Grant: Operating Grant

Protein-coding messenger RNA (mRNA) in humans is synthesized as a precursor molecule (pre-mRNA) containing both coding (exon) and non-coding (intron) sequences. Intron sequences are removed from the pre-mRNA in a process, referred to as splicing, which is catalyzed by a large RNA/protein machine: the spliceosome. Proper expression of the genetic material depends on precise splicing and alternative splicing events are critical in development and tissue differentiation. Over 95% of human pre-mRNAs are spliced and ~2/3 of human genetic diseases are due to splicing errors. Our understanding of the mechanisms by which pre-mRNAs are recognized and then processed by the spliceosome is incomplete. This proposal describes a series of experiments to investigate the assembly, activation, and transitions that occur within the fully assembled spliceosome. They are focused on two proteins, SF3b14 and PRP8, both of which have been shown to form part of the core of the splicing machinery. Our recent determination, using X-ray crystallography, of the structures of human SF3b14 and Domain IV of PRP8 provides the basis for a set of experiments to investigate further the function of these molecules. Using a combination of biophysical, biochemical, and cell biological methods we propose to study the role of these proteins in the spliceosome. The experiments outlined here will provide important insights into the mechanisms of a fundamental mode of gene regulation in humans.