Saturday, September 7, 2024
9/7/2024
PROJECT SUMMARY The long-term goal of this proposal is to understand the regulatory mechanisms of U1 small nuclear ribonucleoprotein (U1 snRNP) in snRNP biogenesis and its role in protein-coding gene regulation. U1 snRNP, the most abundant RNP particle among the spliceosomal snRNP, plays a key role in excising introns (splicing) and preventing premature termination of nascent transcripts (telescripting). Both activities are ensured by the RNA:RNA base-pairing between U1 snRNA and 5’ splice site (5’ss). Pan-cancer patients possess multiple mutations in the U1 snRNA, including 5’ss and U1 snRNP-specific protein binding sequences, which result in alternative splicing of oncogenes and tumor suppressors through novel 5’ss recognition. However, our preliminary data showed that those U1 snRNA mutations do not form a stable Sm core, a key intermediate assembled by the SMN (survival of motor neuron) complex, which determines the stability and abundance of the U1 snRNP in cells. Moreover, we identified that a U1 snRNP-specific U1C protein, previously known to stabilize 5’ss:U1 snRNA base-pairing, plays a critical role in regulating Sm core assembly of all snRNAs. The objective of this proposal is to investigate the mechanism of the newly discovered role of U1C as a gatekeeper in snRNP biogenesis, its potential contribution to the quality control of spliceosomes, and the regulatory mechanism in splicing and telescripting activity. This project will simultaneously address both functions of U1C by harnessing two established components to dissect the role of U1C in snRNP biogenesis as well as its involvement in intronic polyadenylation and 3’UTR length changes, molecular characteristics of the oncogenes in cancer. We propose to pursue three specific aims: 1) Elucidate the role of U1C with SMN complex as a gatekeeper in snRNP biogenesis. 2) Investigate the effects of U1 snRNA mutations found in pan-cancer patients on Sm core assembly. 3) Examine the U1C interaction with mRNA 3’-end termination machinery in telescripting. Using biochemical approaches, we will investigate the molecular connections between U1C-SMN complex and U1C-U1 snRNA. By examining these connections, we can better understand how U1C controls the snRNP repertoire and ultimately impact the function of spliceosome. Moreover, we will delineate the U1C’s interaction with mRNA 3’- end processing machinery. This interaction is a key switch that can convert U1 snRNP from productive splicing to premature mRNA termination or 3’UTR shortening through the loss of 5’ss binding. To elucidate the multi- faceted regulation mechanism of U1C, we will employ in vitro Sm core assembly, RNA-affinity purification, in vitro mRNA processing assays, and in-cell formaldehyde-crosslinking coupled with immunoprecipitation methods. The expected outcome of this project will identify the role of U1C as a in snRNP biogenesis and mRNA metabolism, thus explaining the consequence of the U1 snRNA mutations in pan-cancer and providing evidence of gene regulation by the loss of telescripting activity.
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