: Expressing, Purification, Quantitative RT-PCR, Electron Microscopy

    Stimulation of IRES-mediated translation by 2A pro coexpression. (A) HeLa cells were transfected with CBV3 IRES reporter RNA alone (mock) or cotransfected with capped PV, CBV3 2A pro RNAs, or a CBV3 2A pro active-site mutant. (B) Expression and proteolytic

    Journal:

    Article Title: Competitive Translation Efficiency at the Picornavirus Type 1 Internal Ribosome Entry Site Facilitated by Viral cis and trans Factors

    doi: 10.1128/JVI.80.7.3310-3321.2006

    Figure Lengend Snippet: Stimulation of IRES-mediated translation by 2A pro coexpression. (A) HeLa cells were transfected with CBV3 IRES reporter RNA alone (mock) or cotransfected with capped PV, CBV3 2A pro RNAs, or a CBV3 2A pro active-site mutant. (B) Expression and proteolytic

    Article Snippet: Twenty-four hours after DNA transfection, cells were washed with serum-free medium, infected with CBV3 at an MOI of 10 or mock infected, transfected with reporter RNA, and subjected to the luc assay and Western blotting as described above. α-c- myc antibody 9E10 (Sigma) and biotinylated α-mouse immunoglobulin G (Vector Laboratories) were used to detect exogenous eIF4GI.

    Techniques: Transfection, Mutagenesis, Expressing

    Stability of reporter RNAs with different 3′ termini in HeLa cells. w/o (A); 3′UTR (B); A 12 (C); 3′UTR+A 12 (D); A 50 (E); 3′UTR+A 50 (F). Total RNA from transfected cells was separated by gel electrophoresis

    Journal:

    Article Title: Competitive Translation Efficiency at the Picornavirus Type 1 Internal Ribosome Entry Site Facilitated by Viral cis and trans Factors

    doi: 10.1128/JVI.80.7.3310-3321.2006

    Figure Lengend Snippet: Stability of reporter RNAs with different 3′ termini in HeLa cells. w/o (A); 3′UTR (B); A 12 (C); 3′UTR+A 12 (D); A 50 (E); 3′UTR+A 50 (F). Total RNA from transfected cells was separated by gel electrophoresis

    Article Snippet: Twenty-four hours after DNA transfection, cells were washed with serum-free medium, infected with CBV3 at an MOI of 10 or mock infected, transfected with reporter RNA, and subjected to the luc assay and Western blotting as described above. α-c- myc antibody 9E10 (Sigma) and biotinylated α-mouse immunoglobulin G (Vector Laboratories) were used to detect exogenous eIF4GI.

    Techniques: Transfection, Nucleic Acid Electrophoresis

    Effect of concomitant EV infection on IRES-driven translation. (A) One-step growth curves of CBV3 and PV1(M) in HeLa cells. (B) rLuc activity in mock-infected cells transfected with reporter RNA carrying the CBV3 IRES and CBV3 3′UTR+A

    Journal:

    Article Title: Competitive Translation Efficiency at the Picornavirus Type 1 Internal Ribosome Entry Site Facilitated by Viral cis and trans Factors

    doi: 10.1128/JVI.80.7.3310-3321.2006

    Figure Lengend Snippet: Effect of concomitant EV infection on IRES-driven translation. (A) One-step growth curves of CBV3 and PV1(M) in HeLa cells. (B) rLuc activity in mock-infected cells transfected with reporter RNA carrying the CBV3 IRES and CBV3 3′UTR+A

    Article Snippet: Twenty-four hours after DNA transfection, cells were washed with serum-free medium, infected with CBV3 at an MOI of 10 or mock infected, transfected with reporter RNA, and subjected to the luc assay and Western blotting as described above. α-c- myc antibody 9E10 (Sigma) and biotinylated α-mouse immunoglobulin G (Vector Laboratories) were used to detect exogenous eIF4GI.

    Techniques: Infection, Activity Assay, Transfection

    Cumulative effects of EV cis -acting sequence elements on EV IRES translation in vivo. Efficiency of rLuc expression from specified CBV3 IRES-containing reporter RNAs is shown in comparison with a capped reporter RNA containing the β-globin 5′

    Journal:

    Article Title: Competitive Translation Efficiency at the Picornavirus Type 1 Internal Ribosome Entry Site Facilitated by Viral cis and trans Factors

    doi: 10.1128/JVI.80.7.3310-3321.2006

    Figure Lengend Snippet: Cumulative effects of EV cis -acting sequence elements on EV IRES translation in vivo. Efficiency of rLuc expression from specified CBV3 IRES-containing reporter RNAs is shown in comparison with a capped reporter RNA containing the β-globin 5′

    Article Snippet: Twenty-four hours after DNA transfection, cells were washed with serum-free medium, infected with CBV3 at an MOI of 10 or mock infected, transfected with reporter RNA, and subjected to the luc assay and Western blotting as described above. α-c- myc antibody 9E10 (Sigma) and biotinylated α-mouse immunoglobulin G (Vector Laboratories) were used to detect exogenous eIF4GI.

    Techniques: Sequencing, In Vivo, Expressing

    Northern blot analysis of ins-33 mRNA levels in poly(A)-selected RNA from wild-type or lin-14 ( lf ) mutant animals at the indicated stages that had been grown at the indicated temperatures. mRNA samples from either wild-type (lanes 1 to 3) or lin-14 ( lf

    Journal:

    Article Title: The Caenorhabditis elegans Heterochronic Regulator LIN-14 Is a Novel Transcription Factor That Controls the Developmental Timing of Transcription from the Insulin/Insulin-Like Growth Factor Gene ins-33 by Direct DNA Binding

    doi: 10.1128/MCB.25.24.11059-11072.2005

    Figure Lengend Snippet: Northern blot analysis of ins-33 mRNA levels in poly(A)-selected RNA from wild-type or lin-14 ( lf ) mutant animals at the indicated stages that had been grown at the indicated temperatures. mRNA samples from either wild-type (lanes 1 to 3) or lin-14 ( lf

    Article Snippet: RNA samples were stored at −80°C as an ethanol precipitate. mRNA was prepared from total worm RNA using oligo(dT) cellulose (Sigma) and following published procedures ( ). mRNA samples were shipped as dried pellets on dry ice to Stuart Kim's laboratory at Stanford University for cDNA synthesis and microarray hybridization (procedures available at ).

    Techniques: Northern Blot, Mutagenesis

    drb7.2 mutant plants display altered accumulation of endoIR-siRNA. Wild-type (Col-0) and drb7.2 mutant plants were subjected to high throughput sequencing. ( A ) Size distribution of small RNA reads that perfectly match the Arabidopsis nuclear genome, excluding rRNA and tRNA. The proportion of each size of small RNA is indicated by a color code: 21 nt (blue), 22 nt (green), 23 nt (pink), 24 nt (red) and grey for 18 to 20 nt and 25 to 26 nt. ( B ) Normalized small RNA abundance of the three major classes of endogenous siRNAs. Small RNAs from Col-0 and drb7.2 mutant plants were classified as ta-siRNAs (upper panel), p4-siRNAs (middle panel) or endoIR-siRNAs (lower panel) based on published annotation and their abundance was normalized to the total amount of conserved miRNAs. The color code of small RNA size is the same as in (A). ( C ) Normalized size distribution of endoIR-siRNAs from three IR loci (IR71, IR2039 and AT5G22960). Size distribution of TAS3-derived ta-siRNAs is shown here as a control. Numbers of normalized small RNA reads that perfectly match the aforementioned loci are represented under bracket. The color code of small RNA size is the same as in (A). Graphs in (B) were obtained using the mean value of the two biological replicates and error bars indicate the variation observed between the two replicates.

    Journal: Nucleic Acids Research

    Article Title: A specific dsRNA-binding protein complex selectively sequesters endogenous inverted-repeat siRNA precursors and inhibits their processing

    doi: 10.1093/nar/gkw1264

    Figure Lengend Snippet: drb7.2 mutant plants display altered accumulation of endoIR-siRNA. Wild-type (Col-0) and drb7.2 mutant plants were subjected to high throughput sequencing. ( A ) Size distribution of small RNA reads that perfectly match the Arabidopsis nuclear genome, excluding rRNA and tRNA. The proportion of each size of small RNA is indicated by a color code: 21 nt (blue), 22 nt (green), 23 nt (pink), 24 nt (red) and grey for 18 to 20 nt and 25 to 26 nt. ( B ) Normalized small RNA abundance of the three major classes of endogenous siRNAs. Small RNAs from Col-0 and drb7.2 mutant plants were classified as ta-siRNAs (upper panel), p4-siRNAs (middle panel) or endoIR-siRNAs (lower panel) based on published annotation and their abundance was normalized to the total amount of conserved miRNAs. The color code of small RNA size is the same as in (A). ( C ) Normalized size distribution of endoIR-siRNAs from three IR loci (IR71, IR2039 and AT5G22960). Size distribution of TAS3-derived ta-siRNAs is shown here as a control. Numbers of normalized small RNA reads that perfectly match the aforementioned loci are represented under bracket. The color code of small RNA size is the same as in (A). Graphs in (B) were obtained using the mean value of the two biological replicates and error bars indicate the variation observed between the two replicates.

    Article Snippet: RNA analysis Total RNA was extracted from Arabidopsis tissues with Tri-Reagent (Sigma, St Louis, MO, USA) according to manufacturer's instructions.

    Techniques: Mutagenesis, Next-Generation Sequencing, Derivative Assay

    For figure legend see page . Figure 1. (see previous page) Lacto-rpoB and 23S methyl RNA elements are regulated by RNase III in S. pyogenes. A. Northern blot analysis (polyacrylamide gel electrophoresis) of Lacto-rpoB and 23S-methyl RNA expression in WT (SF370), ΔRNase III (Δ rnc ) and ΔRNase Y (Δ rny ) strains grown to early logarithmic (EL), mid logarithmic (ML) and early stationary (ES) phases. 5S rRNA is used as loading control. B. Expression profiles of Lacto-rpoB and the 23S-methyl motif with surrounding genes captured using the Integrative Genomics Viewer (IGV) software. The sequence coverage was calculated using BEDTools-Version-2.15.0 and the scale is given in number of reads per million. The distribution of reads starting (5') and ending (3') at each nucleotide position is represented in blue and orange, respectively. The position of the oligonucleotide probes (OLEC) used in Northern blot analysis is indicated. C. Prediction of RNA secondary structure using RNAfold (rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). The arrows represent putative cleavages by RNase III (nucleotides determined by analyzing the 5' and 3' ends of the sRNAs in sRNA sequencing data).

    Journal: RNA Biology

    Article Title: RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes

    doi: 10.1080/15476286.2015.1110674

    Figure Lengend Snippet: For figure legend see page . Figure 1. (see previous page) Lacto-rpoB and 23S methyl RNA elements are regulated by RNase III in S. pyogenes. A. Northern blot analysis (polyacrylamide gel electrophoresis) of Lacto-rpoB and 23S-methyl RNA expression in WT (SF370), ΔRNase III (Δ rnc ) and ΔRNase Y (Δ rny ) strains grown to early logarithmic (EL), mid logarithmic (ML) and early stationary (ES) phases. 5S rRNA is used as loading control. B. Expression profiles of Lacto-rpoB and the 23S-methyl motif with surrounding genes captured using the Integrative Genomics Viewer (IGV) software. The sequence coverage was calculated using BEDTools-Version-2.15.0 and the scale is given in number of reads per million. The distribution of reads starting (5') and ending (3') at each nucleotide position is represented in blue and orange, respectively. The position of the oligonucleotide probes (OLEC) used in Northern blot analysis is indicated. C. Prediction of RNA secondary structure using RNAfold (rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). The arrows represent putative cleavages by RNase III (nucleotides determined by analyzing the 5' and 3' ends of the sRNAs in sRNA sequencing data).

    Article Snippet: RNA extraction Total RNA from S. pyogenes SF370 wild type, deletion mutants and complemented strains was prepared using TRIzol (Sigma-Aldrich)/chloroform extraction and isopropanol precipitation from samples collected at several phases of growth (early logarithmic phase, EL; mid logarithmic phase, ML, and early stationary phrase, ES).

    Techniques: Polyacrylamide Gel Electrophoresis, Northern Blot, RNA Expression, Expressing, Software, Sequencing

    RT-PCR for aquaporins and canalicular solute transporters in WIF-B cells . Total RNA from WIF-B cells was reverse transcribed using random primers and then PCR-amplified with primers designed to amplify a nonconserved region of each AQP and solute transporter cDNA. Isolated rat hepatocyte cDNA were used as positive controls. Lane 1, negative control; lane 2, positive control; lane 3, WIF-B cells.

    Journal: BMC Physiology

    Article Title: Expression and subcellular localization of aquaporin water channels in the polarized hepatocyte cell line, WIF-B

    doi: 10.1186/1472-6793-5-13

    Figure Lengend Snippet: RT-PCR for aquaporins and canalicular solute transporters in WIF-B cells . Total RNA from WIF-B cells was reverse transcribed using random primers and then PCR-amplified with primers designed to amplify a nonconserved region of each AQP and solute transporter cDNA. Isolated rat hepatocyte cDNA were used as positive controls. Lane 1, negative control; lane 2, positive control; lane 3, WIF-B cells.

    Article Snippet: RNA isolation Total RNA was extracted from WIF-B cells or freshly isolated rat hepatocytes using Tri-Reagent (Sigma).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Isolation, Negative Control, Positive Control

    DEX treatment down-regulates CBG mRNA and protein levels in hepatoma cell lines. The effect of DEX on CBG mRNA and protein levels was investigated in a human hepatoma cell line, HepG2 (A B) and in a mouse hepatoma cell line, BWTG3 (C D). Both cell lines (HepG2 BWTG3) were incubated with vehicle control (0.1% EtOH) or 1 nM DEX for 8 hrs to determine CBG mRNA (A C) and protein expression (B D). Total RNA was isolated and reversed transcribed to cDNA. Real-time quantitative PCR (qPCR) was performed to determine the mRNA expression levels of CBG and internal standards (18S for HepG2 and GAPDH for BWTG3 cells). CBG protein expression was analyzed by means of Western blotting. GAPDH protein expression was used as loading control. Statistical analysis was done relative to the corresponding vehicle control (0.1% EtOH), using students unpaired t -test (*: p

    Journal: PLoS ONE

    Article Title: Inhibition of Corticosteroid-Binding Globulin Gene Expression by Glucocorticoids Involves C/EBPβ

    doi: 10.1371/journal.pone.0110702

    Figure Lengend Snippet: DEX treatment down-regulates CBG mRNA and protein levels in hepatoma cell lines. The effect of DEX on CBG mRNA and protein levels was investigated in a human hepatoma cell line, HepG2 (A B) and in a mouse hepatoma cell line, BWTG3 (C D). Both cell lines (HepG2 BWTG3) were incubated with vehicle control (0.1% EtOH) or 1 nM DEX for 8 hrs to determine CBG mRNA (A C) and protein expression (B D). Total RNA was isolated and reversed transcribed to cDNA. Real-time quantitative PCR (qPCR) was performed to determine the mRNA expression levels of CBG and internal standards (18S for HepG2 and GAPDH for BWTG3 cells). CBG protein expression was analyzed by means of Western blotting. GAPDH protein expression was used as loading control. Statistical analysis was done relative to the corresponding vehicle control (0.1% EtOH), using students unpaired t -test (*: p

    Article Snippet: For CBG mRNA analysis in both BWTG3 and HepG2 cells, cells were plated at a density of 1×105 cells/well in a 12-well plate, treated with vehicle control (01% EtOH) or 1 nM DEX for 8 hrs, after which total RNA was isolated using TRI Reagent (Sigma-Aldrich) as per the manufacture’s protocol.

    Techniques: Incubation, Expressing, Isolation, Real-time Polymerase Chain Reaction, Western Blot

    Figure 1. The tau IRES is restricted to 240 nucleotides and contributes to total translation levels. ( A ) A dicistronic RNA transfection was done in SK-N-SH neuroblastoma cells for 4 hrs. The BACE 1 5′ leader, β-globin 5′ leader, 240 nucleotide tau, 320 nucleotide tau and EMCV 5′ leaders were inserted upstream of the Photinus luciferase gene and tested for IRES activity. IRES activity is reported as a ratio of Photinus luciferase /Renilla luciferase. The P/R ratio for the negative control BACE 1 was normalized to 1 (n=5). ( B ) An A-capped monocistronic RNA containing the β-globin 5′ leader, 240 nucleotide tau, or 320 nucleotide tau was transfected along with an m7G-capped Renilla luciferase gene to control for transfection efficiency. The P/R ratio for β-globin was normalized to 1 and the P/R ratios for tau were normalized accordingly (n=4). ( C,D,F ) Semi-quantitative PCR of transfected RNAs amplified with gene specific primers to Photinus luciferase or GAPDH. ( E ) A-capped and m7G capped monocistronic RNAs were transfected separately into SK-N-SH cells along with a co-transfected m7G capped Renilla luciferase. The P/R ratios for the m7G capped mRNAs were normalized to 1. The P/R ratios of the A-capped messages were then normalized to the m7G counterpart to obtain a relative amount of IRES activity (n=4). All transfections were performed in triplicate and the average ± SEM is plotted in all cases.

    Journal: RNA Biology

    Article Title: Secondary RNA structure and nucleotide specificity contribute to internal initiation mediated by the human tau 5? leader

    doi: 10.4161/rna.22181

    Figure Lengend Snippet: Figure 1. The tau IRES is restricted to 240 nucleotides and contributes to total translation levels. ( A ) A dicistronic RNA transfection was done in SK-N-SH neuroblastoma cells for 4 hrs. The BACE 1 5′ leader, β-globin 5′ leader, 240 nucleotide tau, 320 nucleotide tau and EMCV 5′ leaders were inserted upstream of the Photinus luciferase gene and tested for IRES activity. IRES activity is reported as a ratio of Photinus luciferase /Renilla luciferase. The P/R ratio for the negative control BACE 1 was normalized to 1 (n=5). ( B ) An A-capped monocistronic RNA containing the β-globin 5′ leader, 240 nucleotide tau, or 320 nucleotide tau was transfected along with an m7G-capped Renilla luciferase gene to control for transfection efficiency. The P/R ratio for β-globin was normalized to 1 and the P/R ratios for tau were normalized accordingly (n=4). ( C,D,F ) Semi-quantitative PCR of transfected RNAs amplified with gene specific primers to Photinus luciferase or GAPDH. ( E ) A-capped and m7G capped monocistronic RNAs were transfected separately into SK-N-SH cells along with a co-transfected m7G capped Renilla luciferase. The P/R ratios for the m7G capped mRNAs were normalized to 1. The P/R ratios of the A-capped messages were then normalized to the m7G counterpart to obtain a relative amount of IRES activity (n=4). All transfections were performed in triplicate and the average ± SEM is plotted in all cases.

    Article Snippet: Following the RNA transfection, cells were harvested and lysed with 250 µL TRI Reagent® (Sigma Aldrich cat#93289–100 ML).

    Techniques: Transfection, Luciferase, Activity Assay, Negative Control, Real-time Polymerase Chain Reaction, Amplification

    Figure 4. Long range interaction in domain I. ( A ) Schematic representation of mutated residues within the structure. Mutant 18–25 was mutated to 5′-TATCCAA. Mutant 59–65 was mutated to 5′-TTGGATA. Mutations were inserted upstream of the Photinus luciferase gene to test for IRES activity ( B ) P/R ratios are normalized to the negative control β-globin. A-capped transfections were done in SK-N-SH cells for 4 h and performed in triplicate, (n = 5 ± S.E.M.) ( C ) Semi quantitative PCR of isolated RNAs from transfected cells. Amplification was done with Photinus luciferase and GAPDH specific primers. ( D ) Native gel of the mutant RNAs. 1 µg of RNA was run on a 10% native gel for 8 h and stained with EtBr. Different migration rates correspond to the overall change in structure.

    Journal: RNA Biology

    Article Title: Secondary RNA structure and nucleotide specificity contribute to internal initiation mediated by the human tau 5? leader

    doi: 10.4161/rna.22181

    Figure Lengend Snippet: Figure 4. Long range interaction in domain I. ( A ) Schematic representation of mutated residues within the structure. Mutant 18–25 was mutated to 5′-TATCCAA. Mutant 59–65 was mutated to 5′-TTGGATA. Mutations were inserted upstream of the Photinus luciferase gene to test for IRES activity ( B ) P/R ratios are normalized to the negative control β-globin. A-capped transfections were done in SK-N-SH cells for 4 h and performed in triplicate, (n = 5 ± S.E.M.) ( C ) Semi quantitative PCR of isolated RNAs from transfected cells. Amplification was done with Photinus luciferase and GAPDH specific primers. ( D ) Native gel of the mutant RNAs. 1 µg of RNA was run on a 10% native gel for 8 h and stained with EtBr. Different migration rates correspond to the overall change in structure.

    Article Snippet: Following the RNA transfection, cells were harvested and lysed with 250 µL TRI Reagent® (Sigma Aldrich cat#93289–100 ML).

    Techniques: Mutagenesis, Luciferase, Activity Assay, Negative Control, Transfection, Real-time Polymerase Chain Reaction, Isolation, Amplification, Staining, Migration

    Figure 2. The majority of the tau 5′ leader is required for IRES activity. ( A ) Schematic representation of deletions made from the 5′ or 3′ ends of the tau 5′ leader. Each deletion was inserted upstream of the Photinus luciferase gene. All RNAs were co-transfected with an m7G capped Renilla luciferase mRNA as a transfection control. ( B ) Transfections were done in SK-N-SH cells for 4 hrs. Photinus to Renilla luciferase ratios are normalized to the negative control, β-globin, which is given a value of 1. BACE 1 was also used as a negative control and EMCV IRES serves as the positive control for IRES activity. All transfections were performed in triplicate. Normalized P/R ratios are reported, (n = 6 ± S.E.M.). ( C ) Semi quantitative PCR of RNA isolated from the transfected cells was amplified with gene specific primers to either the Photinus luciferase or GAPDH ORF.

    Journal: RNA Biology

    Article Title: Secondary RNA structure and nucleotide specificity contribute to internal initiation mediated by the human tau 5? leader

    doi: 10.4161/rna.22181

    Figure Lengend Snippet: Figure 2. The majority of the tau 5′ leader is required for IRES activity. ( A ) Schematic representation of deletions made from the 5′ or 3′ ends of the tau 5′ leader. Each deletion was inserted upstream of the Photinus luciferase gene. All RNAs were co-transfected with an m7G capped Renilla luciferase mRNA as a transfection control. ( B ) Transfections were done in SK-N-SH cells for 4 hrs. Photinus to Renilla luciferase ratios are normalized to the negative control, β-globin, which is given a value of 1. BACE 1 was also used as a negative control and EMCV IRES serves as the positive control for IRES activity. All transfections were performed in triplicate. Normalized P/R ratios are reported, (n = 6 ± S.E.M.). ( C ) Semi quantitative PCR of RNA isolated from the transfected cells was amplified with gene specific primers to either the Photinus luciferase or GAPDH ORF.

    Article Snippet: Following the RNA transfection, cells were harvested and lysed with 250 µL TRI Reagent® (Sigma Aldrich cat#93289–100 ML).

    Techniques: Activity Assay, Luciferase, Transfection, Negative Control, Positive Control, Real-time Polymerase Chain Reaction, Isolation, Amplification

    Examples of leaderless mRNAs. (A), (B), (C), (D) and (E) correspond to E. coli genes dicA (Qin prophage; b1570), pgpA (b0418), racR (Rac prophage; b1356), rhlB (b3780) and ymfK (e14 prophage; b1145) respectively. (F) corresponds to an unnamed paralogue (SCO1678) of the S. coelicolor gene whiH . The panels are modified screenshots from the UCSC Microbial Genome Browser (Chan et al ., 2012 ). In each panel the tracks depict from top to bottom, the genome position, location of annotated genes, the positions of TSSs identified by the analysis of M-A scatterplots (Fig. 1 ), and the number of times each position on the corresponding strand was sequenced following fragmentation of the transcriptome (gRNA-seq). The numbers at the left of the RNA-seq tracks indicate the scale of the sequencing reads.

    Journal: Molecular Microbiology

    Article Title: A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing

    doi: 10.1111/mmi.12810

    Figure Lengend Snippet: Examples of leaderless mRNAs. (A), (B), (C), (D) and (E) correspond to E. coli genes dicA (Qin prophage; b1570), pgpA (b0418), racR (Rac prophage; b1356), rhlB (b3780) and ymfK (e14 prophage; b1145) respectively. (F) corresponds to an unnamed paralogue (SCO1678) of the S. coelicolor gene whiH . The panels are modified screenshots from the UCSC Microbial Genome Browser (Chan et al ., 2012 ). In each panel the tracks depict from top to bottom, the genome position, location of annotated genes, the positions of TSSs identified by the analysis of M-A scatterplots (Fig. 1 ), and the number of times each position on the corresponding strand was sequenced following fragmentation of the transcriptome (gRNA-seq). The numbers at the left of the RNA-seq tracks indicate the scale of the sequencing reads.

    Article Snippet: Isolation of bacterial RNA and transcriptome analysis RNA for RNA-seq analysis was isolated from S. coelicolor grown in YEME broth (Kieser et al ., ), as described previously for P. acnes (Lin et al ., ), and from E. coli BW25113 grown in Luria–Bertani broth (Sigma), as described previously for this organism (Kime et al ., ).

    Techniques: Modification, RNA Sequencing Assay, Sequencing

    Association of P body proteins with HIV-1 and 7SL RNA. (A) Immunoprecipitation of cellular RNA-protein complexes using anti-Flag antibody. 293T cells were cotransfected with pHDV-EGFP (7.0 μg) and empty pcDNA3.1 (control), pF-A3G (3.0 μg),

    Journal: Journal of Virology

    Article Title: P Body-Associated Protein Mov10 Inhibits HIV-1 Replication at Multiple Stages ▿P Body-Associated Protein Mov10 Inhibits HIV-1 Replication at Multiple Stages ▿ †

    doi: 10.1128/JVI.00585-10

    Figure Lengend Snippet: Association of P body proteins with HIV-1 and 7SL RNA. (A) Immunoprecipitation of cellular RNA-protein complexes using anti-Flag antibody. 293T cells were cotransfected with pHDV-EGFP (7.0 μg) and empty pcDNA3.1 (control), pF-A3G (3.0 μg),

    Article Snippet: The remaining 90% of the cells were lysed in the presence of RNaseOUT (Invitrogen) to minimize RNA degradation and used for Western blotting and immunoprecipitation using anti-Flag M2 affinity gel (Sigma).

    Techniques: Immunoprecipitation

    Mechanism of FXN activation by repeat-targeted duplex RNAs. ( a ) RIP examining the association of Ago2 with FXN pre-mRNA after treatment with 50 nM duplex RNA and analysis by real-time PCR. An arrow marks the PCR product of FXN pre-mRNA, which was confirmed by sequencing ( Supplementary Fig. 7 ) ( b ) Anti-GAA duplex RNA with central mismatches (siGAA 9,10 mm with mismatches on both strands; 25 nM) activates FXN expression at a level similar to the analogous fully complementary duplex RNA ( n =3). siExon3 is a positive control for transfection efficiency targeting exon 3 of FXN . ( c ) ChIP for RNAP2 using four different primer sets ( n =4). ( d ) ChIP for transcription-associated histone modification markers H3K4me3, H3K9me2, H3K9me3, H3K9Ac, H3K27me3 and H4Ac ( n =4–8). ( e ) FXN mRNA stability assay. Cells were transfected with duplex RNAs siGAA or CM at 25 nM ( n =3). Actinomycin D (5 μg ml −1 ) was added with fresh media 3 days after transfection and cells were collected at the indicated time points. HPRT expression was measured for normalization. All experiments were performed in GM03816 patient-derived cells. All data are presented as mean±STDEV. * P

    Journal: Nature Communications

    Article Title: Activating frataxin expression by repeat-targeted nucleic acids

    doi: 10.1038/ncomms10606

    Figure Lengend Snippet: Mechanism of FXN activation by repeat-targeted duplex RNAs. ( a ) RIP examining the association of Ago2 with FXN pre-mRNA after treatment with 50 nM duplex RNA and analysis by real-time PCR. An arrow marks the PCR product of FXN pre-mRNA, which was confirmed by sequencing ( Supplementary Fig. 7 ) ( b ) Anti-GAA duplex RNA with central mismatches (siGAA 9,10 mm with mismatches on both strands; 25 nM) activates FXN expression at a level similar to the analogous fully complementary duplex RNA ( n =3). siExon3 is a positive control for transfection efficiency targeting exon 3 of FXN . ( c ) ChIP for RNAP2 using four different primer sets ( n =4). ( d ) ChIP for transcription-associated histone modification markers H3K4me3, H3K9me2, H3K9me3, H3K9Ac, H3K27me3 and H4Ac ( n =4–8). ( e ) FXN mRNA stability assay. Cells were transfected with duplex RNAs siGAA or CM at 25 nM ( n =3). Actinomycin D (5 μg ml −1 ) was added with fresh media 3 days after transfection and cells were collected at the indicated time points. HPRT expression was measured for normalization. All experiments were performed in GM03816 patient-derived cells. All data are presented as mean±STDEV. * P

    Article Snippet: Quantitative PCR Identical volumes of RNA (representing approximately the same number of cells and ranging from 1 to 2 μg of RNA) were treated with 2 units of DNase I (Worthington) in DNase I buffer (10 mM Tris-HCl, pH 7.0, 10 mM NaCl, 2 mM MgCl2 and 0.5 mM CaCl2 ) for 15 min at room temperature to degrade any genomic DNA contamination.

    Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Sequencing, Expressing, Positive Control, Transfection, Chromatin Immunoprecipitation, Modification, Stability Assay, Derivative Assay

    Duplex RNAs alter splicing of luciferase pre-mRNA in HeLa Luc/705 cells. ( a ) Left: schematic showing splicing of pre-mRNA. Boxes represent sequences derived from luciferase exons. The intervening region is derived from human β-globin inton 2 with T/G mutation at nucleotide 705. The 3′ cryptic site and target site for duplex 705 is noted. Right: target sites for duplex RNAs within the β-globin inton 2. The mutation site is in bold. ( b ) PCR amplification followed by gel electrophoresis to separate aberrant and correct splice products upon the addition of duplex RNAs. ( c ) Increase in luciferase activity upon the addition of duplex RNAs. ( d and e ) Effect on splicing and luciferase activity from increasing concentrations of duplex RNA 709. ( f ) PCR amplification followed by gel electrophoresis to separate aberrant and correct splice products after adding mismatch-containing RNA duplexes. ( g ) Effect on splicing of multiple or seed sequence mismatches. Duplex RNAs were transfected into HeLa-derived pLUC/705 cells at 50 nM unless otherwise noted. CM: non-complementary control duplex. Error bars represent standard deviation (SD). Experiments were performed in triplicate or quadruplicate.

    Journal: Nucleic Acids Research

    Article Title: Expanding the action of duplex RNAs into the nucleus: redirecting alternative splicing

    doi: 10.1093/nar/gkr780

    Figure Lengend Snippet: Duplex RNAs alter splicing of luciferase pre-mRNA in HeLa Luc/705 cells. ( a ) Left: schematic showing splicing of pre-mRNA. Boxes represent sequences derived from luciferase exons. The intervening region is derived from human β-globin inton 2 with T/G mutation at nucleotide 705. The 3′ cryptic site and target site for duplex 705 is noted. Right: target sites for duplex RNAs within the β-globin inton 2. The mutation site is in bold. ( b ) PCR amplification followed by gel electrophoresis to separate aberrant and correct splice products upon the addition of duplex RNAs. ( c ) Increase in luciferase activity upon the addition of duplex RNAs. ( d and e ) Effect on splicing and luciferase activity from increasing concentrations of duplex RNA 709. ( f ) PCR amplification followed by gel electrophoresis to separate aberrant and correct splice products after adding mismatch-containing RNA duplexes. ( g ) Effect on splicing of multiple or seed sequence mismatches. Duplex RNAs were transfected into HeLa-derived pLUC/705 cells at 50 nM unless otherwise noted. CM: non-complementary control duplex. Error bars represent standard deviation (SD). Experiments were performed in triplicate or quadruplicate.

    Article Snippet: RT–PCR To generate cDNA, total RNA was extracted and treated with DNase I (Worthington Biochemical) at 25 µM for 10 min.

    Techniques: Luciferase, Derivative Assay, Mutagenesis, Polymerase Chain Reaction, Amplification, Nucleic Acid Electrophoresis, Activity Assay, Sequencing, Transfection, Standard Deviation

    RNAs alter splicing of SMN2 in SMA type 1 fibroblast cells. ( a ) Left: schematic of SMN2 splicing. The dark bar within exon 7 shows the position of the C to T transition in SMN2 relative to SMN1. Right: schematic showing the binding sites for duplex RNAs. Sequences underlined indicate exonic splicing silencer (ESS) or intronic splicing sliencer (ISS). Intron sequences are lower case, and exon sequences are upper case. The C to T transition is bold. ( b and c ) Semiquantitative RT–PCR showing the effect of fully complementary duplex RNAs (100 nM) on splicing of SMN2. ( d ) Semiquantitative RT–PCR comparing effects of fully complementary and mismatch-containing complementary duplex RNAs (100 nM) on splicing of SMN2. ISS: intronic splicing silencer; ESS: exonic splicing silencer. CM is a non-complementary RNA duplex. Student's t -test was performed for evaluating statistical significance between two study groups. ** P

    Journal: Nucleic Acids Research

    Article Title: Expanding the action of duplex RNAs into the nucleus: redirecting alternative splicing

    doi: 10.1093/nar/gkr780

    Figure Lengend Snippet: RNAs alter splicing of SMN2 in SMA type 1 fibroblast cells. ( a ) Left: schematic of SMN2 splicing. The dark bar within exon 7 shows the position of the C to T transition in SMN2 relative to SMN1. Right: schematic showing the binding sites for duplex RNAs. Sequences underlined indicate exonic splicing silencer (ESS) or intronic splicing sliencer (ISS). Intron sequences are lower case, and exon sequences are upper case. The C to T transition is bold. ( b and c ) Semiquantitative RT–PCR showing the effect of fully complementary duplex RNAs (100 nM) on splicing of SMN2. ( d ) Semiquantitative RT–PCR comparing effects of fully complementary and mismatch-containing complementary duplex RNAs (100 nM) on splicing of SMN2. ISS: intronic splicing silencer; ESS: exonic splicing silencer. CM is a non-complementary RNA duplex. Student's t -test was performed for evaluating statistical significance between two study groups. ** P

    Article Snippet: RT–PCR To generate cDNA, total RNA was extracted and treated with DNase I (Worthington Biochemical) at 25 µM for 10 min.

    Techniques: Binding Assay, Reverse Transcription Polymerase Chain Reaction

    Validation by RIP-qPCR of a set of ARA-lincRNAs, which were detected in the RIP-seq assay as associated to AR. LNCaP cells treated with 0.1 nM androgen (red lines) or with vehicle control (blue lines) were assayed as indicated on the x -axis. The amount of the indicated lincRNA that was co-immunoprecipitated with antiAR antibody or with IgG from non-immunized rabbit (negative control) was measured by RT-qPCR in three different biological replicates (each represented with a different symbol), and the corresponding points for antiAR and IgG for each replicate are connected with a straight line. The results are shown as % input RNA (mean ± SEM) of three technical replicates for each individual biological replicate. For the four lincRNA genes in the experiment with androgen whose experimental points are connected with red dotted lines, the amount of RIP material was only enough for two technical replicates each, and the enrichment t -test was not applied. Red and blue solid lines = significant difference between antiAR and IgG ( p

    Journal: Frontiers in Genetics

    Article Title: Chromatin Landscape Distinguishes the Genomic Loci of Hundreds of Androgen-Receptor-Associated LincRNAs From the Loci of Non-associated LincRNAs

    doi: 10.3389/fgene.2018.00132

    Figure Lengend Snippet: Validation by RIP-qPCR of a set of ARA-lincRNAs, which were detected in the RIP-seq assay as associated to AR. LNCaP cells treated with 0.1 nM androgen (red lines) or with vehicle control (blue lines) were assayed as indicated on the x -axis. The amount of the indicated lincRNA that was co-immunoprecipitated with antiAR antibody or with IgG from non-immunized rabbit (negative control) was measured by RT-qPCR in three different biological replicates (each represented with a different symbol), and the corresponding points for antiAR and IgG for each replicate are connected with a straight line. The results are shown as % input RNA (mean ± SEM) of three technical replicates for each individual biological replicate. For the four lincRNA genes in the experiment with androgen whose experimental points are connected with red dotted lines, the amount of RIP material was only enough for two technical replicates each, and the enrichment t -test was not applied. Red and blue solid lines = significant difference between antiAR and IgG ( p

    Article Snippet: For RNA-seq experiments poly(A)+ RNA was extracted from LNCaP cells treated for 24 h with 1 nM of the synthetic androgen R1881 (Sigma) ( n = 2) or with an equivalent volume of vehicle (ethanol) ( n = 2) using FastTrack MAG Maxi mRNA Isolation Kit (Invitrogen) as described in , which essentially included a modification of the kit protocol to have a larger amount of DNase I and a longer DNase treatment time ( ) in order to ensure the elimination of contaminant genomic DNA from the polyA+ RNA fraction.

    Techniques: Real-time Polymerase Chain Reaction, Acetylene Reduction Assay, Immunoprecipitation, Negative Control, Quantitative RT-PCR