The tRNA synthetases are most prominently known for their aminoacylation of tRNA during protein synthesis. However, these enzymes play a number of diverse roles that are also essential to cells. One of these examples is tRNA synthetase-dependent RNA splicing. A novel ternary splicing complex has been identified in yeast where a group I intron called b14 is aided by two proteins, a maturase and a leucyl (Leu)-tRNA synthetase. Tyr-tRNA synthetase (CYT- 18) is the only other tRNA synthetase that has been found to facilitate RNA splicing. However, our preliminary data suggests that the molecular mechanisms by which these two related enzymes promote ribozyme self-splicing activity are quite distinct. Moreover, the Leu-tRNA synthetase-dependent ribozyme is the first example of a two protein: one RNA splicing complex and presents an intriguing elementary model to not only investigate RNA-protein interactions, but also potentially protein-protein interactions which might aid a ribozyme self-splicing reaction. We propose to investigate the Leu-tRNA synthetase-dependent ribozyme splicing reaction and identify discrete determinants that confer this protein synthesis enzyme's unique role in RNA splicing. We have established three-hybrid and RNA-dependent two-hybrid models that show for the first time that the Leu-tRNA synthetase and bI4 maturase can directly, independently, and simultaneously interact with the b14 intron. We have also developed a yeast nucleus-based assay via RT-PCR methods demonstrating that at least one of these protein partners must be bound to faciliate ribozyme splicing activity. We propose to map and identify specific interactions between Leu-tRNA synthetase and the b14 intron that dictate complex assembly and RNA splicing activity. Completion of the proposed specific aims will delineate the undefined splicing role of Leu-tRNA synthetase at the molecular level and provide insight into its inherent determinants that solicited cellular recruitment of this secondary, but also essential activity. Characterization of this simple ternary model offers an important stepping stone in understanding how catalytic ribozymes evolved to more complicated RNP complexes to enhance function and maintain essential biological processes. Moreover, since protein-dependent RNA splicing is critical to the health of human cells as well as to the life cycles of viral, protozoa, and fungal pathogens, elucidation of important molecular determinants required for RNA processing may identify new drug targets for the treatment of infectious disease.
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