How Did It Get There? Understanding tRNAHis Modifications in Caenorhabditis elegans

Histidyl-tRNA (tRNAHis) has the essential role of acting as an adaptor molecule in translation and is required for addition of histidine to growing protein chains by the ribosome. To maintain fidelity of protein synthesis, the histidyl-tRNA synthetase (HisRS) must ensure the correct attachment of the histidine residue to its cognate tRNAHis and not to other tRNAs. For nearly all eukaryotic organisms, the identifying element on a tRNAHis utilized by HisRS to ensure this fidelity is a guanosine nucleotide at the minus one position (G-1) that is present on the 5’ end of a mature tRNAHis, but not on any other tRNA. In many eukaryotes, incorporation of G-1 occurs by a two-step mechanism. First, Ribonuclease P (RNase P) cleaves to remove the 5’-leader sequence from pre-tRNAHis, leaving a guanosine at the plus one position. Then, tRNAHis guanylyltransferase (Thg1) catalyzes the unusual 3’ to 5’ addition of a guanosine nucleotide to the 5’ end of tRNAHis. Consistent with the importance of this role, Thg1 is widely conserved among eukaryotes. Recently, we identified an exception to this pathway in Caenorhabditis elegans. C. elegans tRNAHis contains a G-1 residue and this is still the identifying element for C. elegans HisRS. However, the genome of C. elegans does not encode any identifiable Thg1 enzyme, raising the question of how this G-1 element is incorporated into tRNAHis in this species. Interestingly, tRNAHis genes in C. elegans contain an encoded G-1 nucleotide in their 5'-leader sequences, suggesting that G-1 might be incorporated during transcription in the worm, and retained by an altered activity of RNase P, constituting a mechanism for incorporation of G-1 that has so far only been observed to occur in Bacteria. To test whether this altered RNase P cleavage activity might be responsible for retention of G-1 on tRNAHis, we performed yeast complementation in vivo involving tRNAHis genes from C. elegans, as well as in vitro 5'-maturation assays using transcripts corresponding to C. elegans pre-tRNAHis. Results showing implications for non-canonical recognition of tRNA by C. elegans RNase P will be discussed.A five-year embargo was granted for this item.Academic Major: Biochemistr