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.
The chemistry of life relies on biological catalysts called enzymes, which allow biochemical reactions to proceed under physiological conditions. Enzymes are required for breaking down the food we eat, for building cellular structures from simple molecules, and for reading the genetic code. Most enzymes are made of protein molecules, which are well suited to this purpose because they can form intricate structures that impart specific functions. However, the most ancient enzymes are made of a very different type of molecule, called RNA. RNA is closely related to the DNA double-helix, but with one critical difference: RNA generally exists as just a single-strand. This allows RNA to fold into complex three-dimensional structures, and these structures can imbue an RNA molecule with enzymatic activity. RNA-based enzymes are called "ribozymes" and are at the heart of the machinery that interprets the genetic code. Ribozymes can catalyze diverse biochemical reactions and have great potential for applications in the biomedical and biotechnological arena. Naturally-occurring ribozymes don’t act alone, however: they require intimate interactions with proteins to be fully active. The identity of the "protein helpers" and how these helpers enhance ribozyme function are poorly understood. This project explored the protein-RNA interactions that promote ribozyme activity. To do this, we took advantage of ribozyme/protein partners found in chloroplasts, the entities in plant cells that carry out the process of photosynthesis. Chloroplasts house ribozymes called group II introns, whose activity is required for plants to be green and photosynthetically-competent. The greenness of the plant provided a visual readout of ribozyme activity; we took advantage of this to identify proteins that enhance ribozyme function. In this way, we identified 13 different proteins that are required for group II intron ribozyme activity, and we were able to infer the functions of many unstudied proteins due to their similarity to the proteins we discovered. We made progress in understanding how each protein influences the shape and function of the RNA with which it interacts, but a deep understanding of these processes will require continuing effort. This research at once addresses the gene complement required for the formation of the chloroplast (and thus for photosynthesis), and the mechanisms by which protein and RNA molecules cooperate to achieve catalysis. Fundamental knowledge of this type will enhance our ability to develop tools for modulating traits of biomedical, agronomic, and biotechnological relevance.
