Identification of eight proteins that cross-link to pre-mRNA in the yeast commitment complex

Abstract

In the yeast commitment complex and the mammalian E complex, there is an important base-pairing interaction between the 5′ end of U1 snRNA and the conserved 5′ splice site region of pre-mRNA. But no protein contacts between splicing proteins and the pre-mRNA substrate have been defined in or near this region of early splicing complexes. To address this issue, we used 4-thiouridine-substituted 5′ splice site-containing RNAs as substrates and identified eight cross-linked proteins, all of which were identified previously as commitment complex components. The proteins were localized to three domains: the exon, the six nucleotides of the 5′ ss region, and the downstream intron. The results indicate that the 5′ splice site region and environs are dense with protein contacts in the commitment complex and suggest that some of them make important contributions to formation or stability of the U1 snRNP–pre-mRNA complex.

Keywords

• Yeast commitment complex
• 5′ splice site
• pre-mRNA
• 4-thiouridine
• cross-link