Plant mitochondria use two pathways for the biogenesis of tRNA His

Abstract

All tRNAHis possess an essential extra G–1 guanosine residue at their 5′ end. In eukaryotes after standard processing by RNase P, G–1 is added by a tRNAHis guanylyl transferase. In prokaryotes, G–1 is genome-encoded and retained during maturation. In plant mitochondria, although trnH genes possess a G–1 we find here that both maturation pathways can be used. Indeed, tRNAHis with or without a G–1 are found in a plant mitochondrial tRNA fraction. Furthermore, a recombinant Arabidopsis mitochondrial RNase P can cleave tRNAHis precursors at both positions G+1 and G–1. The G–1 is essential for recognition by plant mitochondrial histidyl-tRNA synthetase. Whether, as shown in prokaryotes and eukaryotes, the presence of uncharged tRNAHis without G–1 has a function or not in plant mitochondrial gene regulation is an open question. We find that when a mutated version of a plant mitochondrial trnH gene containing no encoded extra G is introduced and expressed into isolated potato mitochondria, mature tRNAHis with a G–1 are recovered. This shows that a previously unreported tRNAHis guanylyltransferase activity is present in plant mitochondria.

Life without post-transcriptional addition of G−1: two alternatives for tRNAHis identity in Eukarya

“…These observations indicate a division of labor in A. castellanii, wherein two independent and coexisting mechanisms for tRNA His identity have evolved that are strictly compartmentalized. Inconsistencies in the occurrence of even the encoded G −1 on tRNA His have been previously reported in plant mitochondria and chloroplast (Marechal-Drouard et al 1996a,b;Placido et al 2005Placido et al , 2010.…”

Doing it in reverse: 3′-to-5′ polymerization by the Thg1 superfamily

“…His species from several of these organisms (M. acetivorans, M. barkeri, B. thuringiensis, and M. xanthus) do not predictably require post-transcriptional addition of G À1 , although the possibility of a role in tRNA His maturation, owing to loss of the encoded G À1 from the transcript during maturation by RNase P, as has been observed in the case of tRNA His in some plants (Placido et al 2010), cannot be excluded. As with yeast Thg1, TLPs catalyze efficient Watson-Crick template-dependent 39-to-59 nucleotide addition, attaching G À1 to C 73 -containing tRNA His in vitro and in vivo in yeast (Abad et al 2010;Heinemann et al 2010;Rao et al 2011).…”

“…Among the possibilities, an encoded G À1 could be generated by processing, as in bacteria, or a separate, non-Thg1 reverse polymerase could substitute for Thg1 in these species. Notably, in plant mitochondria, a tRNA His guanylyltransferase activity has been found, but neither of the two Thg1 orthologs that have been identified in plants appears to localize to this organelle (Placido et al 2010). Alternatively, G À1 may be dispensable in some cases, as it is in a-proteobacteria.…”

Differential substrate recognition by isozymes of plant protein-only Ribonuclease P

“…These data suggest a difference in substrate recognition among the PRORPs with regards to cleavage site selection. While the frequency of PRORP-catalyzed pre-tRNA miscleavage in vivo is unknown, PRORP catalyzes phosphodiester bond hydrolysis between −1 nt and +1 nt (correct) and between −2 nt and −1 nt (miscleavage) both in vivo and in vitro with the atypically processed plant mitochondrial pre-tRNA His (Placido et al 2010). Furthermore, PRORPs can correct miscleavage by catalyzing the removal of the miscleaved 1-nt leader, albeit slower than the rate constant for the initial cleavage step (Supplemental Fig.…”