In vitro studies of pre-mRNA splicing

The removal of introns from primary transcripts is an essential step for eukaryotic gene expression. Though exon/intron boundaries are defined by highly degenerate sequences, pre-mRNA splicing is a high-fidelity process, which is under tight control of developmental and/or cellular signaling events. The specificity of splice-site recognition is partially derived from the fine balance between two families of splicing regulatory factors, SR proteins and hnRNP proteins. Whereas SR proteins are essential splicing factors required for proper spliceosome assembly, hnRNPs are factors mainly involved in splicing repression.; SR proteins consist of one or two RNA-recognition motif and a characteristic arginine/serine-rich C-terminal domain. The RS domain, which is extensively phosphorylated, mediates the subcellular localization of individual SR proteins and functions as a splicing activation module by engaging in protein-protein interactions. I show in Chapter 2 that the RS domain of SF2/ASF is dispensable for splicing of several substrates, including constitutive and enhancer-dependent pre-mRNAs. The requirement for this RS domain is substrate-specific, and correlates with the strength of the 3' splice site signals. These data suggest the existence of an RS domain-independent function of SR proteins in pre-mRNA splicing, and mechanisms for their role in enhancer function besides U2AF recruitment.; I further investigated the molecular mechanism by which a splicing silencer (ESS3) in HIV tat exon 3 blocks splicing promoted by SC35 but not by SF2/ASF. I show that splicing silencing is mediated by hnRNP A1, which binds to a high affinity site in ESS3, and appears to bind cooperatively along the exon. Both SC35 and SF2/ASF recognize upstream ESE motifs, but only SF2/ASF can block the propagation of hnRNP A1 towards the exon 5' end. The difference between SF2/ASF and SC35 in splicing activation of tat exon 3 is determined by their intrinsic binding affinity for the exon. This differential antagonism between a negative and two positive regulators exemplifies how an alternative exon can be used to different extents, e.g., in different cell types.