Poster abstracts

Poster number 57 submitted by Fuad Mohammad

A role for 3’-5’ polymerization in tRNA editing

Fuad Mohammad (Departments of Chemistry and Biochemistry, The Ohio State University), Maria G Abad (Departments of Chemistry and Biochemistry, The Ohio State University), Yicheng Long (Departments of Chemistry and Biochemistry, The Ohio State University), Jane E. Jackman (Departments of Chemistry and Biochemistry, The Ohio State University)

Abstract:
Transfer RNAs play a critical function in the cell as translators of information embedded in messenger RNA during protein synthesis. To ensure the fidelity of translation, each tRNA must be correctly recognized by its respective aminoacyl-tRNA synthetase and the ribosome. Therefore, tRNAs undergo various post-transcriptional modifications, some of which maintain and stabilize their universally conserved L-shaped tertiary structure. The discovery of the tRNA^His guanylyltransferase (Thg1) family of enzymes expands the repertoire of tRNA modifications to include the 3’-5’ addition of nucleotides to various tRNA substrates. Some Thg1 homologues, known as Thg1-like proteins (TLPs), catalyze the repair of 5’ truncated tRNAs, and may play a role in amending mismatches observed at the acceptor arm of several mitochondrially-encoded tRNAs in certain lower eukarya. Of the 18 mitochondrially-encoded tRNAs in Dictyostelium discoideum, 9 tRNAs contain such mismatches. Similar mismatches in other organisms, such as Acanthamoeba castellanii, have been shown to be edited to restore Watson Crick base pairing. Here, we focus on two Thg1 homologues in D. discoideum, DdiTLP3 and DdiTLP4. Using truncated tRNA substrates, both enzymes catalyze the repair of the 9 tRNAs proposed to be edited in D. discoideum, and are likely to participate in the tRNA editing mechanism. However, DdiTLP4 adds nucleotides beyond the full-length 5'-end of several tRNAs. This extra addition activity is inhibited at higher ATP concentrations, suggesting a mechanism for controlling the repair activity in vivo. Furthermore, DdiTLP4 is able to repair larger 5'-end truncations than DdiTLP3, which exhibits a more limited ability to repair these substrates and depends on the presence of a pre-activated 5'-end. Sequencing of one of the repair products reaffirms the preference for templated nucleotide addition over non-templated additions by DdiTLP3-4. These results point to an underlying 3’-5’ RNA polymerase activity of DdiTLP3-4 that has been fine tuned to participate in mitochondrial tRNA editing, and may suggest further roles for 3’-5’ nucleotide addition.

Keywords: Thg1, tRNA editing, 3-5 polymerization