Arabidopsis Encodes Four tRNase Z Enzymes

Functional transfer RNA (tRNA) molecules are a prerequisite for protein biosynthesis. Several processing steps are required to generate the mature functional tRNA from precursor molecules. Two of the early processing steps involve cleavage at the tRNA 5′ end and the tRNA 3′ end. While processing at the tRNA 5′ end is performed by RNase P, cleavage at the 3′ end is catalyzed by the endonuclease tRNase Z. In eukaryotes, tRNase Z enzymes are found in two versions: a short form of about 250 to 300 amino acids and a long form of about 700 to 900 amino acids. All eukaryotic genomes analyzed to date encode at least one long tRNase Z protein. Of those, Arabidopsis (Arabidopsis thaliana) is the only organism that encodes four tRNase Z proteins, two short forms and two long forms. We show here that the four proteins are distributed to different subcellular compartments in the plant cell: the nucleus, the cytoplasm, the mitochondrion, and the chloroplast. One tRNase Z is present only in the cytoplasm, one protein is found exclusively in mitochondria, while the third one has dual locations: nucleus and mitochondria. None of these three tRNase Z proteins is essential. The fourth tRNase Z protein is present in chloroplasts, and deletion of its gene results in an embryo-lethal phenotype. In vitro analysis with the recombinant proteins showed that all four tRNase Z enzymes have tRNA 3′ processing activity. In addition, the mitochondrial tRNase Z proteins cleave tRNA-like elements that serve as processing signals in mitochondrial mRNA maturation.     

Unstructured RNA is a substrate for tRNase Z

tRNase Z, which exists in almost all cells, is believed to be working primarily for tRNA 3' maturation. In Escherichia coli, however, the tRNase Z gene appears to be dispensable under normal growth conditions, and its physiological role is not clear. Here, to investigate a possibility that E. coli tRNase Z cleaves RNAs other than pre-tRNAs, we tested several unstructured RNAs for cleavage. Surprisingly, all these substrates were cleaved very efficiently at multiple sites by a recombinant E. coli enzyme in vitro. tRNase Zs from Bacillus subtilis and Thermotoga maritima also cleaved various unstructured RNAs. The E. coli and B. subtilis enzymes seem to have a tendency to cleave after cytidine or before uridine, while cleavage by the T. maritima enzyme inevitably occurred after CCA in addition to the other cleavages. Assays to determine optimal conditions indicated that metal ion requirements differ between B. subtilis and T. maritima tRNase Zs. There was no significant difference in the observed rate constant between unstructured RNA and pre-tRNA substrates, while the K(d) value of a tRNase Z/unstructured RNA complex was much higher than that of an enzyme/pre-tRNA complex. Furthermore, eukaryotic tRNase Zs from yeast, pig, and human cleaved unstructured RNA at multiple sites, but an archaeal tRNase Z from Pyrobaculum aerophilum did not.     

Two archaeal tRNase Z enzymes: similar but different

The endoribonuclease tRNase Z plays an essential role in tRNA metabolism by removal of the 3' trailer element of precursor RNAs. To investigate tRNA processing in archaea, we identified and expressed the tRNase Z from Haloferax volcanii, a halophilic archaeon. The recombinant enzyme is a homodimer and efficiently processes precursor tRNAs. Although the protein is active in vivo at 2-4 M KCl, it is inhibited by high KCl concentrations in vitro, whereas 2-3 M (NH(4))(2)SO(4) do not inhibit tRNA processing. Analysis of the metal content of the metal depleted tRNase Z revealed that it still contains 0.4 Zn(2+) ions per dimer. In addition tRNase Z requires Mn(2+) ions for processing activity. We compared the halophilic tRNase Z to the homologous one from Pyrococcus furiosus, a thermophilic archaeon. Although both enzymes have 46% sequence similarity, they differ in their optimal reaction conditions. Both archaeal tRNase Z proteins process mitochondrial pre-tRNAs. Only the thermophilic tRNase Z shows in addition activity toward intron containing pre-tRNAs, 5' extended precursors, the phosphodiester bis(p-nitrophenyl)phosphate (bpNPP) and the glyoxalase II substrate S-D: -lactoylglutathion (SLG).     

Metal requirements and phosphodiesterase activity of tRNase Z enzymes

The endonuclease tRNase Z from A. thaliana (AthTRZ1) was originally isolated for its tRNA 3' processing activity. Here we show that AthTRZ1 also hydrolyzes the phosphodiester bond in bis(p-nitrophenyl) phosphate (bpNPP) with a kcat of 7.4 s-1 and a KM of 8.5 mM. We analyzed 22 variants of AthTRZ1 with respect to their ability to hydrolyze bpNPP. This mutational mapping identified fourteen variants that lost the ability to hydrolyze bpNPP and seven variants with reduced activity. Surprisingly, a single amino acid change (R252G) resulted in a ten times higher activity compared to the wild type enzyme. tRNase Z enzymes exist in long and short forms. We show here that in contrast to the short tRNase Z enzyme AthTRZ1, the long tRNase Z enzymes do not have bpNPP hydrolysis activity pointing to fundamental differences in substrate cleavage between the two enzyme forms. Furthermore, we determined the metal content of AthTRZ1 and analyzed the metal requirement for bpNPP hydrolysis. AthTRZ1 shows a high affinity for Zn2+ ions; even upon incubation with metal chelators, 0.76 Zn2+ ions are retained per dimer. In contrast to bpNPP hydrolysis, pre-tRNA processing requires additional metal ions, Mn2+ or Mg2+, as Zn2+ ions alone are insufficient.     

Tethered Domains and Flexible Regions in tRNase ZL,the Long Form of tRNase Z

tRNase Z, a member of the metallo-β-lactamase family, endonucleolytically removes the pre-tRNA 3′ trailer in a step central to tRNA maturation. The short form (tRNase Z^S) is the only one found in bacteria and archaebacteria and is also present in some eukaryotes. The homologous long form (tRNase Z^L), exclusively found in eukaryotes, consists of related amino- and carboxy-domains, suggesting that tRNase Z^L arose from a tandem duplication of tRNase Z^S followed by interdependent divergence of the domains. X-ray crystallographic structures of tRNase Z^S reveal a flexible arm (FA) extruded from the body of tRNase Z remote from the active site that binds tRNA far from the scissile bond. No tRNase Z^L structures have been solved; alternative biophysical studies are therefore needed to illuminate its functional characteristics. Structural analyses of tRNase Z^L performed by limited proteolysis, two dimensional gel electrophoresis and mass spectrometry establish stability of the amino and carboxy domains and flexibility of the FA and inter-domain tether, with implications for tRNase Z^L function.     

Human cytosolic tRNase Z can downregulate gene expression through miRNA

A long form of tRNase Z (tRNase Z^L) can cleave any target RNA at any desired site under the direction of artificial small guide RNA including ∼25-nucleotide hook-shaped RNA. Here we show that human miR-103 can form a hook structure to guide target RNA cleavage by human cytosolic tRNase Z^L in vitro. In vivo analyses using luciferase mRNAs modified to contain miR-103 target sequences in their 3′ untranslated regions indicated that miR-103 downregulates gene expression through directing mRNA cleavage by tRNase Z^L. The present data suggest the possibility that human cytosolic tRNase Z^L modulates gene expression through a subset of microRNAs in the cells.     

tRNase Z catalysis and conserved residues on the carboxy side of the His cluster

tRNAs are transcribed as precursors and processed in a series of required reactions leading to aminoacylation and translation. The 3'-end trailer can be removed by the pre-tRNA processing endonuclease tRNase Z, an ancient, conserved member of the beta-lactamase superfamily of metal-dependent hydrolases. The signature sequence of this family, the His domain (HxHxDH, Motif II), and histidines in Motifs III and V and aspartate in Motif IV contribute seven side chains for the coordination of two divalent metal ions. We previously investigated the effects on catalysis of substitutions in Motif II and in the PxKxRN loop and Motif I on the amino side of Motif II. Herein, we present the effects of substitutions on the carboxy side of Motif II within Motifs III, IV, the HEAT and HST loops, and Motif V. Substitution of the Motif IV aspartate reduces catalytic efficiency more than 10,000-fold. Histidines in Motif III, V, and the HST loop are also functionally important. Strikingly, replacement of Glu in the HEAT loop with Ala reduces efficiency by approximately 1000-fold. Proximity and orientation of this Glu side chain relative to His in the HST loop and the importance of both residues for catalysis suggest that they function as a duo in proton transfer at the final stage of reaction, characteristic of the tRNase Z class of RNA endonucleases.     

Crystal structure of the tRNA 3' processing endoribonuclease tRNase Z from Thermotoga maritima

The maturation of the tRNA 3' end is catalyzed by a tRNA 3' processing endoribonuclease named tRNase Z (RNase Z or 3'-tRNase) in eukaryotes, Archaea, and some bacteria. The tRNase Z generally cuts the 3' extra sequence from the precursor tRNA after the discriminator nucleotide. In contrast, Thermotoga maritima tRNase Z cleaves the precursor tRNA precisely after the CCA sequence. In this study, we determined the crystal structure of T. maritima tRNase Z at 2.6-A resolution. The tRNase Z has a four-layer alphabeta/betaalpha sandwich fold, which is classified as a metallo-beta-lactamase fold, and forms a dimer. The active site is located at one edge of the beta-sandwich and is composed of conserved motifs. Based on the structure, we constructed a docking model with the tRNAs that suggests how tRNase Z may recognize the substrate tRNAs.     

The tRNase Z family of proteins: physiological functions, substrate specificity and structural properties

tRNase Z is the endoribonuclease that generates the mature 3'-end of tRNA molecules by removal of the 3'-trailer elements of precursor tRNAs. This enzyme has been characterized from representatives of all three domains of life (Bacteria, Archaea and Eukarya), as well as from mitochondria and chloroplasts. tRNase Z enzymes come in two forms: short versions (280-360 amino acids in length), present in all three kingdoms, and long versions (750-930 amino acids), present only in eukaryotes. The recently solved crystal structure of the bacterial tRNase Z provides the structural basis for the understanding of central functional elements. The substrate is recognized by an exosite that protrudes from the main protein body and consists of a metallo-beta-lactamase domain. Cleavage of the precursor tRNA occurs at the binuclear zinc site located in the other subunit of the functional homodimer. The first gene of the tRNase Z family was cloned in 2002. Since then a comprehensive set of data has been acquired concerning this new enzyme, including detailed functional studies on purified recombinant enzymes, mutagenesis studies and finally the determination of the crystal structure of three bacterial enzymes. This review summarizes the current knowledge about these exciting enzymes.     

The tRNA 30-end Processing Enzyme tRNase Z2 Contributes to Chloroplast Biogenesis in Rice

tRNase Z (TRZ) is a ubiquitous endonuclease that removes the 30-trailer from precursor tRNAs during maturation. In yeast and animals, TRZ regulates the cell cycle via its (t)RNA processing activity;how...     

Analysis of the functional modules of the tRNA 3' endonuclease (tRNase Z)

tRNA 3' processing is one of the essential steps during tRNA maturation. The tRNA 3'-processing endonuclease tRNase Z was only recently isolated, and its functional domains have not been identified so far. We performed an extensive mutational study to identify amino acids and regions involved in dimerization, tRNA binding, and catalytic activity. 29 deletion and point variants of the tRNase Z enzyme were generated. According to the results obtained, variants can be sorted into five different classes. The first class still had wild type activity in all three respects. Members of the second and third class still formed dimers and bound tRNAs but had reduced catalytic activity (class two) or no catalytic activity (class three). The fourth class still formed dimers but did not bind the tRNA and did not process precursors. Since this class still formed dimers, it seems that the amino acids mutated in these variants are important for RNA binding. The fifth class did not have any activity anymore. Several conserved amino acids could be mutated without or with little loss of activity.     

The N-terminal half-domain of the long form of tRNase Z is required for the RNase 65 activity

Transfer RNA (tRNA) 3′ processing endoribonuclease (tRNase Z) is an enzyme responsible for the removal of a 3′ trailer from pre-tRNA. There exists two types of tRNase Z: one is a short form (tRNase ZS) that consists of 300–400 amino acids, and the other is a long form (tRNase ZL) that contains 800–900 amino acids. Here we investigated whether the short and long forms have different preferences for various RNA substrates. We examined three recombinant tRNase ZSs from human, Escherichia coli and Thermotoga maritima, two recombinant tRNase ZLs from human and Saccharomyces cerevisiae, one tRNase ZL from pig liver, and the N- and C-terminal half regions of human tRNase ZL for cleavage of human micro-pre-tRNA^Arg and the RNase 65 activity. All tRNase ZLs cleaved the micro-pre-tRNA and showed the RNase 65 activity, while all tRNase ZSs and both half regions of human tRNase ZL failed to do so with the exception of the C-terminal half, which barely cleaved the micro-pre-tRNA. We also show that only the long forms of tRNase Z can specifically cleave a target RNA under the direction of a new type of small guide RNA, hook RNA. These results indicate that indeed tRNase ZL and tRNase ZS have different substrate specificities and that the differences are attributed to the N-terminal half-domain of tRNase ZL. Furthermore, the optimal concentrations of NaCl, MgCl₂ and MnCl₂ differed between tRNase ZSs and tRNase ZLs, and the K_m values implied that tRNase ZLs interact with pre-tRNA substrates more strongly than tRNase ZSs.     

The flexible arm of tRNase Z is not essential for pre-tRNA binding but affects cleavage site selection

tRNase Z is an enzyme responsible for removing a 3′ trailer from pre-tRNA. Although most tRNase Zs cleave pre-tRNAs immediately after the discriminator nucleotide with the exception of Thermotoga maritima tRNase Z, which cleaves after the ₇₄CCA₇₆ sequence, our knowledge was limited about how the cleavage site in pre-tRNA is selected. Bacterial tRNase Zs contain a unique domain termed flexible arm, which extends from the core domain. Using various tRNase Z variants, here we examined how the flexible arm affects the cleavage site selection. T. maritima tRNase Z variants with modified flexible arms shifted the cleavage site and a Bacillus subtilis tRNase Z variant with no flexible arm showed an anomalous cleavage activity. Some of the T. maritima/B. subtilis chimeric enzymes had both properties: they recognized ₇₄CCA₇₆-containing pre-tRNA and cleaved it after the discriminator. Taken together, the present data indicate that the flexible arm is not essential for pre-tRNA binding but affects the cleavage site selection probably by pushing the distal region of the T arm in such a way that the discriminator nucleotide becomes closer to the catalytic site.     

Identification by Mn2+ rescue of two residues essential for the proton transfer of tRNase Z catalysis

Thermotoga maritima tRNase Z cleaves pre-tRNAs containing the ₇₄CCA₇₆ sequence precisely after the A₇₆ residue to create the mature 3′ termini. Its crystal structure has revealed a four-layer αβ/βα sandwich fold that is typically found in the metallo-β-lactamase superfamily. The well-conserved six histidine and two aspartate residues together with metal ions are assumed to form the tRNase Z catalytic center. Here, we examined tRNase Z variants containing single amino acid substitutions in the catalytic center for pre-tRNA cleavage. Cleavage by each variant in the presence of Mg²⁺ was hardly detected, although it is bound to pre-tRNA. Surprisingly, however, Mn²⁺ ions restored the lost Mg²⁺-dependent activity with two exceptions of the Asp52Ala and His222Ala substitutions, which abolished the activity almost completely. These results provide a piece of evidence that Asp-52 and His-222 directly contribute the proton transfer for the catalysis.     

细菌和人短型tRNase Z~S与粟酒裂殖酵母长型tRNase Z~L功能保守性研究

tRNase Z是一种参与tRNA前体3'末端加工的核酸内切酶,根据氨基酸残基数目分为长型(tRNase ZL)和短型(tRNase ZS)两类.细菌只有短型,而真核生物既可以有短型也可以有长型,并且绝大部分真核生物只有一个长型.为了研究短型tRNase Z和长型tRNase Z的功能保守性,分别构建表达编码枯草芽孢杆菌(Bacillus subtilis)短型tRNase Z(BsuTrz1)、人短型tRNase Z(ELAC1)以及海栖热袍菌(Thermotoga maritima)短型tRNase Z(TmaTrz1)的酵母低表达载体,并转化tRNase ZL温敏型粟酒裂殖酵母(Schizosaccharomyces pombe)进行互补救活实验.结果显示,与粟酒裂殖酵母tRNase ZL(SpTrz1)相比,ELAC1能在37℃完全救活酵母tRNase ZL温敏型菌株,而BsuTrz1的救活能力略弱.但具有不同底物特异性的TmaTrz1则完全不能救活.此外,催化活性受损的ELAC1组氨酸模体突变体(ELAC1-H62A和ELAC1-H64A)救活酵母tRNase ZL温敏型菌株的能力极大受损.这些结果表明,枯草芽孢杆菌和人的tRNase ZS能替代粟酒裂殖酵母的tRNase ZL,并且这些蛋白具有功能保守性.这些实验进一步支持了长型tRNase Z基因是由短型tRNase Z基因复制后序列进化形成的观点.     

Substrate recognition ability differs among various prokaryotic tRNase Zs

There exists a significant difference in pre-tRNA preference among prokaryotic tRNase Zs. This is an enigma, because pre-tRNAs should form the common L-shaped structure and tRNase Zs should form the common structure based on the alphabeta/betaalpha-fold. To address this issue, we examined six different eubacterial and archaeal tRNase Zs including two newly isolated tRNase Zs for cleavage of 18 different pre-tRNA substrates. Two Thermotoga maritima, one Thermus thermophilus, one Bacillus subtilis, one Thermoplasma acidophilum, and one Pyrobaculum aerophilum enzymes were tested. To our surprise, the newly isolated proteins T. maritima and T. thermophilus showed the weak tRNase Z activity, even though their primary amino acid sequences are, on the whole, quite different from those of the typical tRNase Zs. We confirmed that substrate recognition ability is quite different among those tRNase Zs. In addition, we found that the optimal conditions as a whole differ significantly among the enzymes. From these results, we provided several clues to solve the enigma by showing the potential importance of the 74th-76th nucleotide sequence of pre-tRNA, the flexible arm length of tRNase Z, the divalent metal ion species, and the histidine corresponding His222 in T. maritima tRNase Z.     

RloC: a wobble nucleotide-excising and zinc-responsive bacterial tRNase

The conserved bacterial protein RloC, a distant homologue of the tRNA^Lys anticodon nuclease (ACNase) PrrC, is shown here to act as a wobble nucleotide-excising and Zn⁺⁺-responsive tRNase. The more familiar PrrC is silenced by a genetically linked type I DNA restriction-modification (R-M) enzyme, activated by a phage anti-DNA restriction factor and counteracted by phage tRNA repair enzymes. RloC shares PrrC's ABC ATPase motifs and catalytic ACNase triad but features a distinct zinc-hook/coiled-coil insert that renders its ATPase domain similar to Rad50 and related DNA repair proteins. Geobacillus kaustophilus RloC expressed in Escherichia coli exhibited ACNase activity that differed from PrrC's in substrate preference and ability to excise the wobble nucleotide. The latter specificity could impede reversal by phage tRNA repair enzymes and account perhaps for RloC's more frequent occurrence. Mutagenesis and functional assays confirmed RloC's catalytic triad assignment and implicated its zinc hook in regulating the ACNase function. Unlike PrrC, RloC is rarely linked to a type I R-M system but other genomic attributes suggest their possible interaction in trans . As DNA damage alleviates type I DNA restriction, we further propose that these related perturbations prompt RloC to disable translation and thus ward off phage escaping DNA restriction during the recovery from DNA damage.     

Functional analyses for tRNase Z variants: an aspartate and a histidine in the active site are essential for the catalytic activity

We performed functional analyses for various single amino-acid substitution variants of Escherichia coli, Bacillus subtilis, and human tRNase Zs. The well-conserved six histidine, His(I)-His(VI), and two aspartate, Asp(I) and Asp(II), residues together with metal ions are thought to form the active site of tRNase Z. The Mn(2+)-rescue analysis for Thermotoga maritima tRNase Z(S) has suggested that Asp(I) and His(V) directly contribute the proton transfer for the catalysis, and a catalytic mechanism has been proposed. However, experimental evidence supporting the proposed mechanism was limited. Here we intensively examined E. coli and B. subtilis tRNase Z(S) variants and human tRNase Z(L) variants for cleavage activities on pre-tRNAs in the presence of Mg(2+) or Mn(2+) ions. We observed that the Mn(2+) ions cannot rescue the activities of Asp(I)Ala and His(V)Ala variants from each species, which are lost in the presence of Mg(2+). This observation may support the proposed catalytic mechanism.