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 enhancers (MSEs) that flank the alternative exon 5 and promote muscle-specific exon inclusion. To understand the function of the MSEs in muscle-specific splicing, we sought to identify trans-acting factors that bind to these elements. MSE3, which is located 66-81 nucleotides downstream of exon 5, assembles a complex that is both sequence- and muscle-specific. Purification and characterization of the MSE3 complex identified one component as 5-aminoimidazole-4-carboxamide ribonucleotideformyltransferase/IMP cyclohydrolase (PurH), an enzyme involved in de novo purine synthesis. Recombinant human PurH protein directly binds MSE3 RNA and PurH is the primary determinant of sequence-specific binding in the native complex. Furthermore, we show a direct correlation between the in vitro binding affinity of both the MSE3 complex and recombinant PurH with functional activation of exon inclusion in vivo. Together, these results strongly suggest that PurH performs a second function as a component of a complex that regulates MSE3-dependent exon inclusion.
Ryan
K J
KJ
Department of Pathology, Baylor College of Medicine, Houston, Texas 77030, USA.
Charlet-B
N
N
Cooper
T A
TA
D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D002642:Chick Embryo; D004268:DNA-Binding Proteins / Q000235:genetics*; D005091:Exons / Q000235:genetics*; D005347:Fibroblasts; D006801:Humans; D019877:Hydroxymethyl and Formyl Transferases / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism*; D009132:Muscles / Q000196:embryology; D009687:Nuclear Proteins / Q000378:metabolism; D009709:Nucleotide Deaminases / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020539:Sequence Analysis, Protein; D014157:Transcription Factors / Q000235:genetics*; D020107:Troponin T / Q000235:genetics*; D014466:Ultraviolet Rays
D004268:DNA-Binding Proteins; C408202:MEF-3 protein, human; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D010446:Peptide Fragments; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D020107:Troponin T; C113120:inosine monophosphate synthase; D019877:Hydroxymethyl and Formyl Transferases; D009709:Nucleotide Deaminases