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, 7 (9), e44264

Identification and Sequence Analysis of Metazoan tRNA 3'-end Processing Enzymes tRNase Zs

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Identification and Sequence Analysis of Metazoan tRNA 3'-end Processing Enzymes tRNase Zs

Zhikang Wang et al. PLoS One.

Abstract

tRNase Z is the endonuclease responsible for removing the 3'-trailer sequences from precursor tRNAs, a prerequisite for the addition of the CCA sequence. It occurs in the short (tRNase Z(S)) and long (tRNase Z(L)) forms. Here we report the identification and sequence analysis of candidate tRNase Zs from 81 metazoan species. We found that the vast majority of deuterostomes, lophotrochozoans and lower metazoans have one tRNase Z(S) and one tRNase Z(L) genes, whereas ecdysozoans possess only a single tRNase Z(L) gene. Sequence analysis revealed that in metazoans, a single nuclear tRNase Z(L) gene is likely to encode both the nuclear and mitochondrial forms of tRNA 3'-end processing enzyme through mechanisms that include alternative translation initiation from two in-frame start codons and alternative splicing. Sequence conservation analysis revealed a variant PxKxRN motif, PxPxRG, which is located in the N-terminal region of tRNase Z(S)s. We also identified a previously unappreciated motif, AxDx, present in the C-terminal region of both tRNase Z(S)s and tRNase Z(L)s. The AxDx motif consisting mainly of a very short loop is potentially close enough to form hydrogen bonds with the loop containing the PxKxRN or PxPxRG motif. Through complementation analysis, we demonstrated the likely functional importance of the AxDx motif. In conclusion, our analysis supports the notion that in metazoans a single tRNase Z(L) has evolved to participate in both nuclear and mitochondrial tRNA 3'-end processing, whereas tRNase Z(S) may have evolved new functions. Our analysis also unveils new evolutionarily conserved motifs in tRNase Zs, including the C-terminal AxDx motif, which may have functional significance.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Schematic representation of the predicted alternative splice variants of mammalian and nematode tRNase ZL genes (drawn to scale).
(A) Mammalian tRNase ZLs are from H. sapiens (Hsa), C. jacchus (Cja), C. familiaris (Cfa), O. cuniculus (Ocu), B. taurus (Bta), M. musculus (Mmu) and R. norvegicus (Rno). HsaTRZ2i1, HsaTRZ2i2, CjaTRZ2i1, CjaTRZ2i2, CjaTRZ2i3, CfaTRZ2i, OcuTRZ2i, BtaTRZ2i, MmuTRZ2i, and RnoTRZ2i correspond to alternatively spliced variants. Constitutive exons and introns are represented by the filled boxes and intervening horizontal lines respectively. Alternative exons are colored as follows: exon skipping in red, exon with alternative donors in blue and exon with alternative acceptors in green. The locations of the alternative exons in the tRNase ZL genes and its splice variants are shown on the top of the exon. (B) Nematode tRNase Zs are from C. elegans (Cel), C. remanei (Cre) and C. briggsae (Cbr). CelTRZ1i1, CelTRZ1i2, CreTRZ1i1, CreTRZ1i2, CbrTRZ1i1 and CbrTRZ1i2 are splice variants. Exons are shaded black, introns are indicated by lines and the 5′-untranslated region (5′-UTR) sequences are indicated by gray boxes. The exon number indicated on top. The ATG start codon and TAA stop codon are indicated.
Figure 2
Figure 2. Bayesian phylogenetic tree of predicted metazoan tRNase Zs.
This analysis is based on the sequence alignment of the full-length metazoan tRNase ZSs with the C-terminal half of metazoan tRNase ZLs. The accession number for each candidate tRNase Z is identified in Table S1. Bayesian posterior probabilities are indicated at the nodes. The scale bar indicates 0.1 nucleotide substitutions per site. Taxonomic designations are indicated on the right side of the tree.
Figure 3
Figure 3. Multiple sequence alignment of representatives of N-terminal halves of the metazoan and non-metazoan tRNase ZLs.
Representative metazoan tRNase ZLs are from ten deuteromes including C. familiaris (Cfa), H. sapiens (Hsa), L. Africana (Laf), M. musculus (Mmu), X. tropicalis (Xtr), D. rerio (Dre), S. salar (Ssa), C. intestinalis (Cin), B. floridae (Bfl), and S. purpuratus (Spu), six protostomes including B. mori (Bmo), D. melanogaster (Dme), T. castaneum (Tca), C. elegans (Cel), L. gigantean (Lgi), and C. teleta (Cte), and four basal metazoans including N. vectensis (Nve), H. magnipapillata (Hma), A. queenslandica (Aqu), and T. adhaerens (Tad). Non-metazoan tRNase ZLs are from the unicellular choanoflagellate M. brevicollis (Mbr), green plant A. thaliana (Ath) and fungal S. pombe (Spo). See Table 1 for more information. The alignment was constructed using the computer program Clustal W . Identical residues are on a black ground and conserved residues shaded in gray. The conserved motifs of tRNase Zs indicated above the alignment are labeled according to references , , . The numbers in brackets indicate the length of the region in the protein, which are species-specific and could not be correctly aligned. Hyphens represent gaps introduced into sequences for maximum alignment. Amino acid residues predicted to be critical for activity are indicated by a star.
Figure 4
Figure 4. Multiple sequence alignment of representatives of C-terminal halves of the metazoan tRNase ZLs.
Same legend as in Figure 3.
Figure 5
Figure 5. Multiple sequence alignment of representatives of the metazoan and non-metazoan tRNase ZSs.
Metazoan tRNase ZSs are from ten deuteromes including C. familiaris (Cfa), L. africana (Laf), H. sapiens (Hsa), M. musculus (Mmu), X. tropicalis (Xtr), D. rerio (Dre), S. salar (Ssa), C. intestinalis (Cin), B. floridae (Bfl), and S. purpuratus (Spu), one protostome, L. gigantean (Lgi), and three basal metazoans, N. vectensis (Nve), H. magnipapillata (Hma), and A. queenslandica (Aqu). Non-metazoan tRNase ZSs are from the unicellular choanoflagellate M. brevicollis (Mbr), B. subtilis (Bsu) and E. coli (Eco). The annotation of the alignment is as described in the legend to Figure 3.
Figure 6
Figure 6. Sequence logos for the predicted PxRxRN, PxPxRG and AxDx motifs.
The sequence logo of the PxRxRN motif was derived from 174 tRNase ZLs from 75 fungi, 21 plants and 68 metazoans. The sequence log of the PxPxRG motif is derived from alignment of 44 metazoan tRNase ZSs. The sequence logo of the AxDx motif is derived from 240 alignments including 178 tRNase ZLs from 71 metazoan, 21 green plants, 77 fungi, and 62 tRNase ZSs from 30 metazoans, 9 fungi, 2 bacteria and M. brevicollis. The sequence logos were created using WebLogo (http://weblogo.berkeley.edu). The height of each amino acid indicates the level of conservation at that position. Amino acids are colored as follows: red, basic; blue, acidic; orange, polar; and green, hydrophobic.
Figure 7
Figure 7. Growth complementation analysis of S. pombe Trz1 with a mutation in the AxDx motif.
A S. pombe strain bearing a temperature-sensitive allele of trz1 (trz1-1) was transformed with empty vector (4X) or vector expressing the wild-type Trz1 or the Trz1D772A mutant, and equal amounts of cells were spotted on EMM medium supplemented with leucine and adenine and grown at 26°C or 37°C.
Figure 8
Figure 8. Depiction of the predicted hydrogen bonding network in the AxDx strand/loop region of human and B. subtilus tRNase ZSs.
The atomic coordinates of human and B. subtilus tRNase ZSs were obtained from the Protein Data Bank, www.rcsb.org (PDB accession codes 3ZWF and 1Y44, respectively). Images were made with Swiss-PdbViewer . Potential hydrogen bonds were determined using the Swiss-PdbViewer and Insight II. Secondary structure elements are colored green. The AxDx strand/loop is colored yellow and the PxPxRG or PxKxRN loop is colored red. Hydrogen bonds are represented by a dashed green line. (A) In the human tRNase ZS, the AxDx strand/loop-centered hydrogen bond network involves Ala351 (O) and Arg19 (NH1), Asp353 (OD2) and Gly20 (HN), Ala351 (HN) and Leu311 (O), Asp353 (OD1) and Thr10 (HN and OG1), and Gly20 (O) and Thr10 (HN). (B) In B. subtilus, the AxDx strand/loop-centered hydrogen bond network includes Ala296 (O) and Arg17 (NH1), Asp298 (OD2) and Asn18 (HN), Ala 296 (HN) and Leu267 (O), Asp298 (OD2) and Thr8 (HN and OG1), and Asn18 (O) and Thr8 (HN).

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This work was supported in part by grants from the National Science Foundation of China (31070703) (http://www.nsfc.gov.cn/Portal0/default152.htm) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (http://jsycw.ec.js.edu.cn/) and Nanjing Normal University (2007104XGQ0148). The funders had no role in study design, data colletion and analysis, decision to publish, or preparation of the manuscript.
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