Naturally Occurring Mutations in Human Mitochondrial Pre-tRNASer(UCN) Can Affect the Transfer Ribonuclease Z Cleavage Site, Processing Kinetics, and Substrate Secondary Structure

Hua Yan,Neela Zareen,Louis Levinger

doi:10.1074/jbc.m509822200

Abstract

tRNAs are transcribed as precursors with a 5' end leader and a 3' end trailer. The 5' end leader is processed by RNase P, and in most organisms in all three kingdoms, transfer ribonuclease (tRNase) Z can endonucleolytically remove the 3' end trailer. Long ((L)) and short ((S)) forms of the tRNase Z gene are present in the human genome. tRNase Z(L) processes a nuclear-encoded pre-tRNA approximately 1600-fold more efficiently than tRNase Z(S) and is predicted to have a strong mitochondrial transport signal. tRNase Z(L) could, thus, process both nuclear- and mitochondrially encoded pre-tRNAs. More than 150 pathogenesis-associated mutations have been found in the mitochondrial genome, most of them in the 22 mitochondrially encoded tRNAs. All the mutations investigated in human mitochondrial tRNA(Ser(UCN)) affect processing efficiency, and some affect the cleavage site and secondary structure. These changes could affect tRNase Z processing of mutant pre-tRNAs, perhaps contributing to mitochondrial disease.

Highlights

Precursor tRNA transcripts have a 5Ј end leader and a 3Ј end trailer
Efficiency and Kinetics of Human Nuclear-encoded pre-tRNAArg Processing by transfer ribonuclease (tRNase) ZL and tRNase ZS—To choose the best enzyme for analysis of pre-tRNA 3Ј end processing in vitro, human tRNase ZL and tRNase ZS were characterized using nuclear-encoded pre-tRNAArg as a substrate under optimized reaction conditions (Fig. 1)
Kinetic analyses were performed to determine the relative contributions made by changes in kcat and km to the more efficient processing of pre-tRNAArg by tRNase ZL than by tRNase ZS (Fig. 2, Table 1)

Summary

Introduction

Precursor tRNA transcripts have a 5Ј end leader and a 3Ј end trailer. Eukaryotic nuclear-encoded tRNAs are transcribed from RNA polymerase III promoters with short 5Ј end leaders and 3Ј end trailers ending with a U3 tail. In most organisms in all three kingdoms and in extranuclear organelles, CCA is not transcriptionally encoded and can be added to the discriminator (the unpaired nucleotide after the 3Ј end of the acceptor stem, which is retained in mature tRNA) after precise endonucleolytic removal of the 3Ј end trailer by tRNase Z (9 –14). Human tRNase ZL was first characterized as a prostate cancer susceptibility gene [22], and yeast tRNase Z (a long form) has been suggested to have notable mitochondrial functions [23]. Human tRNase ZL is demonstrated to process nuclear-encoded pre-tRNA 1600-fold more efficiently than tRNase ZS, suggesting that it is the important enzyme for pre-tRNA 3Ј end processing in organisms such as human, in which both are present

Methods

Results

Conclusion