Poster abstracts
Poster number 45 submitted by Catherine Edgington
Assessing tRNA substrate specificity of archaeal homologs of the conserved tRNA methyltransferase Trm10
Catherine B. Edgington (The Ohio State University ), Jesse Leavitt (University of California, Santa Cruz), Todd Lowe (University of California, Santa Cruz), Jane E. Jackman (The Ohio State University )
Abstract:
Of all RNA species, tRNA is the most heavily modified with an average of 9-13 bases being modified per tRNA. One such modification, m1G9 and/or m1A9 is catalyzed by the tRNA methyltransferase 10 (Trm10), which is highly conserved throughout Archaea and Eukarya. Higher eukaryotes, including vertebrates, have multiple Trm10 paralogs. We recently demonstrated the biological functions for two of these paralogs in humans and zebrafish, with each enzyme exhibiting a unique pattern of activity and substrate specificity for introducing m1G or m1A into specific tRNAs. However, in these studies we also showed that purified human and zebrafish enzymes are capable of binding to and modifying tRNAs that are not modified by the enzymes in vivo. Thus, determining biological patterns of modification in diverse eukaryotes is challenging and cannot be predicted based on in vitro modification activities alone. However, whether archaeal homologs of Trm10 exhibit similar biochemical properties has not been determined. Recently, we determined the position 9 tRNA modification status for the archaeal species Thermococcus kodakarensis and Pyrococcus furiosus, enabling us to address questions about the substrate specificity of archaeal Trm10 enzymes from these organisms for the first time. T. kodakarensis Trm10 is a bifunctional methyltransferase, meaning it can catalyze both m1A9 and m1G9, but the modification activity catalyzed by P. furiosus Trm10 was not previously known. Using in vitro transcribed tRNA species from both organisms, we have begun to characterize the modification and binding preferences of these two enzymes with bona fide archaeal tRNA Trm10 substrates. Our results have demonstrated that P. furiosus Trm10 is another bifunctional modification enzyme, like T. kodakarensis Trm10. Characterization of the tRNA specificity of these two enzymes will reveal whether archaeal Trm10 enzymes share the ability to modify and bind to non-substrate tRNA with their eukaryotic counterparts, and will reveal important insight into the as of yet unknown mechanisms of tRNA substrate selection for these enzymes.
Keywords: tRNA modification