Title: Co-transcriptional splicing
Funder: Medical Research Council

Abstract: The production of messenger RNA (mRNA) in eukaryotic cells involves precursor mRNA (pre-mRNA) synthesis and processingin particular, 5´ capping, splicing, and 3´ processing. During splicing, the spliceosome removes noncoding introns from pre-mRNA in a co-transcriptional manner on the surface of RNA polymerase II (Pol II). Co-transcriptional splicing enhances the efficiency and accuracy of pre-mRNA processing and explains why splicing is at least 10 times faster in vivo than in vitro (in the absence of Pol II). In particular, co-transcriptional splicing has been suggested to be essential in metazoan cells where introns are often several thousand nucleotides long, raising the intriguing question of how the distant intron ends are functionally paired for splicing. U1 snRNP is the first building block of the spliceosome to engage the nascent pre-mRNA by recognizing the 5´-splice site (5´SS). When the branch point sequence emerges on the nascent pre-mRNA, U2 snRNP is recruited, forming the A complex. The subsequent association of the A complex with the U4/U6.U5 tri-snRNP forms the pre-B complex, which then converts to the B complex and is activated for splicing. Our recent cryo-EM structure of the transcribing Pol II-U1 snRNP complex revealed the molecular basis of a direct interaction between the transcription and splicing machineries within a large supercomplex. This interaction positions the pre-mRNA 5´SS near the RNA exit site of Pol II. Retention of the 5´SS near the Pol II surface leads to formation of a growing intron loop as pre-mRNA is extended, facilitating functional pairing of distant intron ends and spliceosome assembly on the Pol II surface. This worked provided a model mechanism for co-transcriptional splicing and established the foundation for our future work on transcription-coupled alternative splicing. Alternative splicing occurs in more than 95% of human genes and allows generation of multiple functionally distinct transcripts from a single gene, greatly expanding the coding potential of the genome. Regulation of alternative splicing is crucial for cell differentiation and normal cell functioning, whereas its dysregulation causes hereditary diseases and cancer. Because of the co-transcriptional nature of splicing, alternative splicing is intricately coupled to transcription and chromatin structure, yet the underlying crosstalk mechanisms remain unknown. To understand the molecular mechanisms of transcription-coupled alternative splicing in human, we will use a multidisciplinary approach combining in vitro biochemical reconstitution, functional assays and structural analysis using cryo-electron microscopy together with in vivo RNA sequencing. In addition, we will tackle the molecular basis of alternative splice site selection and tissue-specific alternative splicing. Our long-term research goal is to address the fundamental question of how the transcription and splicing machineries functionally interact to regulate gene expression.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE