Accurate and efficient processing of primary transcription products is absolutely required for normal gene expression in eukaryotic cells. Integration and coordination among the individual reactions which constitute a functional RNA processing pathway are the subjects of this research. Splicing and transport of tRNA transcripts in yeast and in Xenopus have been chosen as experimental models based on the availability of a large number of tRNA genes for analysis detailed information regarding both tRNA structure and mechanisms for tRNA processing, and the development of a range of assay systems for measuring splicing and transport reactions. Quantitative microinjection of synthetic RNA substrates into oocyte nuclei is used to measure both splicing and transport. Experiments are proposed in two areas. The first is a systematic mutational analysis of tRNA gene mutations affecting IVS + and IVS- tRNA transport. The second is an analysis of the distinct yeast- like and vertebrate tRNA splicing pathways and their function in oocytes. Injection of splicing intermediates and coinjection of yeast tRNA ligase will be used in this second experimental area. Long term goals include analyses of potential regulation through the use of alternate processing pathways and mechanisms for coupling of the splicing and transport steps. %%% Precursor transfer RNA molecules undergo shortening before they become mature molecules that can function in ribosomal protein synthesis. They must also be transported out of the nucleus of eukaryotic cells. These important steps in the gene expression of functional tRNAs is the subject of this research.
