Assembly of the AI5-beta Group I Intron RNP

Turk, Edward

Abstract

This proposal will elucidate the mechanisms of ribonucleoprotein (RNP) biogenesis. RNPs play a central role in nearly all aspects of RNA biology, including transcription, RNA localization, splicing, translation, mRNA degradation, RNA interference and telomere maintenance. Defects in RNP biogenesis are responsible for retinitis pigmentosa and spinal muscular atrophy, both of which are human diseases characterized by tissue-specific degeneration of cells. RNP biogenesis likely involves an assembly- disassembly cycle governed by context-promoted specificity between the RNA and protein components. However, the mechanisms that drive such specificity are poorly understood. This proposal is focused on biogenesis of the al5 beta-splicing RNP, a group I intron within the S. cerevisiae mitochondrial COX1 gene. AI5 beta is self-splicing in vitro, but requires five nuclear-encoded proteins for efficient in vivo splicing. This system combines the power of yeast genetics, the easy of maintaining and scoring mitochondrial defects, and a read-out for in vitro catalytic activity (splicing), that will allow the genetic, biochemical, and biophysical dissection of RNP biogenesis. The goal of aim 1 is to investigate the assembly and function of the Mrs1 p- al5 beta RNP. This will be achieved by: (a) mapping the native aI5 beta RNA structure and the Mrs1p-al5 beta RNP by chemical and nuclease footprinting; (b) determining if the ancestral DNA binding domain of Mrs 1p has evolved to function as the RNA binding domain for al5 beta.
This aim will begin to elucidate the mechanisms of substrate recognition that facilitate RNP assembly by identifying transitions in aI5 beta RNA structure induced by Mrs1 p binding, and by identifying the molecular elements that govern the RNA-protein interactions. The goal of aim 2 is to investigate the relationship between Mrs 1 p and Suv3p in RNP biogenesis. This will be achieved by: (a) determining if purified Suv3p can disassemble the spliced Mrs1p-al5 beta RNP in vitro; (b) immunoprecipitate Suv3p and probe for associations with Mrs 1 p and excised aI5 beta RNA by Co-IP and RT- PCR analysis.
This aim will clarify the role of Suv3p in al5 beta RNP biogenesis and splicing and provide a rare opportunity to examine the activity of a DExH protein on its native target. ? ? Functional RNAs are at the heart of numerous cellular machines that carry out essential functions and, not surprisingly, many human diseases are caused by mutations within such RNAs. Since proteins enhance the functionality of most RNAs, it is essential to understand how they interact with RNAs. Such studies will potentially identify new causes of genetic diseases and may suggest new therapeutic strategies. ? ? ?