The research described in this proposal investigates the function of RNA found in a variety of axons. This RNA is likely to be transfer RNA and is transported axonally in large amounts in axons which are regenerating. Since protein synthesis is not likely to occur in axons, other functions for axonal tRNA must be considered. The proposed experiments investigate the possibility that the function of tRNA in axons is to participate in post-translational protein modification by donating amino acids to axonal proteins. Protocols involve experiments which test this hypothesis in the giant axon of the squid, in regenerating optic axons of goldfish and in intact and regenerating axons of the rat sciatic nerve. The ability of tRNA to transfer an amino acid to an acceptor protein will be determined by assaying for the enzyme peptidyl transferase in the three systems described above. Also, the proteins which are acceptors for these amino acids will be identified by one or two dimensional polyacrylamide slab gel electrophoresis. Since tRNA in axoplasm serves a different function than tRNAs in cell soma it seems possible that structural differences exist between the two. Thus we plan to investigate this possibility by comparing chromatographic profiles of axonal and somal tRNAs when subjected to reverse phase high pressure liquid chromatography. Experiments involving tRNA in other systems have demonstrated that tRNA and the enzyme involved in amino acid addition, aminoacyl-tRNA synthetase, form, """"""""in vivo"""""""", a high molecular weight structurally significant complex. In squid, and rat, we plan to investigate whether this complex exists for axonal tRNA and its enzymes aminoacyl-tRNA synthetase and peptidy transferase, and in rats, whether this complex is transported axonally as part of the slow component of axonal transport. These experiments are proposed to help determine the function of transfer RNA in axons. The fact that tRNA is transported axonally in such large amounts during regeneration, and that much of it is localized to the advancing growth cone of the axon, suggests an important role for tRNA in nerve regeneration. This research may yield important information as to the mechanisms underlying nerve growth and help us to understand the reasons for the failure of nerves to regenerate in the CNS of man.
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