PROJECT INFORMATION :
The human transcriptome is highly complex with large amounts of RNA copied from almost all regions of the genome. Focusing on genes, introns occupy most of their length, most intronic RNA is removed co-transcriptionally, and gene expression is critically regulated via alternative splicing. However, about 3400 human introns are longer than 50 kb, which cannot be reconciled with the idea of their excision as one contiguous piece. Early work in Drosophila unveiled the mechanism of recursive splicing that circumvents this apparent problem: the 3′ end of an exon is joined successively to segments within the following intron before splicing to the next exon. Our group recently described recursive splicing in man for the first time; it proved to be widespread, occur at substantial levels, and use unexpected sites that do not abide to the fruitfly motif. Still, the molecular mechanism responsible for recognizing and processing these intermediate splicing sites, as well as its spatiotemporal dynamics elude us. Here, we build on preliminary work to hypothesize that regulation occurs via the combinatorial action of variant U1 snRNAs and RNA-binding proteins encoded in primary human cells. Hence, we propose a combination of genomics and biochemical approaches to understand the regulatory code cells implement at recursive splicing sites, and to begin addressing the impact of their deregulation in senescence.
KEY TECHNOLOGIES :
- Factory (nascent) RNA-seq
- CRISPR/Cas genome editing
Melnik S, Caudron-Herger M, Brant L, Carr IM, Rippe K, Cook PR, Papantonis A. Isolation of the protein and RNA content of active sites of transcription from mammalian cells. Nat Protoc. 2016 Mar;11(3):553-65. doi: 10.1038/nprot.2016.032.
Caudron-Herger M, Cook PR, Rippe K, Papantonis A. Dissecting the nascent human transcriptome by analysing the RNA content of transcription factories. Nucleic Acids Res. 2015 Aug 18;43(14):e95. doi: 10.1093/nar/gkv390.
Kelly S, Georgomanolis T, Zirkel A, Diermeier S, O'Reilly D, Murphy S, Längst G, Cook PR, Papantonis A. Splicing of many human genes involves sites embedded within introns. Nucleic Acids Res. 2015 May 19;43(9):4721-32. doi: 10.1093/nar/gkv386.
Kelly S, Greenman C, Cook PR, Papantonis A. Exon Skipping Is Correlated with Exon Circularization. J Mol Biol. 2015 Jul 31;427(15):2414-7. doi: 10.1016/j.jmb.2015.02.018.
Larkin JD, Papantonis A, Cook PR, Marenduzzo D. Space exploration by the promoter of a long human gene during one transcription cycle. Nucleic Acids Res. 2013 Feb 1;41(4):2216-27. doi: 10.1093/nar/gks1441.
INFO EXTRA :