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Analysis of the profile of the tRNA modifications in severalArchaeaallowed us to observe a novel modified uridine in the V-loop of several tRNAs from two species:Pyrococcus furiosusandSulfolobus acidocaldarius. Recently, Ohira and colleagues characterized 2′-phosphouridine (Up) at position 47 in tRNAs of thermophilicSulfurisphaera tokodaii, as well as in several other archaea and thermophilic bacteria. From the presence of the genearkIcorresponding to the RNA kinase responsible for Up47 formation, they also concluded that Up47 should be present in tRNAs of other thermophilicArchaea. Reanalysis of our earlier data confirms that the unidentified residue in tRNAs of bothP. furiosusandS. acidocaldariusis indeed 2′-phosphouridine followed by m5C48. Moreover, we find this modification in several tRNAs of otherArchaeaand of the hyperthermophilic bacteriumAquifex aeolicus.

The high conservation of nucleotides of the T-loop, including their chemical identity, are hallmarks of tRNAs from organisms belonging to the three Domains of Life. These structural characteristics allow the T-loop to adopt a peculiar intraloop conformation able to interact specifically with other conserved residues of the D-loop, which ultimately folds the mature tRNA in a unique functional canonical L-shaped architecture. Paradoxically, despite the high conservation of modified nucleotides in the T-loop, enzymes catalyzing their formation depend mostly on the considered organism, attesting for an independent but convergent evolution of the post-transcriptional modification processes. The driving force behind this is the preservation of a native conformation of the tRNA elbow that underlies the various interactions of tRNA molecules with different cellular components., SCOPUS: re.j, info:eu-repo/semantics/published

To improve and complete our knowledge of archaeal tRNA modification patterns, we have identified and compared the modification pattern (type and location) in tRNAs of three very different archaeal species, Methanococcus maripaludis (a mesophilic methanogen), Pyrococcus furiosus (a hyperthermophile thermococcale), and Sulfolobus acidocaldarius (an acidophilic thermophilic sulfolobale). Most abundant isoacceptor tRNAs (79 in total) for each of the 20 amino acids were isolated by two-dimensional gel electrophoresis followed by in-gel RNase digestions. The resulting oligonucleotide fragments were separated by nanoLC and their nucleotide content analyzed by mass spectrometry (MS/MS). Analysis of total modified nucleosides obtained from complete digestion of bulk tRNAs was also performed. Distinct base- and/or ribose-methylations, cytidine acetylations, and thiolated pyrimidines were identified, some at new positions in tRNAs. Novel, some tentatively identified, modifications were also found. The least diversified modification landscape is observed in the mesophilic Methanococcus maripaludis and the most complex one in Sulfolobus acidocaldarius Notable observations are the frequent occurrence of ac4C nucleotides in thermophilic archaeal tRNAs, the presence of m7G at positions 1 and 10 in Pyrococcus furiosus tRNAs, and the use of wyosine derivatives at position 37 of tRNAs, especially those decoding U1- and C1-starting codons. These results complete those already obtained by others with sets of archaeal tRNAs from Methanocaldococcus jannaschii and Haloferax volcanii., SCOPUS: ar.j, info:eu-repo/semantics/published

Place: Oxford Publisher: Oxford Univ Press WOS:000574288800042; The universal L-shaped tertiary structure of tRNAs is maintained with the help of nucleotide modifications within the D- and T-loops, and these modifications are most extensive within hyperthermophilic species. The obligate-commensal Nanoarchaeum equitans and its phylogenetically-distinct host Ignicoccus hospitalis grow physically coupled under identical hyperthermic conditions. We report here two fundamentally different routes by which these archaea modify the key conserved nucleotide U54 within their tRNA T-loops. In N. equitans, this nucleotide is methylated by the S-adenosylmethionine-dependent enzyme NEQ053 to form m(5)U54, and a recombinant version of this enzyme maintains specificity for U54 in Escherichia coli. In N. equitans, m(5)U54 is subsequently thiolated to form m(5)s(2)U54. In contrast, I. hospitalis isomerizes U54 to pseudouridine prior to methylating its N1-position and thiolating the O4-position of the nucleobase to form the previously uncharacterized nucleotide m(1)s(4)Psi. The methyl and thiol groups in m(1)s(4)Psi and m(5)s(2)U are presented within the T-loop in a spatially identical manner that stabilizes the 3'-endo-anti conformation of nucleotide-54, facilitating stacking onto adjacent nucleotides and reverse-Hoogsteen pairing with nucleotide m(1)A58. Thus, two distinct structurally-equivalent solutions have evolved independently and convergently to maintain the tertiary fold of tRNAs under extreme hyperthermic conditions.

The C5-methylation of uracil to form 5-methyluracil (m5U) is a ubiquitous base modification of nucleic acids. Four enzyme families have converged to catalyze this methylation using different chemical solutions. Here, we investigate the evolution of 5-methyluracil synthase families in Mollicutes, a class of bacteria that has undergone extensive genome erosion. Many mollicutes have lost some of the m5U methyltransferases present in their common ancestor. Cases of duplication and subsequent shift of function are also described. For example, most members of the Spiroplasma subgroup, use the ancestral tetrahydrofolate-dependent TrmFO enzyme, to catalyze the formation of m5U54 in tRNA, while a TrmFO paralog (termed RlmFO) is responsible for m5U1939 formation in 23S RNA. RlmFO has replaced the S-adenosyl-l-methionine (SAM)-enzyme RlmD that adds the same modification in the ancestor and which is still present in mollicutes from the Hominis subgroup. Another paralog of this family, the TrmFO-like protein, has a yet unidentified function that differs from the TrmFO and RlmFO homologs. Despite having evolved towards minimal genomes, the mollicutes possess a repertoire of m5U modifying enzymes that is highly dynamic and has undergone horizontal transfer. This emphasizes the necessity for combining bioinformatics predictions with empirical testing and structural information to get a reliable functional annotation of these enzymes.

International audience; RNA methylation is the most abundant and evolutionarily conserved chemical modification of bases or ribose in noncoding and coding RNAs. This rather simple modification has nevertheless major consequences on the function of maturated RNA molecules and ultimately on their cellular fates. The methyl group employed in the methylation is almost universally derived from S-adenosyl-L-methionine via a simple SN2 displacement reaction. However, in some rare cases, the carbon originates from N5,N10-methylenetetrahydrofolate (CH2=THF). Here, a methylene group is transferred first and requires a subsequent reduction step (2e-+H+) via the flavin adenine dinucleotide hydroquinone (FADH-) to form the final methylated derivative. This FAD/folate-dependent mode of chemical reaction, called reductive methylation, is thus far more complex than the usual simple S-adenosyl-L-methionine-dependent one. This reaction is catalyzed by flavoenzymes, now named TrmFO and RlmFO, which respectively modify transfer and ribosomal RNAs. In this review, we briefly recount how these new RNA methyltransferases were discovered and describe a novel aspect of the chemistry of flavins, wherein this versatile biological cofactor is not just a simple redox catalyst but is also a new methyl transfer agent acting via a critical CH2=(N5)FAD iminium intermediate. The enigmatic structural reorganization of these enzymes that needs to take place during catalysis in order to build their active center is also discussed. Finally, recent findings demonstrated that this flavin-dependent mechanism is also employed by enzymatic systems involved in DNA synthesis, suggesting that the use of this cofactor as a methylating agent of biomolecules could be far more usual than initially anticipated.

International audience; We report new crystallographic structures of Thermus thermophilus ribosomes complexed with long mRNAs and native Escherichia coli tRNAs. They complete the full set of combinations of Watson-Crick G•C and miscoding G•U pairs at the first two positions of the codon-anticodon duplex in ribosome functional complexes. Within the tight decoding center, miscoding G•U pairs occur, in all combinations, with a non-wobble geometry structurally indistinguishable from classical coding Watson-Crick pairs at the same first two positions. The contacts with the ribosomal grip surrounding the decoding center are all quasi-identical, except in the crowded environment of the amino group of a guanosine at the second position; in which case a G in the codons may be preferred. In vivo experimental data show that the translational errors due to miscoding by G•U pairs at the first two positions are the most frequently encountered ones, especially at the second position and with a G on the codon. Such preferred miscodings involve a switch from an A-U to a G•U pair in the tRNA/mRNA complex and very rarely from a G = C to a G•U pair. It is concluded that the frequencies of such occurrences are only weakly affected by the codon/anticodon structures but depend mainly on the stability and lifetime of the complex, the modifications present in the anticodon loop, especially those at positions 34 and 37, in addition to the relative concentration of cognate/near-cognate tRNA species present in the cellular tRNA pool.

The presence of tricyclic wyosine derivatives 3′-adjacent to anticodon is a hallmark of tRNAPhe in eukaryotes and archaea. In yeast, formation of wybutosine (yW) results from five enzymes acting in a strict sequential order. In archaea, the intermediate compound imG-14 (4-demethylwyosine) is a target of three different enzymes, leading to the formation of distinct wyosine derivatives (yW-86, imG, and imG2). We focus here on a peculiar methyltransferase (aTrm5a) that catalyzes two distinct reactions: N1-methylation of guanosine and C7-methylation of imG-14, whose function is to allow the production of isowyosine (imG2), an intermediate of the 7-methylwyosine (mimG) biosynthetic pathway. Based on the formation of mesomeric forms of imG-14, a rationale for such dual enzymatic activities is proposed. This bifunctional tRNA:m1G/imG2 methyltransferase, acting on two chemically distinct guanosine derivatives located at the same position of tRNAPhe, is unique to certain archaea and has no homologs in eukaryotes. This enzyme here referred to as Taw22, probably played an important role in the emergence of the multistep biosynthetic pathway of wyosine derivatives in archaea and eukaryotes.

Translation of the isoleucine codon AUA in most prokaryotes requires a modified C (lysidine or agmatidine) at the wobble position of tRNA2(Ile) to base pair specifically with the A of the AUA codon but not with the G of AUG. Recently, a Bacillus subtilis strain was isolated in which the essential gene encoding tRNA(Ile)-lysidine synthetase was deleted for the first time. In such a strain, C34 at the wobble position of tRNA2(Ile) is expected to remain unmodified and cells depend on a mutant suppressor tRNA derived from tRNA1(Ile), in which G34 has been changed to U34. An important question, therefore, is how U34 base pairs with A without also base pairing with G. Here, we show (i) that unlike U34 at the wobble position of all B. subtilis tRNAs of known sequence, U34 in the mutant tRNA is not modified, and (ii) that the mutant tRNA binds strongly to the AUA codon on B. subtilis ribosomes but only weakly to AUG. These in vitro data explain why the suppressor strain displays only a low level of misreading AUG codons in vivo and, as shown here, grows at a rate comparable to that of the wild-type strain.

Post-transcriptional modifications of the wobble uridine (U34) of tRNAs play a critical role in reading NNA/G codons belonging to split codon boxes. In a subset of Escherichia coli tRNA, this wobble uridine is modified to 5-methylaminomethyluridine (mnm5U34) through sequential enzymatic reactions. Uridine 34 is first converted to 5-carboxymethylaminomethyluridine (cmnm5U34) by the MnmE-MnmG enzyme complex. The cmnm5U34 is further modified to mnm5U by the bifunctional MnmC protein. In the first reaction, the FAD-dependent oxidase domain (MnmC1) converts cmnm5U into 5-aminomethyluridine (nm5U34), and this reaction is immediately followed by the methylation of the free amino group into mnm5U34 by the S-adenosylmethionine-dependent domain (MnmC2). Aquifex aeolicus lacks a bifunctional MnmC protein fusion and instead encodes the Rossmann-fold protein DUF752, which is homologous to the methyltransferase MnmC2 domain of Escherichia coli MnmC (26% identity). Here, we determined the crystal structure of the A. aeolicus DUF752 protein at 2.5 Å resolution, which revealed that it catalyzes the S-adenosylmethionine-dependent methylation of nm5U in vitro, to form mnm5U34 in tRNA. We also showed that naturally occurring tRNA from A. aeolicus contains the 5-mnm group attached to the C5 atom of U34. Taken together, these results support the recent proposal of an alternative MnmC1-independent shortcut pathway for producing mnm5U34 in tRNAs.

International audience; The principles of mRNA decoding are conserved among all extant life forms. We present an integrative view of all the interaction networks between mRNA, tRNA and rRNA: the intrinsic stability of codon-anticodon duplex, the conformation of the anticodon hairpin, the presence of modified nucleotides, the occurrence of non-Watson-Crick pairs in the codon-anticodon helix and the interactions with bases of rRNA at the A-site decoding site. We derive a more information-rich, alternative representation of the genetic code, that is circular with an unsymmetrical distribution of codons leading to a clear segregation between GC-rich 4-codon boxes and AU-rich 2:2-codon and 3:1-codon boxes. All tRNA sequence variations can be visualized, within an internal structural and energy framework, for each organism, and each anticodon of the sense codons. The multiplicity and complexity of nucleotide modifications at positions 34 and 37 of the anticodon loop segregate meaningfully, and correlate well with the necessity to stabilize AU-rich codon-anticodon pairs and to avoid miscoding in split codon boxes. The evolution and expansion of the genetic code is viewed as being originally based on GC content with progressive introduction of A/U together with tRNA modifications. The representation we present should help the engineering of the genetic code to include non-natural amino acids.

Translation machinery of mollicutes: economy is possible for proteins, not much for RNAs . Ribosome Structure and Function 2016

MODOMICS is a database of RNA modifications that provides comprehensive information concerning the chemical structures of modified ribonucleosides, their biosynthetic pathways, RNA-modifying enzymes and location of modified residues in RNA sequences. In the current database version, accessible at http://modomics.genesilico.pl, we included new features: a census of human and yeast snoRNAs involved in RNA-guided RNA modification, a new section covering the 5′-end capping process, and a catalogue of ‘building blocks’ for chemical synthesis of a large variety of modified nucleosides. The MODOMICS collections of RNA modifications, RNA-modifying enzymes and modified RNAs have been also updated. A number of newly identified modified ribonucleosides and more than one hundred functionally and structurally characterized proteins from various organisms have been added. In the RNA sequences section, snRNAs and snoRNAs with experimentally mapped modified nucleosides have been added and the current collection of rRNA and tRNA sequences has been substantially enlarged. To facilitate literature searches, each record in MODOMICS has been cross-referenced to other databases and to selected key publications. New options for database searching and querying have been implemented, including a BLAST search of protein sequences and a PARALIGN search of the collected nucleic acid sequences.

In eubacteria, the post-transcriptional modification of the wobble cytidine of the CAU anticodon in a precursor tRNA(Ile2) to a lysidine residue (2-lysyl-cytidine, abbreviated as L) allows the amino acid specificity to change from methionine to isoleucine and the codon decoding specificity to shift from AUG to AUA. The tilS gene encoding the enzyme that catalyses this modification is widely distributed. However, some microbial species lack a tilS gene, indicating that an alternative strategy exists to accurately translate the AUA codon into Ile. To determine whether a TilS-dependent bacterium, such as Bacillus subtilis, can overcome the absence of lysidine in its tRNA(Ile2) (CAU), we analysed the suppressor mutants of a tilS-thermosensitive allele. These tilS-suppressor mutants carry a substitution of the wobble guanosine into thymidine in one of the tRNA(Ile1) genes (the original GAT anticodon is changed to a TAT). In absence of TilS activity, the AUA codons are translated into isoleucine by the suppressor tRNA(Ile1), although a low level of AUA codons is also mistranslated into methionine. Results are in agreement with rare cases of eubacteria (and archaea), which naturally lack the tilS gene (or tiaS in archaea) but contain a tRNA(Ile2) gene containing a TAT instead of a CAT anticodon.

International audience; MODOMICS, a database devoted to the systems biology of RNA modification, has been subjected to substantial improvements. It provides comprehensive information on the chemical structure of modified nucleosides, pathways of their biosynthesis, sequences of RNAs containing these modifications and RNA-modifying enzymes. MODOMICS also provides cross-references to other databases and to literature. In addition to the previously available manually curated tRNA sequences from a few model organisms, we have now included additional tRNAs and rRNAs, and all RNAs with 3D structures in the Nucleic Acid Database, in which modified nucleosides are present. In total, 3460 modified bases in RNA sequences of different organisms have been annotated. New RNA-modifying enzymes have been also added. The current collection of enzymes includes mainly proteins for the model organisms Escherichia coli and Saccharomyces cerevisiae, and is currently being expanded to include proteins from other organisms, in particular Archaea and Homo sapiens. For enzymes with known structures, links are provided to the corresponding Protein Data Bank entries, while for many others homology models have been created. Many new options for database searching and querying have been included. MODOMICS can be accessed at http://genesilico.pl/modomics.

The conserved cytidine residue at position 56 of tRNA contributes to the maintenance of the L-shaped tertiary structure. aTrm56 catalyzes the 2′- O -methylation of the cytidine residue in archaeal tRNA, using S -adenosyl- L -methionine. Based on the amino acid sequence, aTrm56 is the most distant member of the SpoU family. Here, we determined the crystal structure of Pyrococcus horikoshii aTrm56 complexed with S -adenosyl- L -methionine at 2.48 A resolution. aTrm56 consists of the SPOUT domain, which contains the characteristic deep trefoil knot, and a unique C-terminal β-hairpin. aTrm56 forms a dimer. The S -adenosyl- L -methionine binding and dimerization of aTrm56 were similar to those of the other SpoU members. A structure-based sequence alignment revealed that aTrm56 conserves only motif II, among the four signature motifs. However, an essential Arg16 residue is located at a novel position within motif I. Biochemical assays showed that aTrm56 prefers the L-shaped tRNA to the λ form as its substrate.

The Pyrococcus abyssi genome displays two genes possibly coding for S-adenosyl-l-methionine-dependent RNA(uracil, C5)-methyltransferases (PAB0719 and PAB0760). Their amino acid sequences are more closely related to Escherichia coli RumA catalysing the formation of 5-methyluridine (m(5)U)-1939 in 23S rRNA than to E. coli TrmA (tRNA methyltransferase A) methylating uridine-54 in tRNA. Comparative genomic and phylogenetic analyses show that homologues of PAB0719 and PAB0760 occur only in a few Archaea, these genes having been acquired via a single horizontal gene transfer from a bacterial donor to the common ancestor of Thermococcales and Nanoarchaea. This transfer event was followed by a duplication event in Thermococcales leading to two closely related genes. None of the gene products of the two P. abyssi paralogues catalyses in vitro the formation of m(5)U in a P. abyssi rRNA fragment homologous to the bacterial RumA substrate. Instead, PAB0719 enzyme (renamed (Pab)TrmU54) displays an identical specificity to TrmA, as it catalyses the in vitro formation of m(5)U-54 in tRNA. Thus, during evolution, at least one of the two P. abyssi RumA-type enzymes has changed of target specificity. This functional shift probably occurred in an ancestor of all Thermococcales. This study also provides new evidence in favour of a close relationship between Thermococcales and Nanoarchaea.

The study of RNA modification started in 1957 when the first modified nucleoside was discovered in bulk yeast RNA, pseudouridine: a fifth nucleotide, was born. After a long period of arduous and somewhat frustrating works, remarkable results have been obtained in the last two decades, made possible by new technology developed in many laboratories, and have engendered a renaissance of interest in the field. Research on RNA modification has always been and remains a real chest of treasures. I am happy to add my personal comments to this anniversary edition of RNA.

Functional tRNA molecules always contain a wide variety of post-transcriptionally modified nucleosides. These modifications stabilize tRNA structure, allow for proper interaction with other macromolecules and fine-tune the decoding of mRNAs during translation. Their presence in functionally important regions of tRNA is conserved in all domains of life. However, the identities of many of these modified residues depend much on the phylogeny of organisms the tRNAs are found in, attesting for domain-specific strategies of tRNA maturation. In this work we present a new tool, tRNAmodviz web server (http://genesilico.pl/trnamodviz) for easy comparative analysis and visualization of modification patterns in individual tRNAs, as well as in groups of selected tRNA sequences. We also present results of comparative analysis of tRNA sequences derived from 7 phylogenetically distinct groups of organisms: Gram-negative bacteria, Gram-positive bacteria, cytosol of eukaryotic single cell organisms, Fungi and Metazoa, cytosol of Viridiplantae, mitochondria, plastids and Euryarchaeota. These data update the study conducted 20 y ago with the tRNA sequences available at that time.

Pseudouridine (Psi) located at position 55 in tRNA is a nearly universally conserved RNA modification found in all three domains of life. This modification is catalyzed by TruB in bacteria and by Pus4 in eukaryotes, but so far the Psi55 synthase has not been identified in archaea. In this work, we report the ability of two distinct pseudouridine synthases from the hyperthermophilic archaeon Pyrococcus furiosus to specifically modify U55 in tRNA in vitro. These enzymes are (pfu)Cbf5, a protein known to play a role in RNA-guided modification of rRNA, and (pfu)PsuX, a previously uncharacterized enzyme that is not a member of the TruB/Pus4/Cbf5 family of pseudouridine synthases. (pfu)PsuX is hereafter renamed (pfu)Pus10. Both enzymes specifically modify tRNA U55 in vitro but exhibit differences in substrate recognition. In addition, we find that in a heterologous in vivo system, (pfu)Pus10 efficiently complements an Escherichia coli strain deficient in the bacterial Psi55 synthase TruB. These results indicate that it is probable that (pfu)Cbf5 or (pfu)Pus10 (or both) is responsible for the introduction of pseudouridine at U55 in tRNAs in archaea. While we cannot unequivocally assign the function from our results, both possibilities represent unexpected functions of these proteins as discussed herein., Journal Article, Research Support, N.I.H. Extramural, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Formation of 5-methyluridine (ribothymidine) at position 54 of the T-psi loop of tRNA is catalyzed by site-specific tRNA methyltransferases (tRNA:m(5)U-54 MTase). In all Eukarya and many Gram-negative Bacteria, the methyl donor for this reaction is S-adenosyl-l-methionine (S-AdoMet), while in several Gram-positive Bacteria, the source of carbon is N(5), N(10)-methylenetetrahydrofolate (CH(2)H(4)folate). We have identified the gene for Bacillus subtilis tRNA:m(5)U-54 MTase. The encoded recombinant protein contains tightly bound flavin and is active in Escherichia coli mutant lacking m(5)U-54 in tRNAs and in vitro using T7 tRNA transcript as substrate. This gene is currently annotated gid in Genome Data Banks and it is here renamed trmFO. TrmFO (Gid) orthologs have also been identified in many other bacterial genomes and comparison of their amino acid sequences reveals that they are phylogenetically distinct from either ThyA or ThyX class of thymidylate synthases, which catalyze folate-dependent formation of deoxyribothymine monophosphate, the universal DNA precursor., info:eu-repo/semantics/published

N(2)-Monomethylguanosine-10 (m(2)G10) and N(2),N(2)-dimethylguanosine-26 (m(2)(2)G26) are the only two guanosine modifications that have been detected in tRNA from nearly all archaea and eukaryotes but not in bacteria. In Saccharomyces cerevisiae, formation of m(2)(2)G26 is catalyzed by Trm1p, and we report here the identification of the enzymatic activity that catalyzes the formation of m(2)G10 in yeast tRNA. It is composed of at least two subunits that are associated in vivo: Trm11p (Yol124c), which is the catalytic subunit, and Trm112p (Ynr046w), a putative zinc-binding protein. While deletion of TRM11 has no detectable phenotype under laboratory conditions, deletion of TRM112 leads to a severe growth defect, suggesting that it has additional functions in the cell. Indeed, Trm112p is associated with at least four proteins: two tRNA methyltransferases (Trm9p and Trm11p), one putative protein methyltransferase (Mtc6p/Ydr140w), and one protein with a Rossmann fold dehydrogenase domain (Lys9p/Ynr050c). In addition, TRM11 interacts genetically with TRM1, thus suggesting that the absence of m(2)G10 and m(2)(2)G26 affects tRNA metabolism or functioning.

The activation and charging of amino acids onto the acceptor stems of their cognate tRNAs are the housekeeping functions of aminoacyl-tRNA synthetases. The availability of whole genome sequences has revealed the existence of synthetase-like proteins that have other functions linked to different aspects of cell metabolism and physiology. In eubacteria, a paralog of glutamyl-tRNA synthetase, which lacks the tRNA-binding domain, was found to aminoacylate tRNA(Asp) not on the 3'-hydroxyl group of the acceptor stem but on a cyclopentene diol of the modified nucleoside queuosine present at the wobble position of anticodon loop. This modified nucleoside might be a relic of an ancient code.

In most organisms, the AUA triplet codes for isoleucine (Ile), whereas in a few organelles it codes for methionine (Met). In bacteria, this A-ending triplet is decoded by an unusual tRNA harboring a Met anticodon CAU, where cytidine at the wobble position 34 (C34) is posttranscriptionally modified to a 2-lysyl cytidine (lysidine), abbreviated as (k2C). Now, the bacterial gene tilS, which encodes the enzyme catalyzing the lysylation of C34 in the precursor tRNAIle(CAU), thereby leading to the formation of tRNAIle(k2CAU), has been identified. The formation of lysidine by this essential enzyme allows recognition of tRNAIle(k2CAU) by Ile-tRNA synthetase and switches the base pairing of the tRNA from AUG (Met) to AUA (Ile). This base change is reminiscent of C-to-U type of RNA editing of some mitochondrial tRNAs.

A search has been performed on 2878 tRNA sequences from 60 different genomes in order to detect the existence of closely related 'alloacceptor' tRNAs accepting dissimilar amino acids that could be paralogs generated by gene duplications. This has led to the identification of extremely conserved tRNA(Phe)-tRNA(Tyr) pairs displaying as high as 94% identity between them, and also other potentially paralogous tRNA pairs in archaeal species. These paralogous pairs are enriched for amino acid pairs belonging to the same amino acid biosynthetic family, thus providing evidence for the coevolution of genetic code and amino acid biosynthesis. Overall, the genetic distances between alloacceptor tRNAs yield estimates of how closely clustered in sequence space are the tRNAs in a genome. Among 34 Bacteria, 18 Archaea and 8 Eukarya, Methanopyrus kandleri and Aeropyrum pernix have yielded the lowest alloacceptor distances and largest number of paralogous pairs. Based on a cluster-dispersion model of tRNA evolution, such tight alloacceptor clustering is a measure of primitiveness of tRNA genotypes, and places last universal common ancestor (LUCA) between the branches leading to these two archaea in the tRNA phylogenetic tree.

From 50 genomes of the three domains of life (7 eukarya, 13 archaea, and 30 bacteria), we extracted, analyzed, and compared over 4,000 sequences corresponding to cytoplasmic, nonorganellar tRNAs. For each genome, the complete set of tRNAs required to read the 61 sense codons was identified, which permitted revelation of three major anticodon-sparing strategies. Other features and sequence peculiarities analyzed are the following: (1) fit to the standard cloverleaf structure, (2) characteristic consensus sequences for elongator and initiator tDNAs, (3) frequencies of bases at each sequence position, (4) type and frequencies of conserved 2D and 3D base pairs, (5) anticodon/tDNA usages and anticodon-sparing strategies, (6) identification of the tRNA-Ile with anticodon CAU reading AUA, (7) size of variable arm, (8) occurrence and location of introns, (9) occurrence of 3'-CCA and 5'-extra G encoded at the tDNA level, and (10) distribution of the tRNA genes in genomes and their mode of transcription. Among all tRNA isoacceptors, we found that initiator tDNA-iMet is the most conserved across the three domains, yet domain-specific signatures exist. Also, according to which tRNA feature is considered (5'-extra G encoded in tDNAs-His, AUA codon read by tRNA-Ile with anticodon CAU, presence of intron, absence of "two-out-of-three" reading mode and short V-arm in tDNA-Tyr) Archaea sequester either with Bacteria or Eukarya. No common features between Eukarya and Bacteria not shared with Archaea could be unveiled. Thus, from the tRNomic point of view, Archaea appears as an "intermediate domain" between Eukarya and Bacteria.

Mollicutes is a class of parasitic bacteria that have evolved from a common Firmicutes ancestor mostly by massive genome reduction. With genomes under 1 Mbp in size, most Mollicutes species retain the capacity to replicate and grow autonomously. The major goal of this work was to identify the minimal set of proteins that can sustain ribosome biogenesis and translation of the genetic code in these bacteria. Using the experimentally validated genes from the model bacteria Escherichia coli and Bacillus subtilis as input, genes encoding proteins of the core translation machinery were predicted in 39 distinct Mollicutes species, 33 of which are culturable. The set of 260 input genes encodes proteins involved in ribosome biogenesis, tRNA maturation and aminoacylation, as well as proteins cofactors required for mRNA translation and RNA decay. A core set of 104 of these proteins is found in all species analyzed. Genes encoding proteins involved in post-translational modifications of ribosomal proteins and translation cofactors, post-transcriptional modifications of t+rRNA, in ribosome assembly and RNA degradation are the most frequently lost. As expected, genes coding for aminoacyl-tRNA synthetases, ribosomal proteins and initiation, elongation and termination factors are the most persistent (i.e. conserved in a majority of genomes). Enzymes introducing nucleotides modifications in the anticodon loop of tRNA, in helix 44 of 16S rRNA and in helices 69 and 80 of 23S rRNA, all essential for decoding and facilitating peptidyl transfer, are maintained in all species. Reconstruction of genome evolution in Mollicutes revealed that, beside many gene losses, occasional gains by horizontal gene transfer also occurred. This analysis not only showed that slightly different solutions for preserving a functional, albeit minimal, protein synthetizing machinery have emerged in these successive rounds of reductive evolution but also has broad implications in guiding the reconstruction of a minimal cell by synthetic biology approaches., Author Summary In all cells, proteins are synthesized from the message encoded by mRNA using complex machineries involving many proteins and RNAs. In this process, named translation, the ribosome plays a central role. The elements involved in both ribosome biogenesis and its function are extremely conserved in all organisms from the simplest bacteria to mammalian cells. Most of the 260 known proteins involved in translation have been identified and studied in the bacteria Escherichia coli and Bacillus subtilis, two common cellular models in biology. However, comparative genomics has shown that the translation protein set can be much smaller. This is true for bacteria belonging to the class Mollicutes that are characterized by reduced genomes and hence considered as models for minimal cells. Using homology inference approach and expert analyses, we identified the translation apparatus proteins for 39 of these organisms. Although striking variations were found from one group of species to another, some Mollicutes species require half as many proteins as E. coli or B. subtilis. This analysis allowed us to determine a set of proteins necessary for translation in Mollicutes and define the translation apparatus that would be required in a cellular chassis mimicking a minimal bacterial cell.

This chapter reviews the methods that have been successfully used to date for the identification of RNA modification and editing enzymes. The development of recombinant DNA and RNA techniques and techniques for chemical synthesis of DNA and RNA offers adequate alternatives for obtaining synthetic or semisynthetic unmodified or partially modified RNA substrates suitable for analyzing enzymatic posttranscriptional RNA modifications both in vivo and in vitro. Stepwise automated chemical synthesis of DNA oligonucleotides is widely used for producing starting material for gene construction, primers for PCR, DNA splints, or DNA probes for hybridization and deoxyribozymes. Two distinct approaches are generally used to gain the information on the precise location of modification or editing sites in RNA molecules. The first is based on direct sequencing of pure RNA and is applied mostly for the analysis of rather short RNA molecules, while the other is based on the use of reverse transcription with unfractionated RNA mixtures and specific oligonucleotide primers. Microinjection of antisense RNA, with the aim of blocking translation of selected mRNAs in the Xenopus laevis oocyte or fertilized frog eggs, revealed the existence of what was first identified as an unwinding RNA protein. With the recent access to complete genome sequences, it has become possible to search for homologous genes in the different genomes, to clone them into suitable vectors after amplification by PCR, and to express them in appropriate host cells.

The major types of posttranscriptional modification are the isomerization of uracil to pseudouridine, 2′-O-methylation of the ribose moieties and base methylation. In addition to their roles in pre-rRNA modification, both Dim1p and Cbf5p are required for pre-rRNA processing, and Pet56p is required for synthesis of mitochondrial large ribosomal subunits. This chapter discusses data on Dim1p and Cbf5p that provide insights on the type of systems that may act to coordinate the many steps in ribosome synthesis and the origins of the snoRNA-directed rRNA modification systems present in eukaryotic cells. Uncoupling of the dimethylation and prerRNA processing defects showed that Dimlp rather than the dimethylation activity is required for prerRNA processing. This observation did not, however, determine whether Dim1p is itself directly required for pre-rRNA processing. The binding of Dim1p to the pre-rRNA may therefore be monitored to ensure that it fulfills its functions in both modification and assembly. Ribosome synthesis is a highly dynamic process during which a vast number of processing, modification, and assembly reactions occur simultaneously. The requirement for Cbf5p in pre-rRNA processing appears to be quite different from the requirement for Dim1p. Eukaryotic ribosome synthesis involves very complex pre-rRNA processing and assembly pathways. These include numerous steps occurring in different cellular compartments and requiring a plethora of RNA and proteins, many of them in the form of snoRNPs that only transiently associate with the preribosomal particles. The archaea may hold the final clues to understanding the origins of the small nucleolar ribonucleoproteins (snoRNPs)-directed systems of rRNA modification.

The anticodon stem loop of tRNA is one of the most remarkable hairpins in RNAs that present several modified nucleotides arising by posttranscriptional processes. Many different modifications have been identified at position 34. These modifications are considered to be important for the specificity, fidelity, and efficiency of the tRNA functions, mainly in decoding the genetic message during translation on the ribosome. This chapter summarizes the information available about the role of modified nucleotides present at positions 34 and 37 in modulating tRNA anticodon-anticodon interaction. Implications for codon-anticodon interaction and comparison with other types of RNA-RNA interacting systems are also discussed in this chapter. One way to evaluate the effect of modified nucleotides on the stability and dynamic properties of anticodon-anticodon complexes is the temperature-jump relaxation technique. The chapter describes two RNA loop-loop interacting systems that do not involve modified nucleotides. Their comparison with the loop-loop interacting systems involving hairpins of tRNA molecules described in this chapter are interesting to better understand how modified nucleotides can restrict the interaction to only a few selected nucleotides within the interacting RNA loops. Comparison to other types of loop-loop complexes involving RNAs other than tRNAs, the so-called "kissing" complexes, is also made. The main difference between these different types of loop-loop complexes is the extent of base pairing between the nucleotides of the interacting loops.

This chapter discusses the question of coping up of the nucleic acids with high temperature at the polynucleotide level—RNA, DNA, and their ribonucleoprotein derivatives (RNP/DNP). When nucleic acids are heated in aqueous solution, two types of phenomena take place: denaturation of their architecture and chemical degradation of their building blocks. In vivo, the half-lives of both RNA and DNA of thermophilic organisms are usually longer than that estimated in vitro, attesting to cellular strategies protecting the nucleic acids against the deleterious effects of heat. Despite the susceptibility of certain modified bases and of the ribonucleotide chain to thermal degradation, most naturally occurring tRNAs (especially those from hyperthermophilic organisms) appear fairly resistant to heat denaturation. Despite the intrinsic potentiality of nucleic acids to degrade at elevated temperatures, many hyperthermophiles can survive at very high temperatures approaching or even surpassing the boiling point of water. The majority of stable cellular RNAs, such as tRNA and rRNA molecules, contain a variety of modified nucleosides. Stabilizing strategies of RNAs and DNAs may be classified into three major categories: (i) those which are intrinsic to the chemical structures of the nucleic acids; (ii) those which are dependent on extrinsic interactions with other biomolecules; and (iii) those which are dependent on a battery of enzymes for detecting and repairing the DNA damage or to constantly renew functional RNA molecules. Genetic approach using mutant strains mutated in one or more biomolecules supposedly involved directly or indirectly in stabilization of nucleic acids should be more systematically used.

International audience; Efficient protein synthesis in all organisms requires the post-transcriptional methylation of specific ribosomal ribonucleic acid (rRNA) and transfer RNA (tRNA) nucleotides. The methylation reactions are almost invariably catalyzed by enzymes that use S-adenosylmethionine (AdoMet) as the methyl group donor. One noteworthy exception is seen in some bacteria, where the conserved tRNA methylation at m(5)U54 is added by the enzyme TrmFO using flavin adenine dinucleotide together with N(5),N(10)-methylenetetrahydrofolate as the one-carbon donor. The minimalist bacterium Mycoplasma capricolum possesses two homologs of trmFO, but surprisingly lacks the m(5)U54 tRNA modification. We created single and dual deletions of the trmFO homologs using a novel synthetic biology approach. Subsequent analysis of the M. capricolum RNAs by mass spectrometry shows that the TrmFO homolog encoded by Mcap0476 specifically modifies m(5)U1939 in 23S rRNA, a conserved methylation catalyzed by AdoMet-dependent enzymes in all other characterized bacteria. The Mcap0476 methyltransferase (renamed RlmFO) represents the first folate-dependent flavoprotein seen to modify ribosomal RNA.

International audience; Yeast Pus1p catalyzes the formation of pseudouridine (ψ) at specific sites of several tRNAs, but its function is not essential for cell viability. We show here that Pus1p becomes essential when another tRNA:pseudouridine synthase, Pus4p, or the essential minor tRNA for glutamine are mutated. Strikingly, this mutant tRNA, which carries a mismatch in the TψC arm, displays a nuclear export defect. Furthermore, nuclear export of at least one wild-type tRNA species becomes defective in the absence of Pus1p. Our data, thus, show that the modifications formed by Pus1p are essential when other aspects of tRNA biogenesis or function are compromised and suggest that impairment of nuclear tRNA export in the absence of Pus1p might contribute to this phenotype.

Sera of some patients afflicted with the inflammatory disease myositis contain antibodies of the anti-PL-12 type. A fraction of these polyclonal autoantibodies specifically precipitates the fully matured human tRNA(Ala) bearing the anticodon IGC (PL-12 antigen). Earlier work (Bunn & Mathews, 1987, Science 238:116-119) had shown that the epitopes are located entirely within the anticodon stem-loop of the tRNA(Ala). Here we demonstrate that human anti-tRNA(Ala) autoantibodies immunoprecipitate a synthetic polyribonucleotide containing inosine (I) and N1-methylinosine (m1I) separated by 2 nt as in the anticodon stem-loop of human tRNA(Ala). The shortest polyribonucleotide that can be immunoprecipitated corresponds to the pentanucleotide IpGpCpm1IpUp, which corresponds to part of the anticodon loop of human tRNA(Ala) and lacks the stem-loop structure. The efficiency of immunoprecipitation was about four times greater with longer polyribonucleotides capable of forming a stem-loop structure, and was abolished by altering the relative positions of I and m1I within the synthetic polynucleotide. Synthetic oligodeoxyribonucleotide analogs of the tRNA(Ala) stem-loop, containing the sequence dIpdGdCdm1Ip, are not antigenic. Our results show that human anti-tRNA(Ala) autoantibodies selectively recognize chemical details of modified nucleotides (the 6-keto group of inosine-34 and the 6-keto group and the N1-methyl groups of N1-methylinosine-37) within an anticodon loop structure of a tRNA molecule. We also describe the chemical synthesis of the phosphoramidite derivatives corresponding to N1-methylinosine and N1-methyl-2'-deoxyinosine for use in the automatic chemical synthesis of oligonucleotides containing N1-methylinosine and N1-methyl-2'-deoxyinosine.

The modification patterns of in vitro transcripts of two yeast Saccharomyces cerevisiae tRNAs (tRNAPheand tRNAAsp) and one archaeal Haloferax volcanii tRNA (tRNAIle) were investigated in the cell-free extract of Pyrococcus furiosus supplemented with S -adenosyl-l-methionine (AdoMet). The results indicate that the enzymatic formation of 11 distinct modified nucleotides corresponding to 12 enzymatic activities can be detected in vitro. They correspond to the formation of pseudouridines (Psi) at positions 39 and 55, 2' -O- ribose methylations at positions 6 (Am) and 56 (Cm), base methylations at positions 10 (m2G), 26 (m22G), 37 (m1G), 49 (m5C), 54 (m5U) and 58 (m1A) and both the deamination and methylation of adenosine into m1I at position 57. Most of the detected modified nucleotides are common modifications found in other phylogenetic groups, while Am6, Cm56and m1I57are specific modifications found exclusively in Archaea. It is also shown that the enzymatic formation of m5C49, m5U54, Psi55and m1I57does not depend on the three-dimensional architecture of the tRNA substrate, since these modi-fications also occur in fragmented tRNAs as substrate.

International audience; Pseudouridine (Psi) residues were localized in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (UsnRNAs) by using the chemical mapping method. In contrast to vertebrate UsnRNAs, S. cerevisiae UsnRNAs contain only a few Psi residues, which are located in segments involved in intermolecular RNA-RNA or RNA-protein interactions. At these positions, UsnRNAs are universally modified. When yeast mutants disrupted for one of the several pseudouridine synthase genes (PUS1, PUS2, PUS3, and PUS4) or depleted in rRNA-pseudouridine synthase Cbf5p were tested for UsnRNA Psi content, only the loss of the Pus1p activity was found to affect Psi formation in spliceosomal UsnRNAs. Indeed, Psi44 formation in U2 snRNA was abolished. By using purified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze Psi44 formation in the S. cerevisiae U2 snRNA. Thus, Pus1p is the first UsnRNA pseudouridine synthase characterized so far which exhibits a dual substrate specificity, acting on both tRNAs and U2 snRNA. As depletion of rRNA-pseudouridine synthase Cbf5p had no effect on UsnRNA Psi content, formation of Psi residues in S. cerevisiae UsnRNAs is not dependent on the Cbf5p-snoRNA guided mechanism.

For site specific incorporation of 4-thiouridine into oligoribonucleotides a new phosphoramidite is proposed. It makes use of the S-pivaloyloxymethyl group for the protection of the thiol function. This group is easily introduced and removed without modification of the standard protocol for solid phase synthesis of RNA. Three 4-thiouridine-containing oligoribonucleotides (21-mers), corresponding to tRNA minisubstrates of yeast tRNA:pseudouridine-55 synthase (Pus4) were prepared. These 4-thiouridine containing substrates were characterized and used as photoaffinity probe of the enzyme:substrate complex. Irradiation resulted in the specific photocross-linking of these oligoribonucleotides with purified recombinant tRNA:pseudouridine-55 synthase.

tRNA post-transcriptional modification enzymes of Xenopus laevis were proposed previously to belong to two major groups according to their sensitivity to structural perturbations in their substrates. To further investigate the structural variations tolerated by these enzymes, the tRNA-like domain of turnip yellow mosaic virus RNA (88 nucleotides in length) has been microinjected into the oocytes of Xenopus laevis. This RNA possesses 12 potential target nucleotides for modification within a structure including a pseudoknotted folding, an extended anticodon stem, and unusual D-loop/T-loop interactions. Results indicate that only cytosine-42, a position equivalent to C-49 in canonical tRNAs, was quantitatively modified into m5C in the microinjected RNA. Modification was detected to high levels, indicating that at least one enzyme tolerates non-canonical structural features.

The last 82 nucleotides of the 6.3 kb genomic RNA of plant turnip yellow mosaic virus (TYMV), the so-called 'tRNA-like' domain, presents functional, structural and primary sequence homologies with canonical tRNAs. In particular, one of the stem-loops resembles the TPsi(pseudouridine)-branch of tRNA, except for the presence of a guanosine at position 37 (numbering is from the 3'-end) instead of the classical uridine-55 in tRNA (numbering is from the 5'-end). Both the wild-type TYMV-RNA fragment and a variant, TYMV-mut G37U in which G-37 has been replaced by U-37, have been tested as potential substrates for the yeast tRNA modification enzymes. Results indicate that two modified nucleotides were formed upon incubation of the wild-type TYMV-fragment in a yeast extract: one Psi which formed quantitatively at position 65, and one ribothymidine (T) which formed at low level at position U-38. In the TYMV-mutant G37U, besides the quantitative formation of both Psi-65 and T-38, an additional Psi was detected at position 37. Modified nucleotides Psi-65, T-38 and Psi-37 in TYMV RNA are equivalent to Psi-27, T-54 and Psi-55 in tRNA, respectively. Purified yeast recombinant tRNA:Psisynthases (Pus1 and Pus4), which catalyze respectively the formation of Psi-27 and Psi-55 in yeast tRNAs, are shown to catalyze the quantitative formation of Psi-65 and Psi-37, respectively, in the tRNA-like 3'-domain of mutant TYMV RNA in vitro . These results are discussed in relation to structural elements that are needed by the corresponding enzymes in order to catalyze these post-transcriptional modification reactions.

The structural gene pfTRM1 (GenBank accession no. AF051912), encoding tRNA(guanine-26, N 2- N 2) methyltransferase (EC 2.1.1.32) of the strictly anaerobic hyperthermophilic archaeon Pyrococcus furiosus, has been identified by sequence similarity to the TRM1 gene of Saccharomyces cerevisiae (YDR120c). The pfTRM1 gene in a 3.0 kb restriction DNA fragment of P.furiosus genomic DNA has been cloned by library screening using a PCR probe to the 5'-part of the corresponding ORF. Sequence analysis revealed an entire ORF of 1143 bp encoding a polypeptide of 381 residues (calculated molecular mass 43.3 kDa). The deduced amino acid sequence of this newly identified gene shares significant similarity with the TRM1- like genes of three other archaea (Methanococcus jannaschii, Methanobacterium thermoautotrophicum and Archaeoglobus fulgidus), one eukaryon (Caenorhabditis elegans) and one hyperthermophilic eubacterium (Aquifex aeolicus). Two short consensus motifs for S-adenosyl-l-methionine binding are detected in the sequence of pfTrm1p. Cloning of the P.furiosus TRM1 gene in an Escherichia coli expression vector allowed expression of the recombinant protein (pfTrm1p) with an apparent molecular mass of 42 kDa. A protein extract from the transformed E.coli cells shows enzymatic activity for the quantitative formation of N 2, N 2-dimethylguanosine at position 26 in a transcript of yeast tRNAPhe used as substrate. The recombinant enzyme was also shown to modify bulk E.coli tRNAs in vivo.

Cell-free yeast extract has been successfully used to catalyze the enzymatic formation of 11 out of the 14 naturally occurring modified nucleotides in yeast tRNAPhe(anticodon GAA). They are m2G10, D17, m22G26, Cm32, Gm34,psi39, m5C40, m7G46, m5C49, T54 andpsi55. Only D16, Y37 and m1A58 were not formed under in vitro conditions. However, m1G37was quantitatively produced instead of Y37. The naturally occurring intron was absolutely required for m5C40formation while it hindered completely the enzymatic formation of Cm32, Gm34and m1G37. Enzymatic formation of m22G26,psi39, m7G46, m5C49, T54 andpsi55were not or only slightly affected by the presence of the intron. These results allow us to classify the different tRNA modification enzymes into three groups: intron insensitive, intron dependent, and those requiring the absence of the intron. The fact that truncated tRNAPheconsisting of the anticodon stem and loop prolonged with the 19 nucleotide long intron is a substrate for tRNA: cytosine-40 methylase demonstrates that the enzyme is not only strictly intron dependent, but also does not require fully structured tRNA.