In all organisms, tRNAs are matured by a series of post-transcriptional processing events before they can partake in protein synthesis. These include end trimming to generate the correct 5' and 3' ends, chemical modification to ensure proper structure and function, and addition of a 3' CCA tail where amino acids are ultimately attached. In addition a variable number of tRNAs also contain introns; these interrupt the anticodon sequence and as such render a tRNA non-functional. Therefore, splicing is essential for viability. Splicing of tRNA introns differs from mRNA splicing in that it involves several protein-catalyzed steps, including intron cleavage by a splicing-specific endonuclease, followed by exon-end maturation and ligation by a tri-functional ligase (a fusion of three different enzyme domains, depending on the organism) and lastly removal of a ?dangling? phosphate left behind by previous activities. In trypanosomatids, there is a single intron-containing tRNA, tRNATyr, responsible for decoding all the tyrosine codons. To date little is known about the mechanism(s) of tRNA splicing in any early diverging protist. Our studies have have identified putative components for each step of the tRNA splicing reaction, each with unusual features. The intron is edited at several positions and editing is in turn required for splicing. The splicing endonuclease has a sub-unit structure where one of the critical sub-units in other systems is missing. The splicing ligase forms a higher order complex suggesting its association with additional factors. The phosphotransferase has an unusual exonuclease domain suggesting an additional role in intron degradation. This proposal will focus in the characterization of each component of the tRNA splicing pathway in T. brucei and will also explore the contribution of intron editing to splicing specificity. Given that the T. brucei mechanism is peppered with unique features, successful completion of these studies will generate important basic information on the mechanism and evolution of tRNA splicing, a poorly understood area of protist biology. !
Members of the genus Leishmania and Trypanosoma infect millions of people worldwide. In these organisms, there is a single intron-containing tRNA in T. brucei, but removal of that singular intron is still essential for viability. Although, tRNA splicing is not unique to trypanosomes, we have uncovered a divergent splicing machinery and unprecedented intron editing important for tRNA splicing. This project will unveil the mechanism of tRNA splicing in these medically important organisms.