Remove of introns from messenger RNA precursors (pre-mRNA) is an essential process in eukaryotic gene expression. To remove introns, a large RNA-protein complex, known as the spliceosome, must be assembled on pre-mRNA through an ordered multi-step pathway. Although an outline picture of how pre-mRNA is spliced has emerged from the numerous studies, many important knowledge gaps remain. Among the many interesting unanswered questions, the roles of so-called DExH/D box helicase proteins in the spliceosome are particularly intriguing. A number of putative RNA helicases have been shown to function in the spliceosome. However, our knowledge that links a putative helicase to a specific target RNA duplex is currently limited. The goal of this proposal is to understand the biological functions of the p68 RNA helicase that was recently detected interacting with the transient U1:5'ss duplex during the spliceosome assembly (Liu, 1998). Our preliminary data suggest that the p68 RNA helicase is essential for in vitro pre-mRNA splicing. The protein may be functionally involved in mediating the U1 snRNA and pre-mRNA interactions during the spliceosome assembly. P68 may also plays a role in the process of addition of U5.U4/U6 tri-snRNP to the pre-spliceosome. This proposal will investigate the functional roles of p68 RNA helicase in the pre-mRNA splicing process. The studies will focus on two important issues: (1) Does p68 RNA helicase unwind the U1:5'ss duplex. (2) Does p68 plays a role in the addition of the tri-snRNP. We anticipate that understanding the functional roles of p68 would constitute a significant advance in knowledge about the mechanism of the spliceosome assembly. In the process of characterization of enzymatic activity of p68 helicase, we first demonstrated dsRNA-binding and dsRNA stimulated ATPase activity. We propose experiments to elucidate the RNA-binding mechanism and investigate how the RNA-binding is coupled to the enzymatic activities of p68.
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