List of exons and DEXseq analysis in RNAseq data set E-MTAB-6008.

This data set was created using cufflinks and DEXseq, using Ensembl GRCh37 version 72 as input annotation. For detailled description, see associated publication. Columns included are genomic position, Ensembl transcript and exon ID, base mean across conditions, and statistical testing by DEXseq. Exon_class indicated if the exon is an Alu-exon, a cryptic exon, or not. UCSCoverlap and type indicate if the exon is annotated by UCSC, and if it is a constitutive or alternative exon. If the exon is not annotated in UCSC, it's called a cryptic exon. Stand_alone exons are not predicted by cufflinks to overlap with another exon. The table also includes the number of junction reads confirming either splice site. In addition, we predicted premature stop codons in protein coding genes; for PTC+ exons it includes the distance between the PTC and downstream exon-exon junction, the following (second) exon-exon junction, the number of downstream exons. The last two columns show the ID after merging all discovered Alu-exons with UCSC annotated Alu-exons, and the grouping of evolutionary groups shown in Figure 5 of the associated publication

Immunoprecipitation of Psip1/p52 and p75.

<p>A) Immunoblot of NIH 3T3 nuclear extract with antibodies; A300-848 which recognizes only the p75 isoform of Psip1, and A300-847 which detects both p52 and p75. B) IPs with IgG, A300-847 and A300-848 from NIH 3T3 nuclear extracts, immunoblotted with antibodies recognizing p75 (A300-848) or p52 (A300-847). Input is 5% total extract. C) Immunoblot of A300-847 IPs with αSRSF1. IP was also performed in the presence of RNase A. Input is 10% of total extract and IgG served as a control. D) In vitro pulldown of 293T cell expressed T7-SRSF1 using GST-p52 and Psip1-PWWP and immunoblotted with αT7. Input is 5% of T7-SRSF1 and GST alone is control. E) In vitro pulldown with GST-p52 of; T7-SRSF1 and mutants that mimic its hypo-(RG) and hyper-phosporylation (RD), T7-SRSF3 and GFP-SRSF2. Immunoblotting was with αT7 or αGFP. F) ChIP with αH3K36me3 from wild-type (wt) and <i>Psip1</i>^−/− MEFs immunoblotted with antibodies detecting Srsf1, Srsf2, Srsf3, PTB, Psip1 and H3K36me3. G) In vitro pulldown of HeLa core histones by T7-SRSF1 in the presence or absence of Psip1/p52 and immunoblotted with antibodies detecting pan H3, H3K36me3, H3K9me2 and H3K4me3.</p

Alternative splicing in <i>Psip1</i>^gt/gt cells.

<p>RT-PCR to detect; exon inclusion (In) or skipping (Skip) of (A); <i>Ptprc</i>, <i>Ppfibp1</i>, <i>Rapgef6</i>, <i>Rasgrp3</i>, <i>Ogfrl</i>, and <i>Sorb2</i> all of which showed evidence for altered alternative splicing in array analysis, or (B) <i>Csnk1d and Alg9</i>, <i>which were unchanged in the array</i>, in RNAs prepared from wt and <i>Psip1</i>^gt/gt primary MEFs. C) Specific exon-exon junctions (constitutive-constitutive or constitutive-alternative) of <i>Vcan</i>, <i>Tpp2</i> and <i>Diap2</i> in wt and <i>Psip1</i>^gt/gt MEFs. D) Specific exon-exon junctions (constitutive-constitutive) of <i>Vcan</i> (5′ exons) and <i>Diap2</i> (3′exons) and constitutive-constitutive (5′) and constitutive-alternative of <i>Tpp2</i> alternative exon 24 in wt and <i>Psip1</i>^gt/gt MEFs. E) Specific exon-exon junctions (constitutive-constitutive or constitutive-alternative) of <i>Vcan</i>, <i>Tpp2 and Diap2</i>; exon inclusion (In) or skipping (Skip) of <i>Sorb2</i> in wt and <i>Psip1</i>^−/− MEFs, and after transfection of p52 or p75 Psip1 into <i>Psip1</i>^−/− MEFs. F) Exon inclusion (In) or skipping (Skip) of; <i>Csnk1d</i> Alg9, and constitutive-constitutive (5′) and constitutive-alternative of <i>Tpp2</i> alternative exon 24 in RNAs prepared from wt and <i>Psip1</i>^−/− MEFs, and after transfection of p52 or p75 Psip1 into <i>Psip1</i>^−/− MEFs. Sequence and position of primer pairs for each exons are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002717#pgen.1002717.s004" target="_blank">Table S3</a>. Below the gels in panels A to F, the mean ratio of alternative∶constitutive exon (+/− s.e.m.) is shown for three biological replicates. G) Immunoblots of proteins using A300-847 antibodies to detect p75 and p52 in wt and <i>Psip1</i>^−/− MEFs, also <i>Psip1</i>^−/− MEFs transfected with p52 (<i>Psip1</i>^−/− p52Res) and p75 (<i>Psip1</i>^−/− p75Res). Immunoblot with Pcna served as a loading control.</p

Psip1 PWWP domain binds to H3K36me3.

<p>A) Diagram of p52 and p75 Psip1 isoforms showing the position of the; PWWP domain, AT hook-like domains (hatched box), C-terminal 8 a.a. unique to p52 (black box), and the p75-specific IBD. Vertical arrow indicates the site of gene trap integration in <i>Psip1^gt/gt</i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002717#pgen.1002717-Sutherland1" target="_blank">[22]</a>. Horizontal lines indicate the position of epitopes recognized by antibodies A300-847 and A300-848. B) Peptide array containing 384 histone tail modification combinations incubated with GST-Psip1-PWWP and detected with αGST. Spots corresponding to unmodified H3 26–45 peptide (arrow) and H3K36me3 (arrowhead) are indicated. C) Binding specificity (calculated from the intensity of the histone peptide interaction) of Psip1-PWWP (y axis) to the top list of histone modifications arranged according to decreasing specificity (x axis). Data for all the modifications are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002717#pgen.1002717.s002" target="_blank">Table S1</a>. D) Immunoblot of biotinylated H3K36me3 peptide pull-down detecting GST-p52 with αGST antibodies. Corresponding unmodified histone H3 peptide served as control and GST-p52 was loaded as input. E) Immunoblot of A300-847 IPs with antibodies detecting; unmodified H3, H3K36me3, H3K9me2 and H3K4me3. IgG served as control and 5% of NIH3T3 nuclear extract was loaded as input.</p

Sub-cellular localization of Psip1/p52 and p75.

<p>A) Immunofluorescence and wide-field epifluorescence microscopy on human cells with; (upper row) p75-specific antibody A300-848, (lower row) A300-847 which can recognize both p52 and p75. DNA was counterstained with DAPI. B) Co-immunofluorescence of Psip1/p52 (green/A300-847) and SRSF2 (red) analyzed by confocal microscopy in untreated (upper row), or actinomycin D (ActD) treated cells. C) Co-immunofluorescence of Psip1/p75 (green/A300-848) and SRSF2 (red) in ActD treated cells and analyzed by confocal microscopy.</p

Genomic distribution of Psip1/p52 and H3K36me3.

<p>A) Mean log2 ChIP∶input for Psip1/p52 and H3K36me3 in MEFs for an approximately 1.2Mb genomic window from mouse chromosome 5. n = 4 (3 biological and 1 technical replicate). B) Box plots showing the distribution of log2 ChIP∶input for Psip1/p52 and H3K36me3 across exons and introns of expressed or non-expressed genes. Data are deposited in NCBI GEO (Accession no. GSM697402-GSM697411). C, D) Mean log2 ChIP∶input for Psip1/p52 and H3K36me3 in MEFs at (C) c-<i>Myc</i> and (D) <i>Xist</i> loci. H3K4me3 is also shown for XIST. Filled boxes indicate the positions of exons. n = 4 (3 biological and 1 technical replicate) for H3K36me3 and Psip1. NCBI GEO accession number for array platform is GPL13276. n = 2 biological: replicates for H3K4me3.</p

Psip1/p52 interacting partners.

<p>Proteins identified by mass spectrometry of p52 IPs (200 mM KCl).</p>*<p>indicates known proteins of the ‘spliceosomal complex C’. Data on protein domains and putative protein functions were taken from <a href="http://npd.hgu.mrc.ac.uk/" target="_blank">http://npd.hgu.mrc.ac.uk/</a>.</p

ChIP for H3K36me3, Psip1, and Srsf1 in wt and Psip1 mutant MEFs.

<p>A) Mean log2 ChIP∶input for H3K36me3, Psip1 and Srsf1 in wt MEFs across the <i>Vcan</i> (A), <i>Diap2</i> (B) and <i>Ppfibp1</i> (C) loci. Distribution of Srsf1 in chromatin from <i>Psip1</i>^−/− MEFs is also shown. Filled boxes indicate the positions of exons and the arrows indicate the position of alternatively spliced exons whose inclusion into spliced mRNAs is altered in <i>Psip1</i>^gt/gt cells. n = 2 biological replicates that also incorporate a technical (dye-swap) replicate. Array platform number is GPL14175 and the GEO accession numbers for ChIP data are; GSM782590 (Psip1), GSM782591 (H3K36me3), GSM782592 and GSM782593 (Srsf1 in wt), GSM782594 and GSM782595 (Srsf1 in <i>Psip1</i>^−/−).</p

Splicing factor dynamics suggests that accumulation in speckles is due to inefficient recruitment to pre-mRNA.

<p>(A) FRAP analysis of SRSF1 and U2AF65 splicing factors fused to EYFP show that their kinetics of association to speckles are not modified after TSA treatment. For this experiment, C33A cells stably expressing the corresponding EYFP-fusion protein were used. Similar results were obtained for other splicing factors transiently transfected in HeLa cells (not shown). We show a representative cell for each group. The circle shows the speckle area where the laser pulse was applied, and where fluorescence recovery was measured afterwards. Fluorescence intensity is expressed relative to the fluorescence prior photobleaching. The length of TSA treatment was 4 h for U2AF65 and 6 h for SRSF1. The curves are averages from 10 to 15 cells. (B) Direct interaction between splicing factors and histones is detectable but not affected by TSA treatment. Interaction between the mCherry-tagged splicing factor SRSF1 and the EGFP-tagged histone H2B was analyzed by FLIM-FRET technique. A representative cell expressing both tagged proteins and its FRET efficiency map is showed. Also, the FRET efficiency map of a representative cell expressing EGFP-H2B and mCherry-C1 empty plasmid is shown as a control (NO FRET). The pixels histogram shows average FRET efficiency distributions for Control (7 cells) and TSA-treated cells (6 cells). Peaks of FRET are marked by colored arrowheads, indicating interaction between these two proteins. Nuclear regions associated to these distinct FRET populations are also marked by colored arrowheads in the E _FRET map. (C) Nucleoplasmic interaction between splicing factors is not impaired by TSA treatment. The interaction between EGFP-U1-70K and mCherry-SRSF1, a known interaction pair <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048084#pone.0048084-Ellis1" target="_blank">[51]</a> was assessed by FLIM-FRET in Control and TSA-treated cells. Upper panels: control cells, transfected with mCherry instead of mCherry-SRSF1, show no FRET signal in either speckles or nucleoplasm. Lower panels: FRET efficiency between the two splicing factors is intermediate in nucleoplasm (green) with stronger (red) areas in speckles (marked with white arrowheads in control cells). (D) Proposed model to explain how relaxation of chromatin structure affects splicing factor distribution. Nucleoplasmic free splicing factors are in dynamic equilibrium with splicing factors in speckles (<i>a</i>). Free splicing factors can also interact with other splicing factors independently of splicing (<i>b</i>), these complexes being found both in nucleoplasm and speckles. However, what we call nucleoplasmic fraction involves also splicing factors briefly interacting with other molecules (such as histones) through abundant low affinity interactions (<i>c</i>) and splicing factors recruited to RNA and engaged in productive spliceosome assembly (<i>d</i>). For model simplicity, in <i>c</i> we only included interactions with histones and in <i>d</i> we only depicted co-transcriptional splicing. The red lines and arrows symbolize the proposed system response after TSA treatment: decreased efficiency in recruitment cause an excess of free splicing factors that is buffered by the speckles compartment.</p

Die Klosterbibliothek von St. Peter im Schwarzwald: Raum und Programm

<div><p>Background</p><p>Heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP C) is a core component of 40S ribonucleoprotein particles that bind pre-mRNAs and influence their processing, stability and export. Breast cancer tumor suppressors BRCA1, BRCA2 and PALB2 form a complex and play key roles in homologous recombination (HR), DNA double strand break (DSB) repair and cell cycle regulation following DNA damage.</p> <p>Methods</p><p>PALB2 nucleoprotein complexes were isolated using tandem affinity purification from nuclease-solubilized nuclear fraction. Immunofluorescence was used for localization studies of proteins. siRNA-mediated gene silencing and flow cytometry were used for studying DNA repair efficiency and cell cycle distribution/checkpoints. The effect of hnRNP C on mRNA abundance was assayed using quantitative reverse transcriptase PCR.</p> <p>Results and Significance</p><p>We identified hnRNP C as a component of a nucleoprotein complex containing breast cancer suppressor proteins PALB2, BRCA2 and BRCA1. Notably, other components of the 40S ribonucleoprotein particle were not present in the complex. hnRNP C was found to undergo significant changes of sub-nuclear localization after ionizing radiation (IR) and to partially localize to DNA damage sites. Depletion of hnRNP C substantially altered the normal balance of repair mechanisms following DSB induction, reducing HR usage in particular, and impaired S phase progression after IR. Moreover, loss of hnRNP C strongly reduced the abundance of key HR proteins BRCA1, BRCA2, RAD51 and BRIP1, which can be attributed, at least in part, to the downregulation of their mRNAs due to aberrant splicing. Our results establish hnRNP C as a key regulator of <i>BRCA</i> gene expression and HR-based DNA repair. They also suggest the existence of an RNA regulatory program at sites of DNA damage, which involves a unique function of hnRNP C that is independent of the 40S ribonucleoprotein particles and most other hnRNP proteins.</p> </div