Binding of hnRNP H to an exonic splicing silencer is involved in the regulation of alternative splicing of the rat beta-tropomyosin gene

In the rat beta-tropomyosin (beta-TM) gene, exons 6 and 7 are spliced alter natively in a mutually exclusive manner. Exon 6 is included in mRNA encodin g nonmuscle TM-1, whereas exon 7 is used in mRNA encoding skeletal muscle b eta-TM. Previously, we demonstrated that a six nucleotide mutation at the 5 ' end of exon 7, designated as ex-1, activated exon 7 splicing in nonmuscle cells. In this study, we show that the activating effect of this mutation is not the result of creating an exonic splicing enhancer (ESE) or disrupti ng a putative secondary structure. The sequence in exon 7 acts as a bona fi de exonic splicing silencer (ESS), which is bound specifically by a trans-a cting factor. Isolation and peptide sequencing reveal that this factor is h nRNP H, a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) fam ily. Binding of hnRNP H correlates with the ESS activity. Furthermore, addi tion of antibodies that specifically recognizes hnRNP H to the splicing rea ctions or partial depletion of hnRNP H from nuclear extract activates exon 7 splicing in vitro and this effect can be reversed by addition of purified recombinant hnRNP E-I. These results indicate that hnRNP H participates in exclusion of exon 7 in nonmuscle cells. The involvement of hnRNP H in the activity of an ESS may represent a prototype for the regulation of tissue- and developmental-specific alternative splicing.