hnRNP H is a component of a splicing enhancer complex that activates a c-src alternative exon in neuronal cells

The regulation of the c-src N1 exon is mediated by an intronic splicing enh ancer downstream of the N1 5' splice site. Previous experiments showed that a set of proteins assembles onto the most conserved core of this enhancer sequence specifically in neuronal WERI-1 cell extracts. The most prominent components of this enhancer complex are the proteins hnRNP F, KSRP, and an unidentified protein of 58 kDa (p58). This p58 protein was purified from th e WERI-1 cell nuclear extract by ammonium sulfate precipitation, Mono Q chr omatography, and immunoprecipitation with anti-Sm antibody Y12. Peptide seq uence analysis of purified p58 protein identified it as hnRNP II. Immunopre cipitation of hnRNP H cross-linked to the N1 enhancer RNA, as well as gel m obility shift analysis of the enhancer complex in the presence of hnRNP II- specific antibodies, confirmed that hnRNP H is a protein component of the s plicing enhancer complex. Immunoprecipitation of splicing intermediates fro m in vitro splicing reactions with anti-hnRNP H antibody indicated that hnR NP H remains bound to the src pre-mRNA after the assembly of spliceosome. P artial immunodepletion of hnRNP H from the nuclear extract partially inacti vated the splicing of the N1 exon in vitro. This inhibition of splicing can be restored by the addition of recombinant hnRNP II, indicating that hnRNP H is an important factor for N1 splicing. Finally, in vitro binding assays demonstrate that hnRNP H tan interact with the related protein hnRNP F, su ggesting that hnRNPs H and F may exist as a heterodimer in a single enhance r complex. These two proteins presumably cooperate,vith each other and with other enhancer complex proteins to direct splicing to the N1 exon upstream .