In eukaryotic cells, small nuclear and small nucleolar RNAs function in pre-mRNA and pre-rRNA processing.Many of these RNAs are themselves produced from primary transcripts that require modification and processing. In the yeast Saccharomyces cerevisiae, there are five small nuclear RNAs (snRNAs) involved in mRNA splicing and about 60 small nucleolar RNAs (snoRNAs) that function in ribosomal RNA processing and modification. Although a number of proteins are suspected to play a role in the maturation of small RNAs, the maturation pathways have not been well characterized. We identified a nuclear RNA helicase in yeast called Sen1p that is required for the maturation of tRNA, snRNA U5, rRNA, and numerous snoRNAs. Nine proteins, the snRNAs, and the snoRNAs have been shown to co-immunoprecipitate using antibodies against Sen1p, suggesting that the Sen1p RNA helicase assembles into one or more RNP complexes. Our recent studies show that Sen1p binds to the Sm/snRNA RNP involved in snRNA processing. Sen1p functions in conjunction with the endonuclease RNase III to form the 3' end of snRNA US. Sen1p also functions in 5' and 3' end formation of C/D box snoRNAs and may function in conjunction with other proteins including fibrillarin that bind to the C/D box. in this proposal, our specific aims will focus on the overall process of snoRNA maturation. We will screen for mutants that impair maturation, characterize snoRNA maturation pathways in vivo and in vitro, and define protein-protein and protein-RNA interactions that are important for the overall execution of steps in maturation. We will also characterize the biochemical activities of the Sen1p helicase in vitro and identify proteins that associate with Sen1p. The long-term objective of the proposal is to understand the biological implications of how small RNAs are derived from primary transcripts and how RNA helicases such as Sen1p can influence and possibly cross-regulate disparate RNA processing pathways. Further studies of RNA helicases and the processing of small RNAs in yeast are warranted and are ultimately of medical importance because numerous helicases and small RNAs have counterparts in vertebrates including humans and have been implicated in some human genetic disorders.
| Ursic, Doris; Finkel, Jonathan S; Culbertson, Michael R (2008) Detecting phosphorylation-dependent interactions with the C-terminal domain of RNA polymerase II subunit Rpb1p using a yeast two-hybrid assay. RNA Biol 5:1-4 |
| Hendrick, J L; Wilson, P G; Edelman, I I et al. (2001) Yeast frameshift suppressor mutations in the genes coding for transcription factor Mbf1p and ribosomal protein S3: evidence for autoregulation of S3 synthesis. Genetics 157:1141-58 |
