tRNA 3'-end maturation is a process through which the 3'-trailer sequence of precursor tRNAs (pre-tRNAs) is removed, and processed tRNAs acquire the CCA end which is absolutely essential for tRNA aminoacylation and protein synthesis (for reviews, see [1–3]). In prokaryotes, this process can be either exonucleolytic or endonucleolytic depending on whether the 3'-CCA sequence is genomically encoded. CCA-containing pre-tRNAs are generally processed by the exonucleases that tend to stop removing nucleotides from the 3'-end upon encountering the transcriptionally encoded CCA, whereas CCA-less pre-tRNAs are processed by a 3'-endonuclease termed tRNase Z (also termed RNase Z or 3'-tRNase; for reviews, see [4–7]) that cleaves immediately after the N73 discriminator nucleotide (the first unpaired base after the acceptor stem) to allow subsequent addition of the CCA sequence.
Unlike prokaryotic pre-tRNAs, eukaryotic nuclear and organellar pre-tRNAs generally lack the 3'-CCA sequence (which is added post-transcriptionally) and their 3'-trailer sequences are removed by tRNase Z. Also unlike prokaryotic pre-tRNAs, eukaryotic nuclear pre-tRNAs contain oligo (U) at their 3'-ends, which are recognized and bound by the La protein (for reviews see [2, 8]). In the budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe, the endonucleolytic cleavage of nuclear pre-tRNAs requires the presence of the yeast La protein [9, 10]. In the absence of the yeast La protein, the 3'-trailer sequence of nuclear pre-tRNAs is trimmed by 3'-exoribonucleases including Rex1p . However, organellar pre-tRNAs lack terminal oligo (U). Furthermore, unlike nuclear pre-tRNAs which are typically monocistronic, most organellar pre-tRNAs are polycistronic [12, 13].
tRNase Z is present in all kingdoms of life. It exists in two forms: tRNase ZS [300-400 amino acids (aa)] and tRNase ZL (700-800 aa), which are encoded by different genes. It is believed that the tRNase ZL gene has evolved from a tandem duplication of the tRNase ZS gene, followed by divergence of the sequence . In prokaryotes, only tRNase ZS is identified. By contrast, all eukaryotes possess tRNase ZL, and some have both forms.
The species distribution of tRNase Z is complex. The majority of eukaryotic species analyzed to date, including S. cerevisiae, the fruit fly Drosophila melanogaster and the nematode worm Caenorhabditis elegans contain a single tRNase ZL [15–17]. In contrast, S. pombe have two tRNase ZLs [18, 19]. Interestingly, two tRNase ZLs and two tRNase ZSs have been experimentally identified in the flowering plant Arabidopsis thaliana . In humans, one tRNase ZS (also termed ELAC1) and one tRNase ZL (also termed ELAC2) are found . Our BLAST searches against public genomic and expressed sequence tag (EST) databases reveal that with few exceptions, vertebrates contain one tRNase ZL and one tRNase ZS (a detailed description of tRNase Z protein distribution in the animal kingdom will be provided elsewhere).
tRNase Z belongs to the metallo-β-lactamase (MBL) superfamily [14, 21–24]. The typical MBL domain contains five conserved sequence motifs termed Motifs I-V. Motifs I and IV each harbor an invariant Asp, Motif II (HxHxDH), which is also called the His motif, is the signature motif of the superfamily, whereas Motifs III and V each contain a conserved His residue. Structural studies of tRNase ZSs from E. coli, T. maritima and B. subtilis [25–28] and mutation analyses of tRNase Zs from a variety of species [29–35] reveal that the His and Asp residues of Motifs II-V form the active site for coordination of two catalytic zinc ions. In particular, the Asp residue of Motif II may participate in both zinc ion coordination and act as a general base to generate a hydroxide ion for nucleophilic attack on the scissile phosphodiester bond at the cleavage site [25, 29]. The Asp residue of Motif I is also catalytically important and appears to stabilize the catalytic site .
Besides tRNase Zs, some nucleic acid processing enzymes are also members of the MBL superfamily. Most of these proteins belong to the β-CASP (MBL-associated CISF Artemis SNM1/PSO2) subfamily of the MBL . This subfamily includes the 73-kD subunit of the cleavage and polyadenylation specificity factor (CPSF-73) and its yeast homolog Ysh1p, which are involved in endonucleolytic cleavage of pre-mRNA, the Intergrator complex subunit 11 (Int11) involved in the 3'-end formation of small nuclear RNAs (snRNA) , bacterial RNase J, which participates in rRNA 5'-end maturation  and RNA decay , and the eukaryotic Pso/Snm1/Artemis proteins, which function in DNA repair and V(D)J recombination . However, unlike tRNase Zs, β-CASP proteins contain conserved β-CASP sequence motifs in place of Motif V.
tRNase Z is distinguished from other MBL members by their unique substrate binding domain termed the flexible arm (also termed the exosite). Based on flexible arm type, there are two major types (bacterial- and eukaryotic-types) and one minor type [T. maritima (TM)-type] of tRNase Zs . The bacterial-type tRNase Zs, which are present predominantly in bacteria, possess the bacterial-type flexible arm. The bacterial-type flexible arm is ~55 aa in length and contains the Gly- and Pro-rich GP motif (GxPxGP, sometimes GxPPGP) . The eukaryotic-type tRNase Zs, which are found only in eukaryotes, contain the ELAC2-type flexible arm. This type of flexible arm harbors the GP motif and is ~62 aa long, which is slightly longer than the bacterial-type flexible arm.
The TM-type tRNase Z was believed to be the minor type at the time of discovery since it was found only in T. maritima and A. thaliana . The flexible arm found in TM-type tRNase Zs appears to be shorter (~30 aa) and lacks the GP motif but instead contains one short basic residue-rich region . In addition, both the bacterial- and eukaryotic-type tRNase Zs contain the PxKxRN, HEAT and HST motifs, which form part of loop structures, whereas the TM-type tRNase Z lacks these motifs [33, 40, 41]. The PxKxRN motif has been suggested to function in CCA anti-determination (tRNase Z activity is inhibited by 3'-CCA) [25, 33], whereas the HEAT and HST motifs have been suggested to play a role in facilitating proton transfer at the final stage of reaction [25, 29, 40].
tRNase Z has diverse functions besides its primary role in tRNA 3'-end processing. This is perhaps best exemplified by ELAC2, which serves a multitude of functions within cells. Recent studies have shown that ELAC2 is involved in the generation of MALAT1, a cancer-associated long noncoding RNA which participates in regulation of pre-mRNA splicing , tRNA-derived small RNAs [43, 44], and viral microRNAs (miRNAs) [45, 46]. Overexpression of ELAC2 delays cell cycle progression, suggesting that ELAC2 may be involved in cell cycle control either directly or indirectly via its role as tRNA processing enzyme . ELAC2 also potentiates TGF-β(transforming growth factor-β/Smad-induced transcription response, indicating a role for ELAC2 in TGF-β/Smad signaling mediated growth arrest . Interestingly, a recent study has shown that destruction of human mitochondria through depletion of mitochondrial DNA results in down-regulation of ELAC2 and a delay in cell cycle progression . Since ELAC2 may be involved in cell cycle regulation, it is likely that ELAC2 may link mitochondrial function and cell cycle control. It is important to note that ELAC2 is a candidate prostate cancer susceptibility gene as its mutations are associated with prostate cancer . However, the underlying mechanisms are unknown. In S. cerevisiae, either inactivating mutations or overexpression of tRNase ZL causes a petite phenotype, suggesting that the action of tRNase ZL may be related to mitochondrial function . In addition, the S. cerevisiae tRNase ZL has also been suggested to play a role in 35S rRNA processing .
The study of tRNase Z evolution has been facilitated by the increasing availability of genome sequences. A previous study showed that only tRNase ZS is found in bacteria and that its presence in bacteria is widespread . We recently reported on a systematic survey of tRNase Zs in fungi . Our analysis reveals that while the majority of fungal species contain one tRNase ZL, all four sequenced Schizosaccharomyces species contain two distinct tRNase ZLs either demonstrated or predicted to be localized to the nucleus and mitochondria, respectively. In addition, the presence of tRNase ZS in fungi is restricted to the phylum Basidiomycota and the basal fungal phyla.
Green plants (Viridiplantae) represent a monophyletic group of land plants and green algae that evolved near the base of the tree of eukaryotic life. Flowering plants (angiosperms), which are typically polyploidy, represent the largest, most diverse and most evolutionary advanced phylum of land plants making up 90% of the plant kingdom. It can be divided into two major groups: dicotyledons (dicots), which accounts for the majority of the angiosperm species, and monocotyledons (moncots). At present, there are at least 27 sequenced and annotated genomes representing the major taxonomic groups within green plants, although the majority of them are those of flowering plants. The public availability of these genome sequences enabled us to identify tRNase Zs in green plants and to study their evolution.
In this study, we undertook a comprehensive survey of candidate tRNase Zs from annotated green plant genomes. To understand the evolutionary relationships among green plant tRNase Zs, we further conducted a phylogenetic analysis of these newly identified candidates. Finally, we presented a detailed sequence analysis of tRNase Zs with the intent of further delineating the distinct features of green plant tRNase Zs.