Biochemical Mechanisms of Eukaryotic RNA Processing

PROJECT SUMMARY/ABSTRACT The eukaryotic transcriptome is defined by both genomic sequence and pre-mRNA processing steps that define the composition of mature mRNAs. Nearly every human mRNA must be processed by splicing to remove introns and cleavage and polyadenylation to be released from the transcription machinery and prepared for translation. These processes are catalyzed by large, megadalton-sized macromolecular machines [the spliceosome and cleavage and polyadenylation factor (CPF)] that assemble on RNAs and must precisely recognize which regions of the RNA to keep and which to discard. Failure to recognize the proper splice or 3' end cleavage sites can destroy the protein coding potential of a message; alter the amino acid composition of the encoded protein; or dramatically change when, where, or how much protein is produced from a given message. The Hoskins Laboratory is focused on understanding spliceosome and CPF function in biochemical depth to illuminate how they shape the transcriptome. To study these machines, we integrate single molecule fluorescence microscopy with genetic, transcriptomic, and biochemical assays. In the next funding period, we will focus on how splice and cleavage sites are recognized by the spliceosome and CPF. We will pioneer single molecule methods to follow assembly of CPF and 3' end cleavage in real-time to answer fundamental questions about CPF function that have proved elusive for decades. By studying how 5' splice site recognition occurs co-transcriptionally, we will elucidate how splicing factors influence the transcriptional machinery and vice versa. We will use genetics and transcriptomics to probe how 3' splice site recognition is coupled with proofreading to maintain the integrity of genetic information flow. Finally, we will develop new high-throughput assays and microscopy hardware that will provide quantitative biochemical data for splice and cleavage site recognition at the transcriptome-scale. Together, these research directions will reveal fundamental mechanisms of the pre-mRNA processing machineries and insights into how these machines malfunction in human diseases including cancers and neuromuscular disorders.