The primary transcripts of most eukaryotic genes (precursor mRNAs;pre-mRNAs) contain intervening sequences (introns) that are removed by RNA splicing in a two-step pathway. Pre- mRNA splicing occurs in a ribonucleoprotein (RNP) complex called the spliceosome, which is composed of a large number of proteins and multiple U small nuclear RNP particles (U snRNPs). A small subset of introns, called U12-type introns (as opposed to the major class of U2-type introns), are spliced through the conventional two-step pathway but by a different spliceosome. The U2 snRNP Auxiliary Factor (U2AF) is an essential splicing factor, originally identified in our laboratory, that binds to the polypyrimidine (Py)-tract/3'splice site and initiates spliceosome assembly. Several human proteins are related to the small U2AF subunit, U2AF35, including a protein called U2AF35-related protein (Urp). During the past period of funding, we have shown that Urp is required for splicing of U12-type introns, and for the second step of U2- type intron splicing. In both cases, Urp directly contacts the 3'splice site. Using a combination of molecular biological, biochemical and structural approaches, we will continue to study how U2AF and Urp promote splicing. hUAP56, a member of the DExD/H-box family of RNA- dependent ATPases, was originally identified in our laboratory based upon its interaction with the large U2AF subunit (U2AF65). During the past period of funding we have shown that hUAP56 has multiple roles in U2-type splicing complex assembly, including disrupting the U2AF65-branchpoint/Py-tract interaction, interacting with U4 and U6 snRNAs, and unwinding of the U4/U6 snRNA duplex. We have also found that hUAP56 is required for splicing of U12-type introns. Experiments are proposed to understand the detailed mechanism by which hUAP56 facilitates diverse steps in splicing of U2- and U12-type introns. A variety of mammalian protein splicing factors contain an arginine-serine rich (RS) domain required to promote splicing. We have shown that direct contact with the branchpoint and 5'splice site is a general mechanism by which RS domains promote spliceosome assembly and splicing. Our studies have revealed a pathway of sequential interactions between RS domains and splicing signals during mammalian spliceosome assembly. Molecular, biochemical and structural experiments are proposed to understand in greater detail how RS domains are directed to splicing signals and promote spliceosome assembly and splicing, with an emphasis on studying the role of RS domain phosphorylation. Alternative splicing is an important mechanism of gene regulation and is responsible for substantially increasing diversity of the human proteome. However, the mechanisms that regulate alternative splicing remain largely unknown. During the past period of funding we have shown that FRG1, a protein whose over-expression is responsible for facioscapulohumeral muscular dystrophy (FSHD), functions by misregulating alternative splicing. Experiments are proposed to study how FRG1 affects splicing. In addition, we will perform genome-wide loss-of-function RNA interference screens to study mechanisms of splicing repression and to identify new splicing repressors.
Pre-mRNA splicing is an essential step for expression of the vast majority of protein coding genes in higher eukaryotes. Disruption of normal pre-mRNA splicinggdue to mutations in either the cis-acting regulatory sequences required for splicing or the trans- acting components of the splicing machineryghas been shown to result in a variety of human diseases including atypical cystic fibrosis, retinitis pigmentosa, spinal muscular atrophy and facioscapulohumeral muscular dystrophy, and has also been associated with cancer and metastasis. The experiments proposed in this application will increase our understanding of the factors and mechanisms involved in splice-site recognition, splicing complex assembly and splicing regulation, which may lead to the identification of new therapeutic targets and strategies for the treatment of diseases caused by aberrant pre-mRNA splicing. )
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