Binding of PurH to a muscle-specific splicing enhancer functionally correlates with exon inclusion in vivo

Regulated alternative splicing of avian cardiac troponin T (cTNT) pre-mRNA requires multiple intronic elements called muscle-specific splicing enhance rs (MSEs) that flank the alternative exon 5 and promote muscle-specific exo n inclusion. To understand the function of the MSEs in muscle-specific spli cing, we sought to identify trans-acting factors that bind to these element s. MSE3, which is located 66-81 nucleotides downstream of exon 5, assembles a complex that is both sequence- and muscle-specific, Purification and cha racterization of the MSE3 complex identified one component as 5-aminoimidaz ole-4-carboxamide ribonucleotideformyltransferase/IMP cyclohydrolase (PurH) , an enzyme involved in de novo purine synthesis, Recombinant human PurH pr otein directly binds MSE3 RNA and PurH is the primary determinant of sequen ce-specific binding in the native complex. Furthermore, we show a direct co rrelation between the in vitro binding affinity of both the MSE3 complex an d recombinant PurH with functional activation of exon inclusion in vivo. To gether, these results strongly suggest that PurH performs a second function as a component of a complex that regulates MSE3-dependent exon inclusion.