Functional characterisation of a novel arginine/serine-rich splicing factor

Cazalla, Demián (2003). Functional characterisation of a novel arginine/serine-rich splicing factor. PhD thesis The Open University.



Using a modified gene trap strategy, a novel factor involved in mRNA processing, USSRp58, was identified. Analysis of the USSRp58 sequence revealed that it contains an RS domain, common to factors involved in pre-mRNA splicing, and more generally in RNA processing. The subcellular localisation of the endogenous USSRp58 protein, as well of transiently expressed epitope-tagged USSRp58 protein, revealed that this protein localises to nuclear speckles, which suggests a potential role in pre-mRNA splicing. Two-hybrid analysis and immunoprecipitation/We stern blot assays have been used to demonstrate that USSRp58 interacts with different proteins that have either potential or clearly defined functions in splicing. Immunoprecipitation experiments revealed that USSRp58 also interacts with spliceosomal snRNPs, indicating that it is physically associated with the splicing apparatus. Using in vivo and in vitro splicing assays, USSRp58 was shown to be functionally involved in splicing. Transient overexpression of USSRp58 in cultured cells and analysis of the alternative splicing of an adenovirus E1A reporter indicated that the protein can regulate 5' splice site selection in vivo. Furthermore, nuclear extracts immunodepleted of USSRp58 are unable to perform the second step of splicing in vitro, suggesting that this protein is necessary for this step.

A final set of results describing a nuclear retention signal (NRS) in the RS domain of SC35 was also included in this thesis. Chimeric proteins in which the RS domain of a nonshuttling SR protein, SC35, was fused to either hnRNP A1 or SF2/ASF (both shuttling proteins), were used to demonstrate that an NRS was present in the RS domain of SC35. Analysis of deletion mutants of SC35 indicated that the NRS is comprised in the last 30 amino acids of the protein. Finally, fusion of the NRS found in SC35 to SF2/ASF showed that this NRS is both portable and dominant.

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