Abstract
tRNase Z is the endonuclease that is involved in tRNA 3′-end maturation by removal of the 3′-trailer sequences from tRNA precursors. Most eukaryotes examined to date, including the budding yeast Saccharomyces cerevisiae and humans, have a single long form of tRNase Z (tRNase ZL). In contrast, the fission yeast Schizosaccharomyces pombe contains two candidate tRNase ZLs encoded by the essential genes sptrz1+ and sptrz2+. In the present study, we have expressed recombinant SpTrz1p and SpTrz2p in S. pombe. Both recombinant proteins possess precursor tRNA 3′-endonucleolytic activity in vitro. SpTrz1p localizes to the nucleus and has a simian virus 40 NLS (nuclear localization signal)-like NLS at its N-terminus, which contains four consecutive arginine and lysine residues between residues 208 and 211 that are critical for the NLS function. In contrast, SpTrz2p is a mitochondrial protein with an N-terminal MTS (mitochondrial-targeting signal). High-level overexpression of sptrz1+ has no detectable phenotypes. In contrast, strong overexpression of sptrz2+ is lethal in wild-type cells and results in morphological abnormalities, including swollen and round cells, demonstrating that the correct expression level of sptrz2+ is critical. The present study provides evidence for partitioning of tRNase Z function between two different proteins in S. pombe, although we cannot rule out specialized functions for each protein.
- endonuclease
- 3′-end processing
- post-transcriptional processing
- tRNA precursor (pre-tRNA)
- tRNase Z
Abbreviations: aa, amino acids; DAPI, 4′,6-diamidino-2-phenylindole; DIC, differential interference contrast; DTT, dithiothreitol; EGFP, enhanced green fluorescent protein; EMM, Edinburgh minimal medium; GST, glutathione transferase; MBL, metallo-β-lactamase; miRNA, microRNA; MTS, mitochondrial-targeting signal; NLS, nuclear localization signal; nmt1, no message in thiamine; pre-tRNA, tRNA precursor; ScTrz1p, Saccharomyces cerevisiae tRNase ZL; SpTrz1p, S. pombe tRNase ZL; SV40, simian virus 40; tRNase, ZL, long form of tRNase Z; tRNase, ZS, short form of tRNase Z; YES, yeast extract medium plus supplements
- © The Authors Journal compilation © 2011 Biochemical Society