RNA helicases are enzymes which catalyze the separation of strands in RNA duplexes or the removal of secondary structure in single-stranded RNA. The reaction requires hydrolysis of nucleoside triphosphates as an energy source. A growing number of putative RNA helicase genes involved in a variety of physiological functions such as translation initiation, ribosome assembly, mRNA splicing, and germinal line cell differentiation have been found in organisms ranging from E. coli to human. Many of these putative RNA helicases share a series of sequence elements, most noticeably the DEAD-Box motif. In yeast Saccharomyces cerevisiae, there are at least 16 putative RNA helicase genes belong to this DEAD-Box Protein (DBP) gene family. Multiple DBP genes have also been discovered in E. coli, Drosophila, and HeLa cells. Thus, this gene family appears to have evolved very early and is highly conserved in evolution. The unexpected diversity of the putative RNA helicase genes in yeast as well as in other organisms immediately raises a number of intriguing questions regarding their cellular functions and biochemical properties. A crucial question, in particular, is how and what RNAs are functionally regulated by these enzymes. This proposal will address these questions by focusing on studying the biochemistry and cellular functions of two yeast RNA helicases, PRP28 and SPP81/DED1, which are implicated in nuclear pre-mRNA splicing. Other yeast DBP genes with unknown functions will also be investigated. It is anticipated that these studies will provide novel information about the RNA helicases, of which little is known at the moment. Specific aims are: [1] To construct conditional mutants for SPP81/DED1, as well as for other essential DBP genes with unknown functions. [2] To characterize the splicing phenotypes of these conditional mutants. [3] To identify interacting components of the RNA helicases by isolating extragenic suppressors for conditional mutants. [4] To identify the cellular substrates for PRP28 and SPP81/DED1. [5] To study the biochemical properties of PRP28.