REGULATED TISSUE-SPECIFIC EXPRESSION OF ANTAGONISTIC PRE-MESSENGER-RNA SPLICING FACTORS

The SR proteins are essential metazoan pre-mRNA splicing factors that can also influence the selection of alternative 5' splice sites in a c oncentration-dependent manner. Their activity in alternative splicing in vitro is antagonized by members of the hnRNP A/B family of proteins . The opposite effects of members of these two families of antagonisti c splicing factors in vitro and upon overexpression in vivo suggest th at changes in their relative levels may be a natural mechanism for the regulation of alternative splicing in vivo. One prediction of this mo del is that the ratios of these antagonists should vary in different c ell types and in other situations in which cellular or viral transcrip ts are differentially spliced. We raised monoclonal antibodies specifi c for SFS/ASF and used them to measure the abundance of SFS/ASF protei n and its isoforms, its phosphorylation state in vivo and during splic ing in vitro, and its association with the spliceosome. SF2/ASF exists predominantly or exclusively in a highly phosphorylated state in vivo in all cell types examined, and unphosphorylated protein was not dete ctable. Unphosphorylated recombinant SFS/ASF becomes rapidly phosphory lated under splicing conditions in HeLa cell extracts and associates s tably with one or more exons of beta-globin pre-mRNA. This interaction appears to persist through the splicing reaction and SF2/ASF remains bound to spliced mRNA. We compared the distribution of SFS/ASF to that of its antagonist, hnRNP Al, in different rat tissues and in immortal and transformed cell lines. We found that the protein levels of these antagonistic splicing factors vary naturally over a very wide range, supporting the notion that changes in the ratio of these proteins can affect alternative splicing of a variety of pre-mRNAs in vivo.