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, 288 (38), 27415-22

Partitioning of the Nuclear and Mitochondrial tRNA 3'-end Processing Activities Between Two Different Proteins in Schizosaccharomyces Pombe

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Partitioning of the Nuclear and Mitochondrial tRNA 3'-end Processing Activities Between Two Different Proteins in Schizosaccharomyces Pombe

Xiaojie Zhang et al. J Biol Chem.

Abstract

tRNase Z is an essential endonuclease responsible for tRNA 3'-end maturation. tRNase Z exists in a short form (tRNase Z(S)) and a long form (tRNase Z(L)). Prokaryotes have only tRNase Z(S), whereas eukaryotes can have both forms of tRNase Z. Most eukaryotes characterized thus far, including Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and humans, contain only one tRNase Z(L) gene encoding both nuclear and mitochondrial forms of tRNase Z(L). In contrast, Schizosaccharomyces pombe contains two essential tRNase Z(L) genes (trz1 and trz2) encoding two tRNase Z(L) proteins, which are targeted to the nucleus and mitochondria, respectively. Trz1 protein levels are notably higher than Trz2 protein levels. Here, using temperature-sensitive mutants of trz1 and trz2, we provide in vivo evidence that trz1 and trz2 are involved in nuclear and mitochondrial tRNA 3'-end processing, respectively. In addition, trz2 is also involved in generation of the 5'-ends of other mitochondrial RNAs, whose 5'-ends coincide with the 3'-end of tRNA. Thus, our results provide a rare example showing partitioning of the nuclear and mitochondrial tRNase Z(L) activities between two different proteins in S. pombe. The evolution of two tRNase Z(L) genes and their differential expression in fission yeast may avoid toxic off-target effects.

Keywords: Enzymes; Gene Expression; Mitochondria; Post-transcription; RNA Processing; Transfer RNA (tRNA); tRNA 3′-Endonuclease; tRNA Biogenesis.

Figures

FIGURE 1.
FIGURE 1.
Northern blot analysis of nuclear tRNAs from wild-type and trz1-1 mutant strains. Total RNA was isolated at the indicated times (hours) after shifting the cultures to the nonpermissive temperature of 37 °C. 0 h is the time of the temperature shift. RNA was probed for the tRNAAACVal intron-containing (A) or mature (B) species and the tRNACUULys intron-containing (D) or mature (E) species. U1 snRNA was also probed and used as a loading control (C and F). The positions of the probes are indicated by horizontal bars relative to the various tRNA species on the right of A, B, D, and E. The identities of the tRNAs have been confirmed (5, 39, 42). M, single-stranded DNA markers in nucleotides. Black boxes indicate exons, white boxes represent introns, and thick lines denote 5′- and 3′-extensions.
FIGURE 2.
FIGURE 2.
Low level of trz2 expression is required to obtain full suppression of trz2 temperature-sensitive growth defect. Wild-type and trz2-1 strains were spotted on rich medium (YES medium) (A) or Edinburgh minimal medium (EMM) plates supplemented with uracil and leucine (B) and incubated at 26 or 37 °C. Wild-type and trz2-1 strains transformed with the pREP81X empty plasmid (control) or pREP81X-trz2 carrying wild-type trz2 were spotted in 10-fold dilutions on selective medium (Edinburgh minimal medium containing uracil and lacking leucine) either lacking (C) or containing (D) thiamine and incubated at 26 or 37 °C.
FIGURE 3.
FIGURE 3.
Northern blot analysis of mt-tRNALys from the WT and trz2-1 mutant strains. A, schematic representation of a mt-tRNA gene cluster containing the gene for mt-tRNALys. The sizes of the genes within the cluster and the intergenic spacers separating the two genes are shown in base pairs. The relative positions of the probes used for Northern blot analysis are indicated. Total RNA was extracted from the wild-type and trz2-1 mutant strains after a shift to various times (0 h represents the time just before temperature shift). The RNA blot was probed for mt-tRNALys precursors containing a 3′-spacer using the 32P-labeled probe mitPArg (B), followed by stripping and reprobing for mature mt-tRNALys using the 32P-labeled probe mitMLys (C). U1 snRNA was probed as a loading control (D). The identity of each tRNA species is shown on the right. M, single-stranded DNA markers in nucleotides. Black boxes indicate mt-tRNAs, and thick lines denote intergenic spacers. The positions of the probes are indicated by horizontal bars. Asterisks denote bands whose identities could not be determined unambiguously. These bands may be identified by, for example, RNA deep sequencing.
FIGURE 4.
FIGURE 4.
Northern blot analysis of mt-tRNAArg from wild-type and trz2-1 mutant strains. A, schematic diagram showing a mt-tRNA gene cluster containing the mt-tRNAArg gene. The RNA blot was probed sequentially with the 32P-labeled probe mitPArg, which is specific for mt-tRNAArg precursors containing a 3′-spacer (B), and with the 32P-labeled probe mitMArg, which is specific for mature mt-tRNAArg (C). The same blot was also probed for U1 snRNA as a loading control (D). See the legend to Fig. 3 for a more detailed description. M, single-stranded DNA markers in nucleotides.
FIGURE 5.
FIGURE 5.
Northern blot analysis of mt-tRNAHis from wild-type and trz2-1 mutant strains. A, schematic of a cluster of mt-tRNA genes containing the mt-tRNAHis gene. The RNA blot was probed with the 32P-labeled probe mitPHis to detect mt-tRNAHis precursors containing a 3′-spacer (B) and with the 32P-labeled probe mitMHis to detect mature mt-tRNAHis (C). U1 snRNA served as a loading control (D). See the legend to Fig. 3 for a more detailed description. M, single-stranded DNA markers in nucleotides.
FIGURE 6.
FIGURE 6.
Increased accumulation of mitochondrial mRNA and noncoding RNA (other than tRNA) precursors in the trz2-1 mutant. Left, schematic diagrams of the junctions between mt-tRNAs and other transcripts. Black boxes indicate tRNA genes, and white boxes indicate other genes. Arrows indicate the positions of qRT-PCR primers. Right, total RNA was isolated from S. pombe wild-type, trz1-1, and trz2-1 strains. The expression levels of mitochondrial mRNAs and noncoding RNA (rnl and the predicted RNase P RNA rnpB) were normalized to the S. pombe actin gene act1. Values represent the mean ± S.D. of three independent experiments (p < 0.01 for both). The error bars represent the S.D. of triplicates.

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