The splicing factor-associated protein, p32, regulates RNA splicing by inhibiting ASF/SF2 RNA binding and phosphorylation

The cellular protein p32 was isolated originally as a protein tightly assoc iated with the essential splicing factor ASF/SF2 during its purification fr om HeLa cells, ASF/SF2 is a member of the SR family of splicing factors, wh ich stimulate constitutive splicing and regulate alternative RNA splicing i n a positive or negative fashion, depending on where on the pre-mRNA they b ind, Here we present evidence that p32 interacts with ASF/SF2 and SRp30c, a nother member of the SR protein family, We further show that p32 inhibits A SF/SF2 function as both a splicing enhancer and splicing repressor protein by preventing stable ASF/SF2 interaction with RNA, but p32 does not block S Rp30c function, ASF/SF2 is highly phosphorylated in vivo, a modification re quired for stable RNA binding and protein-protein interaction during splice osome formation, and this phosphorylation, either through HeLa nuclear extr acts or through specific SR protein kinases, is inhibited by p32, Our resul ts suggest that p32 functions as an ASF/SF2 inhibitory factor, regulating A SF/SF2 RNA binding and phosphorylation, These findings place p32 into a Ite m group of proteins that control RNA splicing by sequestering an essential RNA splicing factor into an inhibitory complex.