Methionyl-tRNA synthetase from Caenorhabditis elegans: a specific multidomain organization for convergent functional evolution

Methionyl-tRNA synthetase (MetRS) is a multidomain protein that specifically binds tRNAMet and catalyzes the synthesis of methionyl-tRNAMet. The minimal, core enzyme found in Aquifex aeolicus is made of a catalytic domain, which catalyzes the aminoacylation reaction, and an anticodon-binding domain, which promotes tRNA-protein association. In eukaryotes, additional domains are appended in cis or in trans to the core enzyme and increase the stability of the tRNA-protein complexes. Eventually, as observed for MetRS from Homo sapiens, the C-terminal appended domain causes a slow release of aminoacyl-tRNA and establishes a limiting step in the global aminoacylation reaction. Here, we report that MetRS from the nematode Caenorhabditis elegans displays a new type of structural organization. Its very C-terminal appended domain is related to the oligonucleotide binding-fold-based tRNA-binding domain (tRBD) recovered at the C-terminus of MetRS from plant, but, in the nematode enzyme, this domain is separated from the core enzyme by an insertion domain. Gel retardation and tRNA aminoacylation experiments show that MetRS from nematode is functionally related to human MetRS despite the fact that their appended tRBDs have distinct structural folds, and are not orthologs. Thus, functional convergence of human and nematode MetRS is the result of parallel and convergent evolution that might have been triggered by the selective pressure to invent processivity of tRNA handling in translation in higher eukaryotes.     

Related domains in yeast tRNA ligase, bacteriophage T4 polynucleotide kinase and RNA ligase, and mammalian myelin 2',3'-cyclic nucleotide phosphohydrolase revealed by amino acid sequence comparison

Related domains containing the purine NTP-binding sequence pattern have been revealed in two enzymes involved in tRNA processing, yeast tRNA ligase and phage T4 polynucleotide kinase, and in one of the major proteins of mammalian nerve myelin sheath, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase). It is suggested that, similarly to the tRNA processing enzymes, CNPase possesses polynucleotide kinase activity, in addition to the phosphohydrolase one. It is speculated that CNPase may be an authentic mammalian polynucleotide kinase recruited as a structural component of the myelin sheath, analogously to the eye lens crystallins. Significant sequence similarity was revealed also between the N-terminal regions of yeast tRNA ligase and phage T4 RNA ligase. A tentative scheme of the domainal organizations for the three complex enzymes is proposed. According to this model, tRNA ligase contains at least three functional domains, in the order: N-ligase-kinase-phosphohydrolase-C, whereas polynucleotide kinase and CNPase encompass only the two C-terminal domains in the same order.     

Identification of a protein component of a mammalian tRNA(Sec) complex implicated in the decoding of UGA as selenocysteine

This report describes a novel RNA-binding protein, SECp43, that associates specifically with mammalian selenocysteine tRNA (tRNA(Sec)). SECp43, identified from a degenerate PCR screen, is a highly conserved protein with two ribonucleoprotein-binding domains and a polar/acidic carboxy terminus. The protein and corresponding mRNA are generally expressed in rat tissues and mammalian cell lines. To gain insight into the biological role of SECp43, affinity-purified antibody was employed to identify its molecular partners. Surprisingly, the application of native HeLa cell extracts to a SECp43 antibody column results in the purification of a 90-nt RNA species identified by direct sequencing and Northern blot analysis as tRNA(Sec). The purification of tRNA(Sec) by the antibody column is striking, based on the low abundance of this tRNA species. Using recombinant SECp43 as a probe for interacting protein partners, we also identify a 48-kDa interacting protein, which is a possible component of the mammalian selenocysteine insertion (SECIS) pathway. To our knowledge, SECp43 is the first cloned protein demonstrated to associate specifically with eukaryotic tRNA(Sec).     

Mamit-tRNA, a database of mammalian mitochondrial tRNA primary and secondary structures

Mamit-tRNA (http://mamit-tRNA.u-strasbg.fr), a database for mammalian mitochondrial genomes, has been developed for deciphering structural features of mammalian mitochondrial tRNAs and as a helpful tool in the frame of human diseases linked to point mutations in mitochondrial tRNA genes. To accommodate the rapid growing availability of fully sequenced mammalian mitochondrial genomes, Mamit-tRNA has implemented a relational database, and all annotated tRNA genes have been curated and aligned manually. System administrative tools have been integrated to improve efficiency and to allow real-time update (from GenBank Database at NCBI) of available mammalian mitochondrial genomes. More than 3000 tRNA gene sequences from 150 organisms are classified into 22 families according to the amino acid specificity as defined by the anticodon triplets and organized according to phylogeny. Each sequence is displayed linearly with color codes indicating secondary structural domains and can be converted into a printable two-dimensional (2D) cloverleaf structure. Consensus and typical 2D structures can be extracted for any combination of primary sequences within a given tRNA specificity on the basis of phylogenetic relationships or on the basis of structural peculiarities. Mamit-tRNA further displays static individual 2D structures of human mitochondrial tRNA genes with location of polymorphisms and pathology-related point mutations. The site offers also a table allowing for an easy conversion of human mitochondrial genome nucleotide numbering into conventional tRNA numbering. The database is expected to facilitate exploration of structure/function relationships of mitochondrial tRNAs and to assist clinicians in the frame of pathology-related mutation assignments.     

两种具有调节血管生成作用的氨基酰-tRNA合成酶

氨基酰-tRNA合成酶是生物体内蛋白质合成过程中的一类关键酶,它催化体内tRNA的氨基酰化反应.作为一类古老的蛋白质,氨基酰-tRNA合成酶在其漫长的进化过程中,通过其他结构域的插入或融合逐渐演化出许多新的功能.最近的研究结果表明,人酪氨酰-tRNA合成酶的片段具有促进血管生成的功能,而人色氨酰-tRNA合成酶的片段则具有抑制血管生长的功能.在哺乳动物细胞中,蛋白质的生物合成途径与细胞信号转导途径紧密相连.今后,随着对氨基酰-tRNA合成酶研究的不断深入,可以通过它们与细胞因子和信号转导相连的功能治疗人类的疾病.     

Search for characteristic structural features of mammalian mitochondrial tRNAs

A number of mitochondrial (mt) tRNAs have strong structural deviations from the classical tRNA cloverleaf secondary structure and from the conventional L-shaped tertiary structure. As a consequence, there is a general trend to consider all mitochondrial tRNAs as "bizarre" tRNAs. Here, a large sequence comparison of the 22 tRNA genes within 31 fully sequenced mammalian mt genomes has been performed to define the structural characteristics of this specific group of tRNAs. Vertical alignments define the degree of conservation/variability of primary sequences and secondary structures and search for potential tertiary interactions within each of the 22 families. Further horizontal alignments ascertain that, with the exception of serine-specific tRNAs, mammalian mt tRNAs do fold into cloverleaf structures with mostly classical features. However, deviations exist and concern large variations in size of the D- and T-loops. The predominant absence of the conserved nucleotides G18G19 and T54T55C56, respectively in these loops, suggests that classical tertiary interactions between both domains do not take place. Classification of the tRNA sequences according to their genomic origin (G-rich or G-poor DNA strand) highlight specific features such as richness/poorness in mismatches or G-T pairs in stems and extremely low G-content or C-content in the D- and T-loops. The resulting 22 "typical" mammalian mitochondrial sequences built up a phylogenetic basis for experimental structural and functional investigations. Moreover, they are expected to help in the evaluation of the possible impacts of those point mutations detected in human mitochondrial tRNA genes and correlated with pathologies.     

Crucial Optimization of Translational Components towards Efficient Incorporation of Unnatural Amino Acids into Proteins in Mammalian Cells

The ability to site-specifically incorporate unnatural amino acids (UAAs) into proteins is a powerful tool in protein engineering. While dozens of UAAs have been successfully introduced into proteins expressed by Escherichia coli cells, it has been much more challenging to create tRNA and tRNA-Synthetase pairs that enable UAAs incorporation, for use in mammalian systems. By altering the orthogonality properties of existing unnatural pairs, previously evolved pairs for use in E. coli could be used in mammalian cells. This would bypass the cumbersome step of having to evolve mutant synthetases and would allow for the rapid development of new mammalian pairs. A major limitation to the amount of UAA-containing proteins that can be expressed in the cell is the availability of UAA-charged orthogonal suppressor tRNA. By using a natural mammalian tRNA promoter, the amount of functional suppressor tRNA can be greatly increased. Furthermore, increasing recognition of the suppressor tRNA by the mutant synthetase will ultimately lead to the appearance of more UAA-charged tRNA.     

Function of yeast and amphioxus tRNA ligase in IRE1alpha-dependent XBP1 mRNA splicing

During their maturation step, transfer RNAs (tRNAs) undergo excision of their introns by specific splicing. Although tRNA splicing is a molecular event observed in all domains of life, the machinery of the ligation reaction has diverged during evolution. Yeast tRNA ligase 1 (TRL1) is a multifunctional protein that alone catalyzes RNA ligation in tRNA splicing, whereas three molecules [RNA ligase (RNL), Clp1, and PNK/CPDase] are necessary for RNA ligation in tRNA splicing in amphioxi. RNA ligation not only occurs in tRNA splicing, but also in yeast HAC1 mRNA splicing and in animal X-box binding protein 1 (XBP1) mRNA splicing under conditions of endoplasmic reticulum (ER) stress. Yeast TRL1 is known to function as an RNA ligase for HAC1 mRNA splicing, whereas the RNA ligase for XBP1 mRNA splicing is unknown in animals. We examined whether yeast and amphioxus RNA ligases for tRNA splicing function in RNA ligation in mammalian XBP1 splicing. Both RNA ligases functioned in RNA ligation in mammalian XBP1 splicing in vitro. Interestingly, Clp1, and PNK/CPDase were not necessary for exon–exon ligation in XBP1 mRNA by amphioxus RNL. These results suggest that RNA ligase for tRNA splicing might therefore commonly function as an RNA ligase for XBP1 mRNA splicing.     

Adaptation of aminoacylation identity rules to mammalian mitochondria

Many mammalian mitochondrial aminoacyl-tRNA synthetases are of bacterial-type and share structural domains with homologous bacterial enzymes of the same specificity. Despite this high similarity, synthetases from bacteria are known for their inability to aminoacylate mitochondrial tRNAs, while mitochondrial enzymes do aminoacylate bacterial tRNAs. Here, the reasons for non-aminoacylation by a bacterial enzyme of a mitochondrial tRNA have been explored. A mutagenic analysis performed on in vitro transcribed human mitochondrial tRNA^Asp variants tested for their ability to become aspartylated by Escherichia coli aspartyl-tRNA synthetase, reveals that full conversion cannot be achieved on the basis of the currently established tRNA/synthetase recognition rules. Integration of the full set of aspartylation identity elements and stabilization of the structural tRNA scaffold by restoration of D- and T-loop interactions, enable only a partial gain in aspartylation efficiency. The sequence context and high structural instability of the mitochondrial tRNA are additional features hindering optimal adaptation of the tRNA to the bacterial enzyme. Our data support the hypothesis that non-aminoacylation of mitochondrial tRNAs by bacterial synthetases is linked to the large sequence and structural relaxation of the organelle encoded tRNAs, itself a consequence of the high rate of mitochondrial genome divergence.     

The selective reaction of methoxyamine with cytidine residues in mammalian initiator transfer ribonucleic acid

Methoxyamine reacts selectively with tRNA molecules at certain exposed cytosine residues usually located in non base-paired regions of the two dimensional clover leaf structure. Here methoxyamine is used for the first time in a study of a mammalian tRNA structure. One of the sequence abnormalities of myeloma initiator tRNA is a cytosine instead of the usual uracil immediately preceding the anticodon. A study of the reaction of the cytosine residues with methoxyamine indicates that the accessibility of bases to chemical reagents in the anticodon loop of this mammalian initiator tRNA is very similar to that observed for the bacterial initiator tRNA.     

A novel abundant family of retroposed elements (DAS-SINEs) in the nine-banded armadillo (Dasypus novemcinctus)

About half of the mammalian genome is composed of retroposons. Long interspersed elements (LINEs) and short interspersed elements (SINEs) are the most abundant repetitive elements and account for about 21% and 13% of the human genome, respectively. SINEs have been detected in all major mammalian lineages, except for the South American order Xenarthra, also termed Edentata (armadillos, anteaters, and sloths). Investigating this order, we discovered a novel high-copy-number family of tRNA derived SINEs in the nine-banded armadillo Dasypus novemcinctus, a species that successfully crossed the Central American land bridge to North America in the Pliocene. A specific computer algorithm was developed, and we detected and extracted 687 specific SINEs from databases. Termed DAS-SINEs, we further divided them into six distinct subfamilies. We extracted tRNA(Ala)-derived monomers, two types of dimers, and three subfamilies of chimeric fusion products of a tRNA(Ala) domain and an approximately 180-nt sequence of thus far unidentified origin. Comparisons of secondary structures of the DAS-SINEs' tRNA domains suggest selective pressure to maintain a tRNA-like D-arm structure in the respective founder RNAs, as shown by compensatory mutations. By analysis of subfamily-specific genetic variability, comparison of the proportion of direct repeats, and analysis of self-integrations as well as key events of dimerization and deletions or insertions, we were able to delineate the evolutionary history of the DAS-SINE subfamilies.     

Scyl1 Facilitates Nuclear tRNA Export in Mammalian Cells by Acting at the Nuclear Pore Complex

Scyl1 is an evolutionarily conserved N-terminal protein kinase-like domain protein that plays a role in COP1-mediated retrograde protein trafficking in mammalian cells. Furthermore, loss of Scyl1 function has been shown to result in neurodegenerative disorders in mice. Here, we report that Scyl1 is also a cytoplasmic component of the mammalian nuclear tRNA export machinery. Like exportin-t, overexpression of Scyl1 restored export of a nuclear export-defective serine amber suppressor tRNA mutant in COS-7 cells. Scyl1 binds tRNA saturably, and associates with the nuclear pore complex by interacting, in part, with Nup98. Scyl1 copurifies with the nuclear tRNA export receptors exportin-t and exportin-5, the RanGTPase, and the eukaryotic elongation factor eEF-1A, which transports aminoacyl-tRNAs to the ribosomes. Scyl1 interacts directly with exportin-t and RanGTP but not with eEF-1A or RanGDP in vitro. Moreover, exportin-t containing tRNA, Scyl1, and RanGTP form a quaternary complex in vitro. Biochemical characterization also suggests that the nuclear aminoacylation-dependent pathway is primarily responsible for tRNA export in mammalian cells. These findings together suggest that Scyl1 participates in the nuclear aminoacylation-dependent tRNA export pathway and may unload aminoacyl-tRNAs from the nuclear tRNA export receptor at the cytoplasmic side of the nuclear pore complex and channels them to eEF-1A.     

Determinants in tRNA for activation of arginyl-tRNA synthetase: evidence that tRNA flexibility is required for the induced-fit mechanism

Arginyl-tRNA synthetase (ArgRS) catalyzes formation of arginyl-adenylate in a tRNA-dependent reaction. Previous studies have revealed that conformational changes occur upon tRNA binding. In this study, we analyzed the sequence and structural features of tRNA that are essential to activate the catalytic center of mammalian arginyl-tRNA synthetase. Here, tRNA variants with different activator potential are presented. The three regions that are crucial for activation of ArgRS are the terminal adenosine, the D-loop, and the anticodon stem-loop of tRNA. The Add-1 N-terminal domain of ArgRS, which has the very unique property among aminoacyl-tRNA synthetases to interact with the D-loop in the corner of the convex side of tRNA, has an essential role in anchoring tRNA and participating in tRNA-induced amino acid activation. The results suggest that locking the acceptor extremity, the anticodon loop, and the D-loop of tRNA on the catalytic, anticodon-binding, and Add-1 domains of ArgRS also requires some flexibility of the tRNA molecule, provided by G:U base pairs, to achieve the productive conformation of the active site of the enzyme by induced fit.     

Kinetic quality control of anticodon recognition by a eukaryotic aminoacyl-tRNA synthetase

Aminoacyl-tRNA synthetases are an ancient class of enzymes responsible for the matching of amino acids with anticodon sequences of tRNAs. Eukaryotic tRNA synthetases are often larger than their bacterial counterparts, and several mammalian enzymes use the additional domains to facilitate assembly into a multi-synthetase complex. Human cysteinyl-tRNA synthetase (CysRS) does not associate with the multi-synthetase complex, yet contains a eukaryotic-specific C-terminal extension that follows the tRNA anticodon-binding domain. Here we show by mutational and kinetic analysis that the C-terminal extension of human CysRS is used to selectively improve recognition and binding of the anticodon sequence, such that the specificity of anticodon recognition by human CysRS is higher than that of its bacterial counterparts. However, the improved anticodon recognition is achieved at the expense of a significantly slower rate in the aminoacylation reaction, suggesting a previously unrecognized kinetic quality control mechanism. This kinetic quality control reflects an evolutionary adaptation of some tRNA synthetases to improve the anticodon specificity of tRNA aminoacylation from bacteria to humans, possibly to accommodate concomitant changes in codon usage.     

Identification of essential domains for Escherichia coli tRNA(leu) aminoacylation and amino acid editing using minimalist RNA molecules

Escherichia coli leucyl-tRNA synthetase (LeuRS) aminoacylates up to six different class II tRNA(leu) molecules. Each has a distinct anticodon and varied nucleotides in other regions of the tRNA. Attempts to construct a minihelix RNA that can be aminoacylated with leucine have been unsuccessful. Herein, we describe the smallest tRNA(leu) analog that has been aminoacylated to a significant extent to date. A series of tRNA(leu) analogs with various domains and combinations of domains deleted was constructed. The minimal RNA that was efficiently aminoacylated with LeuRS was one in which the anticodon stem-loop and variable arm stem-loop, but neither the D-arm nor T-arm, were deleted. Aminoacylation of this minimal RNA was abolished when the discriminator base A73 was replaced with C73 or when putative tertiary interactions between the D-loop and T-loop were disrupted, suggesting that these identity elements are still functioning in the minimized RNA. The various constructs that were significantly aminoacylated were also tested for amino acid editing by the synthetase. The anticodon and variable stem-loop domains were also dispensable for hydrolysis of the charged tRNA(leu) mimics. These results suggest that LeuRS may rely on identity elements in overlapping domains of the tRNA for both its aminoacylation and editing activities.     

A model for the tertiary structure of mammalian mitochondrial transfer RNAs lacking the entire 'dihydrouridine' loop and stem

The mammalian mitochondrial tRNA(AGY)Ser is unique in lacking the entire dihydrouridine arm. This reduces its secondary structure to a 'truncated cloverleaf'. Experimental evidence on the tertiary structure has been obtained by chemically probing the conformation of both the bovine and human species in their native conformation and at various stages of denaturation. A structural model of the bovine tRNA is presented based on the results of this chemical probing, on a comparison between nine homologous 'truncated cloverleaf' secondary structures and on analogies with the crystal structure of yeast phenylalanine tRNA. The proposed structure is very similar in shape to that of yeast tRNA(Phe) but is slightly smaller in size. It is defined by a unique set of tertiary interactions. Structural considerations suggest that other mammalian mitochondrial tRNAs have smaller dimensions as well.     

Solution structure of the ribosome recycling factor from Aquifex aeolicus

The solution structure of ribosome recycling factor (RRF) from hyperthermophilic bacterium, Aquifex aeolicus, was determined by heteronuclear multidimensional NMR spectroscopy. Fifteen structures were calculated using restraints derived from NOE, J-coupling, and T1/T2 anisotropies. The resulting structure has an overall L-shaped conformation with two domains and is similar to that of a tRNA molecule. The domain I (corresponding to the anticodon stem of tRNA) is a rigid three alpha-helix bundle. Being slightly different from usual coiled-coil arrangements, each helix of domain I is not twisted but straight and parallel to the main axis. The domain II (corresponding to the portion with the CCA end of tRNA) is an alpha/beta domain with an alpha-helix and two beta-sheets, that has some flexible regions. The backbone atomic root-mean-square deviation (rmsd) values of both domains were 0.7 A when calculated separately, which is smaller than that of the molecule as a whole (1.4 A). Measurement of 15N-[1H] NOE values show that the residues in the corner of the L-shaped molecule are undergoing fast internal motion. These results indicate that the joint region between two domains contributes to the fluctuation in the orientation of two domains. Thus, it was shown that RRF remains the tRNA mimicry in solution where it functions.     

Entry into cells and selective degradation of tRNAs by a cytotoxic member of the RNase A family

Onconase (P-30 protein), an enzyme in the ribonuclease A superfamily, exerts cytostatic, cytotoxic, and antiviral activity when added to the medium of growing mammalian cells. We find that onconase enters living mammalian cells and selectively cleaves tRNA with no detectable degradation of rRNA. The RNA specificity of onconase in vitro using reticulocyte lysate and purified RNA substrates indicates that proteins associated with rRNA protect the rRNA from the onconase ribonucleolytic action contributing to the cellular tRNA selectivity of onconase. The onconase-mediated tRNA degradation in cells appears to be accompanied by increased levels of tRNA turnover and induction of tRNA synthesis perhaps in response to the selective toxin-induced loss of tRNA. Degradation products of tRNA(3)(Lys), which acts as a primer for HIV-1 replication, were clearly detected in cells infected with HIV-1 and treated with sublethal concentrations of onconase. However, a new synthesis of tRNA(3)(Lys) also seemed to occur in these cells resulting in plateauing of the steady-state levels of this tRNA. We conclude that the degradation of tRNAs may be a primary factor in the cytotoxic activity of onconase.     

Initiator-Elongator Discrimination in Vertebrate tRNAs for Protein Synthesis

Initiator tRNAs are used exclusively for initiation of protein synthesis and not for the elongation step. We show, in vivo and in vitro, that the primary sequence feature that prevents the human initiator tRNA from acting in the elongation step is the nature of base pairs 50:64 and 51:63 in the TΨC stem of the initiator tRNA. Various considerations suggest that this is due to sequence-dependent perturbation of the sugar phosphate backbone in the TΨC stem of initiator tRNA, which most likely blocks binding of the elongation factor to the tRNA. Because the sequences of all vertebrate initiator tRNAs are identical, our findings with the human initiator tRNA are likely to be valid for all vertebrate systems. We have developed reporter systems that can be used to monitor, in mammalian cells, the activity in elongation of mutant human initiator tRNAs carrying anticodon sequence mutations from CAU to CCU (the C35 mutant) or to CUA (the U35A36 mutant). Combination of the anticodon sequence mutation with mutations in base pairs 50:64 and 51:63 yielded tRNAs that act as elongators in mammalian cells. Further mutation of the A1:U72 base pair, which is conserved in virtually all eukaryotic initiator tRNAs, to G1:C72 in the C35 mutant background yielded tRNAs that were even more active in elongation. In addition, in a rabbit reticulocyte in vitro protein-synthesizing system, a tRNA carrying the TΨC stem and the A1:U72-to-G1:C72 mutations was almost as active in elongation as the elongator methionine tRNA. The combination of mutant initiator tRNA with the CCU anticodon and the reporter system developed here provides the first example of missense suppression in mammalian cells.