Protein-Dependent Splicing of Chloroplast Group II Introns

University Of Oregon Eugene, Eugene OR

Investigators

Abstract

Catalytic ribonucleoprotein particles (RNPs) are at the core of several fundamental cellular processes, including protein synthesis, tRNA processing, and RNA splicing. The contributions of the RNA and protein components are varied. In some instances, RNA subunits harbor catalytic activity and the proteins enhance the formation or stability of the active RNA structure. In others, RNAs provide an assembly scaffold for catalytically-active proteins. RNA and protein molecules are known to cooperate in forming substrate binding surfaces and, in principle, they could cooperate to form an active site. This project uses RNPs harboring "self-splicing" group II introns to explore modes of cooperation between RNA and protein. The chemical mechanism of group II intron splicing is identical to that in the spliceosome and it is speculated that these two splicing machineries evolved from a common ancestor. Nine nucleus-encoded proteins that are required for the splicing of various subsets of the 17 group II introns in maize chloroplasts were identified previously. These proteins provide unique tools for exploring how proteins and RNAs cooperate during RNP assembly and catalysis. Experiments will focus on the subset of intron RNPs containing a protein called CRS2. CRS2 is derived from a bacterial peptidyl-tRNA hydrolase (PTH) and functions in heterodimeric complexes with either of two closely-related proteins, CAF1 or CAF2. Recent data suggest that CRS2/CAF complexes interact intimately with the intron catalytic core in a manner that differs from interactions in previously-studied group II intron RNPs. Structural and phylogenetic data suggest the intriguing possibility that the region of CRS2 derived from the PTH active site may contribute to splicing catalysis; this notion is bolstered by recent biochemical studies of intron RNP architecture, which place the CRS2 active site region near the branchpoint adenosine that initiates splicing. Experiments will take advantage of established protein expression systems and biochemical assays to further dissect the architecture of CRS2/CAF/intron RNPs, to test whether CRS2/CAF binding organizes RNA elements at the catalytic core, and to test whether CRS2 contributes directly to catalysis. The results obtained could impact our understanding of RNA-protein cooperation in the spliceosome, ribosome, and other catalytic RNPs. The most ancient and universal components of the machinery for expressing genetic information consist of large complexes between RNA and protein, macromolecules with distinct biochemical and structural features. The contributions of the RNA and protein moieties have become intertwined during a long period of co-evolution, and understanding of the repertoire of possible modes of cooperation between these molecules is incomplete. This project uses catalytic RNA-protein particles called group II intron RNPs to explore this issue. Prior results suggest that the proteins in these particles play a distinct and more fundamental role than has previously been documented for proteins in catalytic RNPs. Thus, the results obtained could impact our under-standing of RNA-protein cooperation in other macromolecular complexes underlying gene expression. High school, undergraduate and graduate students, including minority/underprivileged students, will be educated through their involvement in this project.