17 research outputs found
沃度保兒謨ニ因セル濕疹
Evaluation of CNA-IC50 correlations by known cancer driver mutations. Distributions of PCCs for cancer cell lines with mutated or wild-type proto-oncogenes or tumor suppressors as depicted in each figure. The rank position of negatively correlated drugs (p < 0.10) is shown. (EPS 975 kb
RSR-2, the <i>Caenorhabditis elegans</i> Ortholog of Human Spliceosomal Component SRm300/SRRM2, Regulates Development by Influencing the Transcriptional Machinery
<div><p>Protein components of the spliceosome are highly conserved in eukaryotes and can influence several steps of the gene expression process. RSR-2, the <i>Caenorhabditis elegans</i> ortholog of the human spliceosomal protein SRm300/SRRM2, is essential for viability, in contrast to the yeast ortholog Cwc21p. We took advantage of mutants and RNA interference (RNAi) to study <i>rsr-2</i> functions in <i>C. elegans</i>, and through genetic epistasis analysis found that <i>rsr-2</i> is within the germline sex determination pathway. Intriguingly, transcriptome analyses of <i>rsr-2(RNAi)</i> animals did not reveal appreciable splicing defects but instead a slight global decrease in transcript levels. We further investigated this effect in transcription and observed that RSR-2 colocalizes with DNA in germline nuclei and coprecipitates with chromatin, displaying a ChIP-Seq profile similar to that obtained for the RNA Polymerase II (RNAPII). Consistent with a novel transcription function we demonstrate that the recruitment of RSR-2 to chromatin is splicing-independent and that RSR-2 interacts with RNAPII and affects RNAPII phosphorylation states. Proteomic analyses identified proteins associated with RSR-2 that are involved in different gene expression steps, including RNA metabolism and transcription with PRP-8 and PRP-19 being the strongest interacting partners. PRP-8 is a core component of the spliceosome and PRP-19 is the core component of the PRP19 complex, which interacts with RNAPII and is necessary for full transcriptional activity. Taken together, our study proposes that RSR-2 is a multifunctional protein whose role in transcription influences <i>C. elegans</i> development.</p></div
RSR-2 interacts with RNAPII and affects its phosphorylation state.
<p>RNAPII phosphoisoform detection by western blot in wild type and <i>rsr-2(RNAi)</i> worms. POL IIo is the abbreviation for the hyperphosphorylated form of the RNAPII whereas POL IIa represents the hypophosphorylated form of the RNAPII. Hyperphosphorylated RNAPII accumulates in <i>rsr-2(RNAi)</i> worms. The actin antibody C4 was used as a loading control. (A) RNAPII phosphoisoforms detected with the N-20 antibody. (B) RNAPII phosphoisoforms co-immunoprecipitate with RSR-2. IP was performed with the anti-RSR-2 antibody using an extract from a mixed-stage worm population. Eluted IP product was checked for the presence of RNAPII phosphorylated isoforms (Ser-2 and Ser-5 phosphorylation). Lane 1: Input (4% of IP), lane 2: IP with unspecific rabbit IgG antibody, lane 3: IP with RSR-2 antibody, lane 4: unspecific binding to beads. Pictures displayed are representative of a series of two experiments.</p
N-terminal conservation of RSR-2 and scheme of <i>rsr-2</i> gene.
<p>(A) N-terminal sequences of SRm300/SRRM2 (<i>H. sapiens</i>), RSR-2 (<i>C. elegans</i>), and the full sequence of CWC21 (<i>S. cerevisiae</i>) were compared using the ClustalW2 program. Identical and similar residues are highlighted in green and yellow, respectively. Black boxes on top of the sequences indicates the cwf21 motif. (B) Scale schematics of <i>rsr-2</i> pre-mRNA. Red boxes represent exons. Connecting lines represent introns. The white box indicates the cwf21 motif. Grey boxes represent regions deleted in <i>rsr-2</i> alleles <i>tm2625</i> and <i>tm2607</i>.</p
RSR-2 binds to intronless genes.
<p>(A) Chromatin-binding profiles of RNAPII and RSR-2, and RNA-Seq reads in intronless genes. RNA-Seq reads correspond to the N2 mid-L4 stage dataset from the modENCODE consortium <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003543#pgen.1003543-Gerstein1" target="_blank">[82]</a>. (B) ChIP-qPCR for intronless genes with mouse anti-RNAPII (8WG16), rabbit anti-RSR-2 (Q5092) and unspecific mouse and rabbit IgG antibodies (sc-2025 and sc-2027 respectively). All qPCR values are represented as the percentage of input immunoprecipitated.</p
RSR-2 is associated with chromatin and modifies RNAPII distribution.
<p>(A) Snapshot of the genome browser (chromosome IV) showing ChIP-Seq data. Chromatin-binding profiles of RNAPII and RSR-2 are similar. RNAPII peaks are represented in black, RSR-2 peaks are represented in red, and input samples are represented in blue. Upon <i>rsr-2</i> RNAi, the RNAPII peak profile at <i>phy-2</i> locus shifts from the 3′ to the 5′end. RSR-2 peaks disappear upon <i>rsr-2</i> RNAi. Data was visualized with the Integrated Genome Browser (IGB) software. (B) ChIP-qPCR showing how RNAPII occupancy changes upon <i>rsr-2</i> RNAi at the <i>phy-2</i> locus. Black bars are a scaled representation of the regions covered by the primer pairs used in this experiment. (C) Distribution of RNAPII and RSR-2 ChIP peaks along five zones within an averaged gene. Statistically called peaks from a single ChIP-Seq experiment were classified with respect to their position relative to the nearest TSS. Y axis indicates the frequency of peaks within each zone. <i>rsr-2</i> RNAi slightly modifies the distribution of RNAPII along an averaged gene.</p
<i>rsr-2</i> expression in the soma and in the germline.
<p>(A) Expression of the transgene <i>cerEx01</i>[<i>rsr-2</i> promoter::GFP::H2B::<i>rsr-2</i> 3′ UTR] in the germline as complex array. Upper panel: detail of the distal part of the germline showing expression of GFP::H2B in the transition and meiotic zone (broken line) but not in the mitotic zone (continuous line). Bottom panel: corresponding Nomarski image. (B) Representative image of <i>rsr-2</i> mRNA in wild type germline detected by <i>in situ</i> hybridization (n = 56). Magnification of the distal part of the germline (left). The broken line labels the transition and meiotic areas whereas the continuous line marks the mitotic zone. (C) Expression of the transgene <i>cerEx04</i>[<i>rsr-2</i> promoter::GFP::<i>rsr-2</i> genomic fragment (exons, introns and 3′ UTR)]. <i>myo-2</i>::mcherry was used as a control of transformation. (D) GFP::RSR-2 forming nuclear speckles in a hypodermal cell nucleus (left) and a neuronal nucleus (right). (E) Confocal images of anti-RSR-2 immunofluorescence in the germline. Nuclei counterstained with DAPI. The three pictures on the right correspond to magnified images of the white boxes.</p
RNA-Seq of <i>rsr-2(RNAi)</i> and <i>prp-8(RNAi)</i> L3 animals.
<p>(A) Venn diagrams of down- and up-regulated transcripts in <i>rsr-2(RNAi)</i> (yellow) and <i>prp-8(RNAi)</i> (blue) L3 animal populations. Overlapping transcripts between the two samples are represented in green. (B) Graph representing the abundance of RNA-Seq reads mapped in intronic regions without internal genes corresponding to control <i>gfp(RNAi)</i>, <i>prp-8(RNAi)</i>, and <i>rsr-2(RNAi)</i> animals. (C) Semiquantitative RT-PCR of wild type and <i>smg-1(r861)</i> worms treated with <i>gfp</i> RNAi control, <i>rsr-2</i> RNAi and <i>prp-8</i> RNAi. <i>rpl-12</i> was used as a positive control for intron retention in NMD-defective animals. <i>act-1</i> was used as an endogenous control. The black box indicates the accumulation of aberrant mRNAs for <i>dpy-2</i> and <i>dpy-8</i> in the <i>smg-1(r861)</i>; <i>prp-8(RNAi)</i> sample, but not in the <i>smg-1(r861)</i>; <i>rsr-2(RNAi)</i> sample.</p
Protein members of RSR-2-containing complexes.
<p>Some of the proteins identified from mass spectra of peptides recovered from complexes immunopurified with an antibody against RSR-2. Two biological replicates are immunoprecipitated (IP). After the immunopurification, the peptide mixture is analyzed by LC/MS coupled to nano-HPLC. The total number of unique peptides identified for each protein is indicated in the columns under the antibody heading. All of unique peptides have been identified in the immunoprecipitated sample but not in the control sample immunopurified with an unspecific rabbit antibody against IgG.</p
Reduction of transcript levels in <i>rsr-2(RNAi)</i> L4 animals.
<p>(A) Comparison of the average signal intensities of transcripts, exons and introns in tiling arrays between <i>gfp(RNAi)</i> and <i>rsr-2(RNAi)</i> animals. Error bars represent standard error of the mean (SEM). (B) mRNA levels of several sex determination genes upon <i>rsr-2</i> RNAi. qPCR expression data was normalized to transcript levels of <i>tbb-2</i>. mRNA levels in <i>rsr-2(RNAi)</i> animals are represented relative to the expression in <i>gfp(RNAi)</i> control animals (arbitrary value of 1.0). Three separate experiments were analyzed. Bars represent the standard deviation within each data set. Student's independent samples t-test was used to study significantly different gene expression between the two conditions: one, two and three asterisks indicate p<0.05, p<0.01 and p<0.001, respectively. (C) A subset of germline-related genes are correctly spliced upon <i>rsr-2</i> inactivation. Semiquantitative RT-PCR analysis of germline transcripts in wild type and <i>smg-5</i>(<i>r680</i>) NMD mutants treated with control <i>gfp</i> RNAi and <i>rsr-2</i> RNAi. <i>rpl-12</i> was used as a control for defective NMD pathway. <i>act-1</i> was used as an endogenous control.</p