Yeast Prp2 ATPase and its associated G-patch protein
One of the major spliceosomal proteins is RNA helicases, whose function is believed to mediate RNA and protein rearrangement in the spliceosome during the splicing cycle. We characterized one such protein called Prp2 in order to elucidate the function of RNA helicases in splicing. We found that yeast spliceosome could recognize the pre-mRNA in the absence of Prp2 but the complex stalled right before the first step of splicing. The block is released and splicing resumed when Prp2 and ATP were provided, indicating that the spliceosome is not active without Prp2 and the activation process requires ATP hydrolysis catalyzed by a spliceosome-bound Prp2. This activation process is likely conserved through evolution since we recently found that human spliceosome also requires a human protein with great similarity to yeast Prp2 for activity (see project 3).
There are eight RNA helicases in yeast Saccharomyces cerevisiae that are involved in pre-mRNA splicing and each of them is needed for cell viability. These proteins have highly conserved central helicase domain, which is involved in ATP hydrolysis and is absolutely required for its splicing function. The role of the less conserved amino- and carboxyl-extensions, however, remains unclear. We found that the C-terminal domain of Prp2 and a couple of other spliceosomal helicases is required for spliceosome binding. Moreover, a G-patch protein interacting with Prp2 through the C-domain was identified, and we showed that this interaction is essential for the binding of Prp2 to the spliceosome. This helicase-associated protein called Spp2 is required for yeast splicing and plays a role in providing specificity to Prp2 during spliceosome interaction. It remains to be investigated whether additional helicase-associated proteins are involved in the spliceosome binding of other RNA helicases.