Many essential biological processes involve intricately structured RNAs that are complexed with proteins in ribonucleoprotein particles (RNPs). The contributions of the RNA and protein components in RNPs are varied. In some instances, RNA subunits harbor catalytic activity and the proteins serve to 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 to form substrate binding surfaces and, in principle, they could cooperate to form an active site. This project uses RNPs involving "self-splicing" group II introns as tools to explore modes of cooperation between RNA and protein in RNPs. Group II introns are highly structured catalytic RNAs that require proteins to function efficiently in vivo. Group II intron RNPs are excellent models for dissecting the roles of RNA and protein components in RNP assembly and function because they are relatively simple, and because proteins that facilitate intron function can be identified genetically and then studied in vitro. This project focuses on three nucleus-encoded proteins, CRS2, CAF1, and CAF2, which are required for the splicing of group II introns in the chloroplast. CRS2 is related to peptidyl-tRNA hydrolase enzymes. CAF1 and CAF2 are closely related to one another, and belong to a diverse family of plant proteins that also includes the group II intron splicing factor CRS1. The defining feature of the CAF1/CAF2/CRS1 family is a previously unrecognized RNA binding domain of ancient origin, recently named the CRM domain. Prior data indicate that CRS2/CAF2 and CRS2/CAF1 complexes mediate the splicing of different intron sets in the chloroplast and are bound in vivo to their genetically-defined intron targets. The following working model has emerged from prior work and will be tested during the course of this project: (A) the CRM domain factors CAF1 and CAF2 harbor intron-specific binding activity and influence intron folding; (B) CRS2 is recruited to specific introns via interaction with a CAF; (C) CRS2's conserved peptidyl-tRNA hydrolase active site may contribute functional groups during splicing catalysis. The varied but complementary experimental approaches take advantage of the availability of mutant strains lacking each of the three factors, antisera to each of the three proteins, and proven recombinant expression systems for each protein.
