Factor-Dependent Splicing of Chloroplast Group II Introns

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BarkanMany essential biological processes involve large, intricatelystructured RNA molecules whose folding is likely to be problematic. In thecell, structured RNAs are complexed with proteins or require transientinteractions with proteins to function efficiently. Such interactions mayserve to enhance the formation or stability of the correctly folded RNA."Self-splicing" group I and group II introns are highly structuredcatalytic molecules that require proteins to function efficiently in vivo.Because proteins that facilitate intron function can be identifiedgenetically and then studied in vitro, these provide convenient models forprobing the role of proteins in RNA catalyzed reactions and RNA folding.Two factors that facilitate the splicing of group I introns in fungalmitochondria have been examined in great detail. However, the study ofgroup II splicing factors has lagged behind. This project concerns twonuclear genes in maize that are required in vivo for the splicing ofspecific chloroplast group II introns. These genes provide unique toolsfor addressing the roles of proteins in facilitating group II intronsplicing. The crs1 gene is required specifically for the splicing of theatpF intron; the crs2 gene is required specifically for the splicing ofnine of the ten subgroup IIB introns in the chloroplast. The current goalof this project is to define the role played by each protein in thesplicing process. The crs1 and crs2 genes were recently cloned bytransposon tagging. CRS1 is a novel, hydrophilic protein. CRS2 isrelated to peptidyl-tRNA hydrolases, which function to recyclepeptidyl-tRNAs that are released as abortive translation products. Invitro and "in E. coli " RNA binding and splicing assays will now be usedto distinguish between several models for the roles of these proteins inthe splicing process. Only a handful of proteins have been shown toenhance the function of catalytic RNAs in vitro or in vivo. It is toosoon to know how large the repertoire of mechanisms will be for suchinteractions. Therefore, the results of these experiments are likely tolead to fundamental insights into the RNA folding problem and the role ofproteins in RNA catalyzed reactions. The results will also elucidate thenature of the regulatory interactions between the nuclear and chloroplastgenome in a higher plant.Lay summary: The chemical reactions that occur inside cells are catalyzed bymolecules called enzymes that facilitate the breaking and forming ofchemical bonds under physiological conditions. Most enzymes are composedof protein but some are composed of RNA, which is similar to DNA, thecarrier of genetic information. Regardless of its chemical makeup, thefolding of the enzyme into its correct three dimensional shape is criticalfor its function. Mechanisms by which proteins fold have been studied formany years. One fundamental concept to arise from this work is that mostproteins cannot fold spontaneously into their correct shape, but ratherrequire help from other protein molecules, dubbed "chaperones". Much lessis known about how catalytic RNA molecules assume their correct structure.This project concerns two proteins, CRS1 and CRS2, that are required forthe function of certain catalytic RNA molecules. The goal of this projectis to come to an understanding of the role these proteins play infacilitating the RNA-catalyzed reactions. In particular, this projectexplores how these proteins modulate the folding or stability of thecatalytic RNA molecules. Only a handful of proteins have been shown toenhance the function of catalytic RNAs. Therefore, the results of theseexperiments are likely to lead to fundamental insights into the RNA folding problem and the role of proteins in RNA catalyzed reactions.