Molecular Recognition During Pre-mRNA Splicing

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SUMMARY The goal of our project is to understand the structural features guiding the initial stages of pre-mRNA splice site recognition, which are critical for accurate pre-mRNA splicing and frequently dysregulated in human diseases. We focus on the U2AF, SF1 and SF3B1 splicing factors directing the U2 small nuclear ribonucleoprotein (U2 snRNP) to the 3´ splice sites of pre-mRNAs. The U2AF subunits, U2AF2 and U2AF1, recognize the polypyrimidine and AG-dinucleotide splice site signals. A third subunit, SF1 initially associates with the branch point sequence of the pre-mRNA then is displaced by the SF3B1 subunit of the U2 snRNP. Dynamic phosphorylation and dephosphorylation of SF3B1 is required for formation of the active spliceosome. Previously, we made progress towards understanding the molecular underpinnings of the initial steps of 3´ splice site selection. Using X-ray crystallography, biophysical techniques, and functional assays for pre-mRNA splicing in human cells, we have shown that representative cancer-associated mutations at the U2AF2 – RNA interface disrupt RNA binding and splicing. We have deciphered structural details showing how U2AF2 accommodates diverse nucleotides of splice site signals. By complementary single molecule Förster resonance energy transfer approaches, we further revealed that the U2AF2 conformation changes in response to different splice site sequences as well as the U2AF1 subunit and its recurrent cancer-associated mutation. We have established important, functional interfaces of U2AF2 with SF1 and SF3B1 during pre-mRNA splicing in human cells and discovered that phosphorylation strongly reduces SF3B1 binding to U2AF2. However, these provocative results raise new questions. First, what are the effects of recurrent U2AF2 mutations in neurodevelopmental disorders compared to those in cancers? Second, how is dynamic SF3B1 phosphorylation and dephosphorylation temporally regulated with U2AF2 dissociation prior to splicing? Third, how are the U2AF2, U2AF1, and SF1 subunits arranged to accurately recognize the splice site signals and ensure the fidelity of splicing? We address these questions in the aims of this proposal by leveraging structural approaches (including X-ray crystallography, cryoelectron microscopy, calorimetry, and fluorescence) and complementary functional assays (including co- immunoprecipitations, pre-mRNA splicing assays, and transcriptome-wide sequencing). Altogether, the results of these aims contribute to understanding the structural and functional underpinnings of 3´ splice site recognition and its dysregulation in human disease.