Accurate pre-mRNA splicing is mediated by the recognition of appropriate splice site elements;however, the primary determinants that characterize mammalian splice site sequences are highly degenerate in nature. Therefore, efficient splice site recognition must be facilitated by intronic/exonic enhancer and silencer elements. Splice site and regulatory element mutations as well as the misregulation of trans-acting factors may perturb specific patterns of splicing resulting in changes in protein expression, localization, or activities and the development of human diseases. Long-term objectives are to investigate the basic molecular interactions that contribute to splice site recognition and pairing. In particular, experiments will address the functional significance of SF1 and SF1 isoforms in the splicing of specific cellular pre-mRNAs and the molecular mechanisms by which SR/hnRNP proteins participate in early complex formation.
The specific aims of this proposal are to (1) investigate intron-specific RNA-protein assemblies at splice site and regulatory determinants and their functional roles in pre-mRNA splicing and (2) examine SF1 isoform-specific interactions and activities. The Tat hybrid assay will be used to investigate SF1 and SF1 isoform interactions at select intronic sequences. With reporter-based mini-gene constructs measuring splicing efficiencies, correlations between binding and splicing will identify introns which will be tested for SF1 dependent splicing using siRNA knockdown of endogenous SF1. SF1 isoform-specific protein interactions with WW domain-containing proteins will initially be analyzed by co-immunoprecipitation, while complementary experiments will examine the association of WW domain-containing proteins with splice site complexes will using the Tat hybrid assay. Additionally, regulatory protein-enhancer/silencer interactions and functions will be examined in cells using Tat hybrid and reporter-based splicing assays. Subsequent experiments will investigate the effects of SR/hnRNP proteins on splicing factor-splice site interactions using established regulatory elements and artificial recruitment strategies in a variation of the Tat hybrid assay followed by biochemical analyses to map possible splicing factor-regulatory protein interactions and test models of regulatory protein function. Relevance: Recent studies estimated that at least 15% of the point mutations associated with human disease are silent mutations that cause defects in splicing, a cellular process that is important in generating the diversity of proteins required for cellular function. Understanding the fundamental mechanisms that mediate RNA splicing may allow disease-associated splicing defects to be corrected.
|D'Orso, Iván; Jang, Gwendolyn M; Pastuszak, Alexander W et al. (2012) Transition step during assembly of HIV Tat:P-TEFb transcription complexes and transfer to TAR RNA. Mol Cell Biol 32:4780-93|