Project description:Alternative splicing of pre-mRNA is a prominent mechanism to generate protein diversity, yet its regulation is poorly understood. Here, we demonstrate a direct role for histone modifications in alternative splicing. We find distinctive histone modification signatures which correlate with splicing outcome in a set of human genes. Modulation of histone modifications causes splice site switching. The mechanism for histone-mediated splice site selection involves a histone mark which is read by a chromatin protein, which in turn recruits a splicing regulator. These results outline an adaptor system for reading of histone marks by the pre-mRNA splicing machinery. To obtain an estimate of how many PTB-dependent alternative splicing events are regulated by SET2/MRG15-mediated recruitment of PTB, we carried out a genomewide comparative analysis of alternative splicing in hMSC cells depleted of either SETD2, MRG15 or PTB using specific siRNAs, or mock-depleted using a control siRNA.
Project description:Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB (HJAY)
Project description:Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB (Exon array)
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes