Global control of co-transcriptional splicing

Harvard Medical School, Boston MA

Investigators

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Abstract

Project Summary Alternative splicing (AS) is a widespread phenomenon in human genes that significantly diversifies the types of proteins and RNAs produced. This process plays an essential role in defining cellular differentiation and identity3,4. Moreover, problems in alternative splicing have been directly linked to a range of human diseases such as muscular dystrophies, neurodegenerative conditions, and cancers. The typical human gene has about eight introns, which are processed by spliceosomal subunits that recognize sequences near splice sites (SSs) and branchpoints. During the splicing process, AS arises as the result of kinetic competition between multiple possible splicing outcomes that is influenced by cis-elements and trans-acting factors. Additionally, AS is influenced by chromatin and transcription rates, as splicing is coupled with gene transcription. The splicing process and its regulation are highly transient. Spliceosomal assembly is initiated by U1 snRNP binding to the 5’ SS and the branch point located upstream of the 3’SS is recognized by the U2 snRNP. Subunits of both U1 and U2 associate with Pol II co-transcriptionally prior to recognizing their respective cis-elements. Additionally, regulatory factors that aid in splice site recognition and control splicing outcomes bind to specific sequences in nascent RNA. Since these cis-elements are usually located within introns, their influence is temporary and lasts only until the introns are removed. Thus, to understand AS requires quantitative in vivo analyses of the dynamics of intron excision within the context of the native genomic context. Our group has pioneered tools that obtain direct, high-resolution, and quantitative views into the endogenous splicing process, both during and after transcription. These methods have revealed that splicing itself occurs far after RNA synthesis14 and that the order of intron excision is largely fixed across cell types and controlled heavily by cis-elements in the nascent RNA13. Here we propose to apply these tools to determine how trans-acting factors affect the relationship between transcription and splicing and how cis-elements in nascent RNA impact splicing dynamics and outcome. We will examine how regulators of splicing and transcription manage the intricate relationship between these two processes. To examine how cis-elements control splicing order, we will investigate how natural variation of the pre-mRNA sequence influence its processing. Additionally, we will use our unique RNA flow technique15 to quantitatively track RNA molecules throughout their life cycle, from synthesis to degradation, giving us insights into how these factors affect the RNA's ultimate fate. In specific aim 1, we will investigate mechanisms of factors connecting splicing to transcription. In specific aim 2, we will study the role of cis-elements in determining splicing order. Overall, our multi-faceted approach aims to provide a comprehensive understanding of the mechanisms that regulate RNA processing, offering critical insights that could inform future research and therapeutic strategies.