Requirements for accurate and efficient processing are common to the transcripts of all classes of eukaryotic genes. The organization and control of eukaryotic RNA processing pathways are the subjects of this research. The contributions of selectivity, kinetics and compartmentation to TRNA processing are being examined in this proposal. Splicing and nuclear transport are being studied as examples of key steps in a well-defined processing pathway. Our prior analyses of the properties of the yeast splicing complex assembly suggested both selectivity and compartmentation may contribute to ordering. Using nuclear microinjection of Xenopus oocytes it has been previously noted that tRNA nuclear transport is selective, requires prior splicing and transport, define kinetic parameters for export, and analyze compartmentation via multifunctional enzymes and the formation of stable complexes. Experimental approaches include: (1) Expression of yeast ligase-dihydrofolate reductase fusion proteins in E. coli to examine structure/function relationships in this multifunctional protein; (2) Microinjection of pre-tRNA and other labeled macromolecules in Xenopus oocytes to examine the requirements for nuclear transport; and (3) Analysis of Xenopus tRNA splicing and processing both in vivo and in vitro.
