Dissertations / Theses on the topic 'Modifications of tRNA'

AN ABSTRACT OF THE DISSERTATION OF MANISHA DEOGHARIA, for the Doctor of Philosophy degree in Molecular Biology, Microbiology and Biochemistry presented on May 16, 2018, at Southern Illinois University, Carbondale TITLE: PSEUDOURIDINE MODIFICATIONS IN HUMAN tRNAs AND ARCHAEAL rRNAs MAJOR PROFESSOR: DR. RAMESH GUPTA RNAs undergo several post-transcriptional modifications inside the cell. The most abundant modification found in RNA is pseudouridine. Pseudouridine is present in all major classes of RNA. The classical TΨC sequence of tRNA reflects T (ribothymidine or 5-methyluridine) at position 54 in most Bacteria and Eukarya, and Ψ and C at positions 55 and 56, respectively, in nearly all tRNAs. TrmA and TruB homologs produce T54 and Ψ55, respectively, in Bacteria and Eukarya. However, archaeal tRNAs commonly have Ψ54 (or m1Ψ54) instead of T54, and Pus10 produces both Ψ54 and Ψ55 in these tRNAs. The pus10 gene is present in nearly all Archaea and most eukaryotes, but not in Bacteria and yeast. This coincides with the presence of Ψ54 in archaeal tRNAs and certain tRNAs (for Gln, Trp, Pro Thr, etc.) of animals, and its absence in the tRNAs of Bacteria and yeast. tRNAs for Trp and Pro that function as primers for replication of retroviruses also contain Ψ54. We found that Pus10 is the Ψ54 synthase in eukaryotes. The Ψ54 activity is specific for certain tRNAs, and it requires a conserved Am1AAU sequence at positions 57-60 of the tRNA for its maximum activity. Recombinant Pus10 can also form Ψ54 in select tRNAs and presence of m1A at position 58 is necessary for its maximum activity. Humans have two paralogs of TruB, TruB1, and TruB2 which are predicted to be the Ψ55 synthases for cytoplasmic and mitochondrial tRNAs, respectively. We found that recombinant human Pus10 can also modify Ψ55 of tRNAs in vitro. This Ψ55 activity of human Pus10 is not selective for specific tRNAs. Another pseudouridine synthase, Cbf5, which functions in guide dependent manner, is necessary for Ψ production in 23S rRNA of H. volcanii. Cbf5 is the catalytic component of the box H/ACA ribonucleoprotein complex that brings about these modifications. It consists of a guide RNA and three core proteins Nop10, Gar1, and L7Ae along with Cbf5. We found that Nop10 is necessary for Ψ production in 23S rRNA.

Stable RNAs undergo a wide variety of post-transcriptional modifications, that add to the functional repertoire of these molecules. Some of these modifications are catalyzed by stand-alone protein enzymes, while some others are catalyzed by RNA-protein complexes. tRNAs from all domains of life contain many such modifications, that increase their structural stability and refine their decoding properties. Certain regions of tRNAs are more frequently modified than others. Two such regions are the anticodon loop, and the TψC stem. In the halophilic euryarchaeon Haloferax volcanii, tRNATrp and tRNAMet, both of which are transcribed as intron-containing pre-tRNA forms, contain Cm34 and ψ54, in addition to other modifications, in these two regions, respectively. The Cm34 modification in both cases is RNP-mediated: tRNATrp Cm34 formation being guided by its own intron, while that of tRNAMet being guided by a unique guide RNA called sR-tMet. We created genomic deletion of H. volcanii tRNATrp intron by homologous recombination based technique, and showed that this strain is viable, and does not demonstrate any observable growth phenotype. However, the corresponding modifications are absent in this intron-deleted strain. Our structural and functional characterizations of sR-tMet revealed that it is unique in its structural properties and deviates considerably from its homologs in other Archaea. We also identified a novel L7Ae (a core protein associated with archaeal methylation guide RNPs) binding motif in sR-tMet. ψ54, the near universal modification found in TψC stem-loop of archaeal tRNAs is catalyzed by the protein Pus10. An earlier study from our laboratory had shown that Pus10 from two different archaea, Methanocaldococcus jannaschii (MjPus10) and Pyrococcus furiosus (PfuPus10) have differential activities towards ψ54 formation. Using the crystal structure of Human Pus10 as template, we created homology models of MjPus10 and PfuPus10 proteins and identified several residues and motifs that might lead to this difference in activity. By a combination of both in vitro and in vivo mutational approaches, we confirmed several previously unidentified residues/motifs that serve as positive determinants of tRNA ψ54 formation. Finally, as an extension to this study, we have identified a novel tRNA ψ54 forming activity in mammalian nuclear extracts, and attributed this activity to Pus10.

Transfer RNAs (tRNAs) are key adaptor molecules that mediate decoding of messenger RNAs (mRNAs) into proteins by complementary pairing of their anticodons with mRNA codons. tRNAs that undergo adenosine to inosine editing at the wobble base, or position 34, display expanded codon decoding capacity as inosine enables pairing not only with uridine, but also with cytosine and adenosine. The essential heterodimeric enzyme Adenosine Deaminase Acting on Transfer RNA (ADAT) catalyzes this post-transcriptional modification in eukaryotes and is comprised of subunits ADAT2 and ADAT3. Emergence of heterodimeric ADAT has been proposed to have shaped both tRNA gene content and codon composition of eukaryotic genomes in such a way that these two features became mirrored. Although the exact contribution of wobble inosine (I34) to translation elongation has not been established, previous reports have suggested that it might play a role in improving translational efficiency and accuracy of genes enriched in codons recognized by I34-modified tRNAs. To further understand the role of the inosine modification in translation, we generated cell lines depleted in the catalytic subunit ADAT2. Silencing of ADAT2 lead to impaired cellular proliferation and had a variable impact on the expression of genes coding for extracellular matrix (ECM) proteins such as mucins. Notably, ADAT2 deficiency did not have major effects on the post-translational glycosylation of mucins, neither did it trigger the unfolded protein response. Supported by the absence of clear defects in decoding rates in ADAT2 depleted cells, as measured by ribosome profiling, our findings suggest that a reduced pool of I34-modified tRNAs might suffice to carry out cellular functions in steady-state conditions. However, we found that, under circumstances involving a high demand for these tRNAs such as airway remodeling, ADAT2 is required for the proper translation of an ECM gene enriched in stretches of codons read by I34-tRNAs. Taken together, our results suggest that the inosine modification is particularly relevant for the synthesis of ECM proteins during specialized processes including neural development and airway remodeling. The importance of the inosine modification has been recently underscored by the identification of pathogenic mutations in the gene encoding ADAT3, all of which share common neurodevelopmental phenotypes. The most prevalent mutation identified to date is a valine to methionine (V144M) substitution that is linked to intellectual disability and strabismus. In the present study we characterized human ADAT in terms of activity and quaternary structure, and investigated the effect of the ADAT3 V144M mutation on the enzyme. We showed that the V144M substitution leads to decreased enzymatic activity of ADAT, which might result from alterations in the tertiary structure and subcellular localization of ADAT3 that were found to be associated to the mutation.
Els ARNs de transferència (ARNt) són molècules que tenen un paper clau en el procés de traducció dels ARN missatgers (ARNm) en proteïnes mitjançant la interacció del seu anticodó amb codons d’ARNm. Els ARNt que passen per un procés d’edició d’adenosina a inosina a la base wobble, o posició 34, són capaços de llegir més d’un codó d’ARNm gràcies a la capacitat de la inosina de reconèixer els tres nucleòtids uridina, citidina i adenosina. L’enzim responsable d’aquesta modificació post-transcripcional en eucariotes s’anomena Adenosina Deaminasa específica per l’ARNt (ADAT), es tracta d’un complex heterodimèric format per les subunitats ADAT2 i ADAT3 que és essencial per a la viabilitat de l’organisme. Estudis previs han proposat que l’aparició d’ADAT va determinar el nombre de còpies gèniques de cada ARNt així com la composició de codons presents als genomes eucariòtics de tal manera que aquests dos factors estiguessin mútuament balancejats. Tot i que la contribució precisa de la inosina 34 (I34) a la traducció de proteïnes durant la fase d’elongació encara s’ha determinat experimentalment, algunes investigacions han suggerit que podria jugar un rol en l’eficiència i fidelitat de traducció de gens enriquits en codons reconeguts per ARNt modificats amb I34. Amb l’objectiu d’investigar el rol de la inosina en la traducció, hem generat línies cel·lulars on el gen codificant per ADAT2 ha estat silenciat. La depleció d’ADAT2 comporta un retard en el creixement cel·lular i té un efecte variable en l’expressió gènica de proteïnes de la matriu extracel·lular. El patró de modificacions post-traduccionals de glicosilació d’aquestes proteïnes no resulta alterat per la deficiència d’ADAT2, que tampoc activa la resposta a proteïnes desplegades. Juntament amb l’absència de defectes en la velocitat d’elongació analitzada per ribosome profiling, aquestes observacions suggereixen que la cèl·lula és capaç de dur a terme les seves funcions amb un nombre reduït d’ARNt modificats amb inosina. Hem vist, però, que en condicions que requereixen majors quantitats d’ARNt inosinats, la depleció d’ADAT2 dóna lloc a la traducció ineficient d’un gen de matriu extracel·lular altament enriquit en codons sensibles llegits per ARNt modificats. Així doncs, els nostres resultats indiquen que la inosina pot exercir un rol important en la síntesi de proteïnes de la matriu extracel·lular, particularment durant processos de desenvolupament neuronal i de remodelat de les vies respiratòries. La rellevància de la modificació I34 s’ha vist reforçada recentment per la identificació de mutacions de caire patogènic localitzades al gen que codifica ADAT3. Totes elles tenen en comú la presència de fenotips relacionats amb el desenvolupament neurològic. La mutació d’ADAT3 més comuna consisteix en la substitució d’un residu valina per un metionina (V144M) i està associada a la manifestació de discapacitat intel·lectual i estrabisme. En el present estudi hem caracteritzat l’activitat enzimàtica i l’estructura quaternària de l’ADAT humà, així com l’impacte de la mutació V144M d’ADAT3 en el complex heterdimèric. Els nostres revelen que la substitució V144M dóna lloc a una menor activitat enzimàtica d’ADAT. És possible que aquesta reducció es vegi influïda per les alteracions en l’estructura terciària i en la localització cel·lular d’ADAT3 que indueix la mutació.

In all the three domains of life, most RNAs undergo post transcriptional modifications both on the bases as well as the ribose sugars of the individual ribonucleotides. 2'-O-methylation of ribose sugars and isomerization of Uridines to Pseudouridines are two most predominant modifications in rRNAs and tRNAs across all domains of life. Besides 2'-O-methylation of ribose sugars, methylation of pseudouridine (Ø) at position 54 of tRNA, producing m1Ø, is a hallmark of many archaeal species but the specific methylase involved in the formation of this modification had yet to be characterized. A comparative genomics analysis had previously identified COG1901 (DUF358), part of the SPOUT superfamily, as a candidate for this missing methylase family. To test this prediction, the COG1901 encoding gene, HVO_1989, was deleted from the Haloferax volcanii genome. Analyses of modified base contents indicated that while m1Ø was present in tRNA extracted from the wild-type strain, it was absent from tRNA extracted from the mutant strain. Expression of the gene encoding COG1901 from Halobacterium sp. NRC-1, VNG1980C, complemented the m1Ø minus phenotype of the ÄHVO_1989 strain. This in vivo validation was extended with in vitro tests. Using the COG1901 recombinant enzyme from Methanocaldococcus jannaschii (Mj1640), purified enzyme Pus10 from M. jannaschii and full-size tRNA transcripts or TØ-arm (17-mer) fragments as substrates, the sequential pathway of m1Ø54 formation in Archaea was reconstituted. The methylation reaction is AdoMet-dependent. The efficiency of the methylase reaction depended on the identity of the residue at position 55 of the TØ-loop. The presence of Ø55 allowed the efficient conversion of Ø54 to m1Ø54, whereas in the presence of C55 the reaction was rather inefficient and no methylation reaction occurred if a purine was present at this position. These results led to renaming the Archaeal COG1901 members as TrmY proteins. Another aim of this study was to investigate the mechanism of target RNA recruitment to a box C/D sRNP. From data obtained, we have made the following hypothesis- aNop5p, either alone or as a heterodimer with Fibrillarin, binds to single stranded bulges and loops of target RNA. This aNop5p bound target is then hybridized to an assembling guide sRNP complex containing the guide RNA and L7Ae or guide RNA, L7Ae and aNop5p. If the guide:target sequences are complementary to each other, they hybridize and the target nucleotide gets modified. We also think that post modification, the guide and target strands separate, the core proteins rearrange themselves on the guide RNA and then prime the guide RNA for next round of modification. Compared to the general archaeal populations, haloarchaea contain significantly fewer number of box C/D guide RNAs. In archaea, previous studies have underscored the importance of a symmetric assembly of the core proteins on the sRNA. This meant that if the core proteins were unable to bind to either the terminal box C/D or the internal box C'/D' motifs, the sRNP was not efficient to carry out the modification of the target RNA. Essentially the only two haloarchaeal box C/D sRNPs known before had a symmetric architecture. In this study we discovered the first naturally occurring asymmetric box C/D sRNP called sR-41 in Haloferax volcannii. The architecture of Haloferax volcanii sR-41 box C/D sRNP seems to be closer in conformation to eukaryal snoRNPs (eukaryal counterparts of archaeal sRNPs) in which the core proteins assemble asymmetrically on the RNA. Till date, no information regarding the catalytic mechanism of an asymmetrically arranged eukaryal box C/D snoRNPs are available, because of unavailability of any assembly systems or crystal structures. Hence, this archaeal sR-41 guide sRNP provides a unique opportunity to study mechanism of modification in an asymmetrically arranged box C/D sRNP molecule.

Transfer RNA from all organisms has modified nucleosides and position 34 (the wobble position) is one of the most extensively modified positions. Some wobble nucleoside modifications restrict codon choice (e.g. 5-methylaminomethyl-2-thiouridine, mnm5s2U) while some extend the decoding capacity (e.g. uridine-5-oxyacetic acid, cmo5U). In this thesis the influence of wobble nucleoside modification on cell physiology and translation efficiency and accuracy is described. A mutant proL tRNA (proL207) was isolated that had an unmodified adenosine in the wobble position. Surprisingly, the proL207 mutant grows normally and is efficiently selected at the non-complementary CCC codon. The explanation of how an A34 containing tRNA can read CCC codon could be that a protonated A can form a base pair with C. cmo5U (uridine-5-oxyacetic acid) is present in the wobble position of five tRNA species in S.enterica. Two genes (cmoA and cmoB) have been identified that are involved in the synthetic pathway of cmo5U. Mutants were constructed in alanine, valine, proline, and threonine codon boxes which left only a cmo5U containing tRNA present in the cell. The influence of cmo5U on growth or on A site selection rates of the ternary complex was found to be tRNA dependent. During the study of the frameshift suppressor sufY of the hisC3737 frameshift mutation, a dominant mutation was found in YbbB protein, a selenouridine synthetase. The frameshifting occurs at CCC-CAA codon contexts and is specific for CAA codons, which are read by tRNAGlncmnm5s2UUG . The sufY204 mutation is a dominant mutation resulting in a change from Gly67 to Glu67 in the YbbB protein, and mediates the synthesis of several novel modified nucleosides/nucleotides (UKs) with unknown structure. The synthesis of these UKs is connected to the synthesis of cmnm5s2U34. The presence of UK on tRNAGlnU*UG reduced aminoacylation and therefore might account for the slow entry at CAA codons which could result in +1 frameshifting by P site tRNA. The selenourdine synthetase activity is not required for the synthesis of UKs. We hypothesize that an intrinsic activity that is low in the wild type protein has been elevated by the single amino acid substitution and results in the synthesis of UKs.

Covalent nucleotide modifications in RNAs affect numerous biological processes, and novel functions are continually being revealed even for well-known modifications. Among all RNA species, transfer RNAs (tRNAs) are highly enriched with diverse modifications, which are known to play roles in decoding and tRNA stability, charging, and cellular trafficking. However, studies of tRNA modifications have been limited in a small scale and performed by groups with different methodologies. To systematically compare the functions of a large set of noncoding RNA modifications in translational regulation, I carried out ribosome profiling in 57 budding yeast mutants lacking nonessential genes involved in tRNA modifications. Deletion mutants with enzymes known to modify the anticodon loop or non-tRNA substrates such as rRNA exhibited the most dramatic translational perturbations, including altered dwell time of ribosomes on relevant codons, and altered ribosome density in protein-coding regions or untranslated regions of specific genes. Several mutants that result in loss of tRNA modifications in locations away from the anticodon loop also exhibited altered dwell time of ribosomes on relevant codons. Translational upregulation of the nutrient-responsive transcription factor Gcn4 was observed in roughly half of the mutants, consistent with the previous studies of Gcn4 in response to numerous tRNA perturbations. This work also discovered unexpected roles for tRNA modifying enzymes in rRNA 2’-O-methylation, and in transcriptional regulation of TY retroelements. Taken together, this work revealed the importance and novel functions of tRNA modifications, and provides a rich resource for discovery of additional links between tRNA modifications and gene regulation.

La modification posttranscriptionnelle des acides ribonucléiques (ARNs) est une étape de maturation conservée dans tous les domaines du vivant. Mes travaux de thèse ont porté sur la caractérisation fonctionnelle et structurale de flavoenzymes impliquées dans la modification des ARN de transfert (ARNt) : les dihydrouridines synthases (Dus) dictant la formation de dihydrouridine via la flavine mononucléotide (FMN) et TrmFO responsable de la méthylation en C5 de l'uridine 54 via la flavine adénosine dinucléotide (FAD) ainsi que le methylènetétrahydrofolate. Afin d'élucider le mécanisme de TrmFO, nous avons élaboré une apoenzyme grâce à une simple mutation qui est efficacement reconstituée in vitro. Nous avons chimiquement synthétisé l'intermédiaire catalytique qui consiste en un FAD-iminium comportant un methylène sur le N5 de l'isoalloxazine. Cette espèce synthétique a été caractérisée par spectrométrie de masse et absorption UV-visible. La reconstitution de TrmFO avec cette molécule restore l'activité in vitro sur un ARNt transcrit prouvant le rôle du FAD comme agent méthylant via une méthylation réductrice. Dus2 réduit spécifiquement U20 et est constituée d'un Dus domaine néanmoins, chez les mammifères un double-stranded RNA-binding domaine (dsRBD) est présent. Afin de comprendre la fonction de cette organisation modulaire, nous avons montré que seule l'enzyme sauvage est active contrairement aux domaines isolés. Nous avons résolu les structures cristallographiques des deux domaines suggérant une redistribution des charges positives en surface. Ce dsRBD dicte la reconnaissance de l'ARNt en se fixant à la tige acceptrice/Tpsi. Ceci est régulé par une extension N-terminal, mis en évidence par des mutations, des titrations RMN ainsi qu’une structure cristallographique en complexe avec un ARN de 22 nucléotides. Ce travail illustre l’acquisition d’un dsRBD au cours de l’évolution dont la fonction est étendu à la reconnaissance des ARNts
Posttranscriptional modification of ribonucleic acids (RNAs) is a crucial maturation step conserved in all domains of life. During my thesis, I have brought structural and functional insights on flavoenzymes involved in transfer RNA (tRNA) modifications: dihydrouridine synthase (Dus) responsible for dihydrouridine formation using flavin mononucleotide (FMN) and TrmFO responsible for C5 methylation of uridine position 54 relying on flavin adenosine dinucleotide (FAD) and methylenetetrahydrofolate. To elucidate the chemical mechanism of TrmFO we designed an apoprotein via a single mutation that could be reconstituted in vitro with FAD. Furthermore, we chemically synthesized the postulated intermediate active species consisting of a flavin iminium harboring a methylene moiety on the isoalloxazine N5 that was further characterized by mass spectrometry and UV-visible spectroscopy. Reconstitution of TrmFO with this molecule restored in vitro activity on a tRNA transcript proving that TrmFO uses FAD as a methylating agent via a reductive methylation.Dus2 reduces U20 and is comprised of a canonical Dus domain however, mammals have an additional double-stranded RNA-binding domain (dsRBD). To bring functional insight for this modular organization, we showed that only full length human Dus2 was active while its isolated domains were not. tRNA recognition is driven by the dsRBD via binding the acceptor and TΨ stem of tRNA with higher affinity then dsRNA as evidenced by NMR. We further solved the X-ray structures for both domains showing redistribution of surface positive charges justifying the involvement of this dsRBD for tRNA recognition in mammalian Dus2. This was attributed to a peculiar N-terminal extension proven by mutational analysis and an X-ray structure of dsRBD in complex with 22-nucleotide dsRNA. Altogether our work illustrates how during evolution, Dus2 enzymes acquired an engineered dsRBD for efficient tRNA binding via a ruler mechanism

Toxicity of the zymocin complex from Kluyveromyces lactis, which causes cell death of Saccharomyces cerevisiae, relies on a specific tRNA anticodon modification in sensitive yeast cells. Primarily tRNAGlu with a highly modified uridine base in the anticodon wobble position (U34) serves as recognition and cleavage site for zymocin, which functions as a tRNA endoribonuclease. Cleavage results in depletion of essential tRNAs, translational breakdown and ultimately cell death. KTI11, URM1 and SIT4 are genes which confer zymocin sensitivity. Interestingly, deletion of each of these genes protects against zymocin due to U34 modification defects. Here, using genetic analysis, data was generated to further our understanding about how KTI11, URM1 and SIT4 function in the process of tRNA modification and zymocin dependent tRNA-cleavage. This work shows that Kti11 associates with two different protein complexes involved in translationally relevant modification pathways. Kti11 partners with the multifunctional Elongator complex and this association is crucial for Elongator functions in tRNA modification, explaining why kti11 mutants phenocopy Elongator minus cells. Furthermore, evidence is provide that Kti11 is subunit of a trimeric complex, Dph1•Dph2•Kti11, which is required to form a unique diphthamide modification on eEF2. Moreover, based on the zymocin resistance of urm1Δ cells and since Urm1 harbours features of prokaryotic sulphur carriers, it was hypothesised that Urm1 may be involved in U34 thio-modification. Data are presented suggesting that Urm1-Uba4 function as a sulphur relay system responsible for thiolation of tRNAs. Finally, this study focussed on the investigation of protein phosphatase Sit4. It is shown that Sit4 and Rrd1 form a functional phosphatase complex supporting Sit4 functions in the Target Of Rapamycin (TOR) pathway. Finally, the data suggest that SIT4, URM1 and KTI11 dependent tRNA anticodon modification has a signalling function and is implicated in the Nitrogen Catabolite Repression (NCR) branch of the TOR pathway.

Thesis (Honors)--Ohio State University, 2007.
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Mon étude a porté sur la spécificité de l'interaction entre deux protéines du parasite du paludisme (Plasmodium), tRip (tRNA import protein) et la tyrosyl-ARNt synthétase apicoplastique (api-TyrRS), avec l'ARN de transfert (ARNt). Plasmodium est un parasite intracellulaire qui conserve une organelle vestigiale, l’apicoplaste, qui possède son propre système de traduction. J’ai adapté la séquence de l’ARN messager pour produire l’api-TyrRS in vitro, et j’ai étudié la spécificité de la reconnaissance de l’ARNtTyr apicoplastique, qui évite les interactions erronées plutôt que de favoriser les correctes. La protéine tRip est située à la surface du parasite et est responsable de l’import des ARNt de l’hôte. Mes résultats suggèrent que cet import à lieu pendant la phase sanguine duparasite. Elle ne reconnait pas tous les ARNt de la même façon. Les modifications posttranscriptionnelles modulent l’affinité de tRip, et potentiellement, le taux d’import de cet ARNt. Finalement, j’ai identifié par SELEX une séquence nucléotidique qui se lie spécifiquement à tRip, un début pour la conception d'une molécule qui ciblerait spécifiquement le parasite du paludisme
My study focused on the specificity of the interaction between two proteins of the malaria parasite (Plasmodium), tRip (tRNA import protein) and the apicoplastic tyrosyl-tRNA synthetase (api-TyrRS), with the transfer RNA (tRNA). Plasmodium is an intracellular parasite with a vestigial organelle, the apicoplast, which has its own translation system. The messenger RNA sequence was adapted to produce api-TyrRS in vitro, and I studied the specificity of apicoplastic tRNATyr recognition, which avoids erroneous interactions rather than favoring the correct ones. The tRip protein is located on the surface of the parasite, and is responsible for importing tRNAs from the host. My results suggest that this import takes place during the blood phase of the parasite. In addition, not all tRNAs are recognized uniformly. The post-transcriptional modifications of the tRNAs define the affinity of tRip, and potentialy, the import rate of this tRNA. Finally, I identified a short nucleotide sequence that binds specifically to tRip. It is a good starting point for designing a molecule that specifically targets the malaria parasite

La traduction représente un processus central au sein de la cellule, elle assure le transfert de l’information génétique de l’ARNm vers les protéines. De nombreux acteurs y sont impliqués directement ou indirectement et parmi eux, chez les eucaryotes, la petite protéine Trm112. Celle-ci participe à la modification de plusieurs acteurs directs en interagissant et en activant quatre MTases. Le facteur de terminaison eRF1 est méthylé par le complexe Mtq2-Trm112, l’ARNr 18S par Bud23-Trm112 et certains ARNt par les complexes Trm9-Trm112 et Trm11-Trm112. Au cours de ce travail, les structures cristallographiques de Trm9-Trm112 et de Bud23-Trm112 de levure ont été résolues. L’étude comparative structurale de ces complexes et de la structure connue de Mtq2-Trm112, a permis de mettre en évidence que dans un même organisme, les séquences des trois protéines ont évolué de manière à conserver l’interaction avec Trm112. Même si les quatre partenaires présentent moins de 20% d’identité de séquence, les résidus clés pour l’interaction avec la petite protéine activatrice sont conservés ou partagent des caractéristiques identiques. En plus de l’analyse structurale, le complexe Trm9-Trm112 a fait l’objet d’une étude fonctionnelle chez S. cerevisiae ce qui a permis de cartographier le site actif de l’enzyme et de proposer un modèle de mécanisme d’action. Enfin, les premières études in vivo réalisées chez Haloferax volcanii suggèrent que cette plateforme serait également présente chez certains organismes procaryotes
Protein synthesis is a central process in the cell; it ensures the transfer of genetic information from mRNA in to protein. A lot of actors are involved directly or indirectly in translation. In Eukaryotes, Trm112, a small protein, interacts with and activates four methyltransferases modifying direct actors of translation. The termination factor eRF1 is methylated by the Mtq2-Trm112 complex, the 18S rRNA by Bud23-Trm112 and some tRNA by the Trm9-Trm112 and Trm11-Trm112 complexes. During this work, the crystal structures of Trm9-Trm112 and Bud23-Trm112 complexes from yeast were solved. The comparative analysis of these two new structures with Mtq2-Trm112 structure highlights the structural plasticity allowing Trm112 to interact through a very similar mode with its partners although those share less than 20% sequence identity. In the same organism, the key residues for the interaction with Trm112 are conserved or share similar characteristics. In addition to the structural analysis, the function of the Trm9-Trm112 complex was studied in S. cerevisiae. This analysis allowed to map the active site of the enzyme and to propose a model of its mechanism of action. Finally, the first data obtained in vivo, with the Archaea Haloferax volcanii suggest that the Trm112 platform might also be present in some prokaryotic organisms

This thesis describes the use of functional genomics screens in yeast to study anticancer drug action and intracellular transport. The yeast Saccharomyces cerevisiae provides a particularly useful model system for global drug screens, due to the availability of knockout mutants for all yeast genes. A complete collection of yeast deletion mutants was screened for sensitivity to monensin, a drug that affects intracellular transport. A total of 63 deletion mutants were recovered, and most of them were in genes involved in transport beyond the Golgi. Surprisingly, none of the V-ATPase subunits were identified. Further analysis showed that a V-ATPase mutant interacts synthetically with many of the monensin-sensitive mutants. This suggests that monensin may act by interfering with the maintenance of an acidic pH in the late secretory pathway. The second part of the thesis concerns identification of the underlying causes for susceptibility and resistance to the anticancer drug 5-fluorouracil (5-FU). In a functional genomics screen for 5-FU sensitivity, 138 mutants were identified. Mutants affecting tRNA modifications were particularly sensitive to 5-FU. The cytotoxic effect of 5-FU is strongly enhanced in these mutants at higher temperature, which suggests that tRNAs are destabilized in the presence of 5-FU. Consistent with this, higher temperatures also potentiate the effect of 5-FU on wild type yeast cells. In a plasmid screen, five genes were found to confer resistance to 5-FU when overexpressed. Two of these genes, CPA1 and CPA2 encode the two subunits of the arginine-specific carbamoyl-phosphate synthase. The three other genes, HMS1, YAE1 and YJL055W are partially dependent on CPA1 and CPA2 for their effects on 5-FU resistance. The specific incorporation of [14C]5-FU into tRNA is diminished in all overexpressor strains, which suggest that they may affect the pyrimidine biosynthetic pathway.

The complexity of all biomolecules in existence today can be attributed to the variation of the amino acid repertoire. In nature, 20 canonical amino acids are translated to form these biomolecules, however, many of these amino acids have revealed posttranslational modifications (i.e. acetylation, methylation) after incorporation. Amino acids that exhibit PTM are known for their involvement in cellular processes such as DNA repair and DNA replication; these PTMs are commonly found on histones within the chromatin complex. Utilization of in vivo site-specific incorporation has recently reported functionality of post-translationally modified amino acids.1 xii Here we report the synthesis and in vivo site-specific incorporation of the histone PTM, 2-hydroxyisobutyrl lysine (Khib), with the pyrrolysyl tRNA/ RNA synthetase system. This translational machine can better serve to probe Khib for functional benefits. Additionally, this thesis focuses much of its attention on the development of unnatural amino acids (UAA) with optogenetic characteristics. These UAAs, if site-specifically incorporated, can be used to control enzymes and proteins through rapid light perturbation (365nm UV light). Furthermore, discussed is the synthesis of photo-caged threonine and photo-caged serine as potential substrates for the pyrrolysyl translational machinery.

La plupart de ARN de transfert (tRNA) subissent des modifications post-transcriptionnelle nécessaires à leur fonction. La modification t6A (N6-threonylcarbamoyladenosine) présente en position 37 des ARNt spécifiques des codons ANN, joue un rôle primordial dans la fidélité de la traduction (appariement correct avec le codon AUG initiateur ; prévention des décalages de phase de lecture etc.). La modification t6A est catalysée en deux étapes par les protéines de la famille Sua5 /YrdC (aboutissant à la synthèse d’un intermédiaire TCA : threonylcarbamoyladenylate) puis transfert de l’entité Carbamoylthreonine du TCA sur l’ARNt via les protéines du complexe KEOPS chez les eucaryotes et archae ou des protéines YgjD, YeaZ et YjeE chez les bactéries ou encore de la protéine Qri7 dans les mitochondries de levures. Le complexe KEOPS comprend les 4 sous-unités suivantes : Kae1, Bud32, Cgi121 et Pcc1 auxquelles s’ajoutent une 5ème sous-unité (Gon7) retrouvée uniquement chez la levure. Alors que YgjD est l’homologue bactérien de la protéine Kae1, YeaZ et YjeE n’ont pas d’homologue chez les eucaryotes ni les archées. Jusqu’à présent, les mécanismes catalytiques responsables de la modification t6A restent peu connus.Nous présentons dans cette thèse une série d’études structure-fonction de plusieurs protéines impliquées dans la biosynthèse de la modification t6A : Sua5 de P. Abyssi ; les sous-complexes Bud32-Cgi121 et Gon7-Pcc1 de S. cerevisiae ainsi que le sous-complexe YgjD-YeaZ de E. coli. Les principaux résultats confirment que Sua5/YrdC est l’acteur majeur de la synthèse de l’intermédiaire TCA via son activité pyrophosphatase. Dans la levure, la protéine Gon7, empêche l’homodimérisation de Pcc1 qui ne peut plus induire de dimérisation du complexe entier (alors que c’est le cas chez les archées pour lesquelles Gon7 est absente). La structure du sous-complexe Bud32-Cgi121 de levure fournit des informations essentielles quant à son rôle de Kinase et d’ATPase au sein du complexe KEOPS. Ensemble, ces deux structures Bud32-Cgi121 et Gon7-Pcc1 nous permettent de proposer un modèle pentamérique du complexe KEOPS. Enfin, concernant les protéines bactériennes, nous montrons que l’activité ATPase de YjeE est stimulée par son association au complexe YgjD-YeaZ et que la formation du complexe ternaire YgjD-YeaZ-YjeE a lieu en présence d’ATP. Nous proposons un modèle structural de ce complexe ternaire pouvant expliquer les rôles des protéines YeaZ et YjeE dans la modification t6A.L’ensemble des études structurales abordées dans cette thèse permet donc de mieux comprendre le mécanisme catalytique de la modification t6A essentielle et ubiquitaire dans les 3 royaumes de la vie
Most tRNAs undergo chemical modifications during their maturation after the transcription. N6-threonylcarbamoyladenosine (t6A) is universally present at position 37 of tRNAs that recognize ANN-codons. tRNA t6A plays an essential role in translational fidelity through enhancing the codon-anticodon interaction. Recently, the tRNA t6A-modifying enzymes have been identified and characterized in bacteria, archaea and yeast. The biosynthesis of tRNA t6A proceeds in two main steps: first, the biosynthesis of an unstable intermediate threonylcarbamoyladenylate (TCA) by Sua5/YrdC family protein, using ATP, L-threonine, bicarbonate as substrates; second, the transfer of threonylcarbamoyl-moiety from TCA onto A37 of cognate tRNAs by a set of other proteins that use Kae1/Qri7/YgjD family proteins as a catalytic component. Though the biosynthesis of tRNA t6A could be accomplished by Sua5 and Qri7 in yeast mitochondria, the t6A biosynthesis in archaea and yeast cytoplasm requires Sua5 and KEOPS protein complex, which consists of Kae1, Bud32, Cgi121, Pcc1 in archaea, and a fifth Gon7 in yeast. In bacteria, it requires YrdC, YgjD, YeaZ and YjeE, of which YeaZ and YjeE are not related to any KEOPS subunits. Presently, the molecular mechanism of Sua5/YrdC in catalyzing the TCA biosynthesis is not well understood; How the KEOPS subunits assembly and cooperatively transfer threonylcarbamoyl-moiety from TCA to tRNA is not known; The contribution of YeaZ and YjeE in t6A biosynthesis in bacteria still remains to be probed.In this study, we report crystal structures of P. abyssi Sua5, S. cerevisiae Gon7/Pcc1 and Bud32/Cgi121 binary complexes, and E. coli YgjD-YeaZ heterodimer. Based on the information revealed by the crystal structures, advanced biochemical characterizations were carried out to validate the hypotheses. We confirm first that Sua5/YrdC is capable of catalyzing the TCA biosynthesis using substrates of ATP, L-threonine, and bicarbonate. The structure of P. abyssi Sua5 in complex with pyrophosphate provides a basis for its ATP-pyrophosphatase activity. Second, the structure of Gon7 reveals that it functions as a structural mimic of Pcc1 and therefore prevents the formation of Pcc1 homodimer, which mediates the formation of a dimer of tetrameric KEOPS from archaea. The structure of Bud32-Cgi121 in complex with ADP provides a basis in support of the dual kinase and ATPase activities of Bud32. We present a structural model of yeast KEOPS that exists as a heteropentamer. Third, we discovered that the weak intrinsic ATPase activity of YjeE is activated by YgjD-YeaZ heterodimer. YgjD, YeaZ and YjeE associate and form a ternary complex that is regulated by both the formation of YgjD-YeaZ heterodimer and the binding of ATP to YjeE. The model of YgjD-YeaZ-YjeE ternary complex provides structural insight into the essential role of YeaZ and YjeE in t6A biosynthesis in bacteria. This work provides structural insights into understanding the biosynthesis of tRNA t6A that is essential and ubiquitous in all three domains of life

The yeast EKC (Endopeptidase-like and Kinase associated to transcribed Chromatin)/KEOPS (Kinase, Endopeptidase and Other Proteins of small Size) complex is composed of five subunits: three small proteins (Pcc1, Pcc2, Cgi121, without known enzymatic signatures), the atypical protein kinase Bud32, and Kae1, whose actual function remains unknown. With the only exception of Pcc2 subunit, which is restricted to Fungi, the whole EKC/KEOPS complex is conserved in Archaea and Eukarya, while Kae1, the most conserved and ancient member of the complex, shows clear orthologs also in Bacteria. Although this complex has been implicated in several cellular processes, including transcription, telomere homeostasis and genomic stability maintenance, its precise mechanism of action or detailed molecular function has never been described so far. My PhD research work has been part of a broad network of collaborations aimed to elucidate the molecular mechanism(s) of action of the complex, adopting several different approaches and using Saccharomyces cerevisiae as model organism. At the beginning of my PhD, several data indicating Kae1 as a Bud32 substrate were available, and I was involved in the study of this enzyme/substrate relationship. In particular, I tried to define the importance and the physiological role of the phosphorylation of Kae1 at Ser367, identified by MS analyses as one of the Kae1 residues phosphorylated by Bud32. Results of an in vitro kinase assay, performed using a recombinant Kae1 mutant protein not phosphorylatable at position 367 as substrate of a recombinant Bud32 kinase, together with the phenotypic analysis of the yeast kae1-S367A mutant strain, showed that Serine367 is not the principal residue of Kae1 phosphorylated by Bud32. To identify and define the importance of other Kae1 residues phosphorylated by Bud32, further analyses will be required. In parallel, transcriptome analyses of EKC/KEOPS mutants (performed by the group directed by Frank Holstege) revealed a specific profile of upregulated genes that is highly enriched in targets of the Gcn4 transcriptional activator. Expression of GCN4, the key player of the general amino acid control, is regulated at the translational level via a well-studied mechanism of re-initiation depending on the presence of four short upstream open reading frames (uORFs 1 to 4) present in the 5’ UTR of the GCN4 mRNA. Using Gcn4-LacZ fusion reporters, we show that translation of GCN4 is derepressed in the EKC/KEOPS mutants due to the defective recognition of the inhibitory uORFs. Other collaborators and also two independent groups showed that EKC/KEOPS mutants are defective for the universally conserved N6-threonylcarbamoyl adenosine modification at position 37 (t6A37) of the anticodon stem-loop of all tRNAs decoding ANN codons, including the initiator tRNAiMet. This modification is required for proper codon-anticodon loop recognition and stability of this interaction. t6A37 defect impairs translation at different levels, in particular at the initiation step, and it is the cause of Gcn4 derepression in EKC/KEOPS mutants. These findings are further supported by strong genetic interactions of EKC/KEOPS mutants with the translation initiation factor eIF5, as well as threonine biosynthesis and tRNA metabolism genes. At this point we wondered if EKC/KEOPS complex is able to modify not only tRNAs, but also other RNA species, by the addition of a threonylcarbamoyl group to adenosine nucleotides. So I tried to re-produce a published experiment of primer extension, which correlated the absence of the t6A modification in EKC/KEOPS mutants to the disappearance/decrease of the “stop band” relative to A37, produced during the retro-transcription of an ANN-decoding tRNA primed by radioabeled oligonucleotides. The idea was that once fine-tuned the technique on a substrate already tested, I could analyze any other RNA species, choosing the appropriate oligonucleotide to extend. Even if I tested different experimental conditions, identical or comparable to those published, I never got the same result. In order to identify RNAs bound by the EKC/KEOPS complex, we chose a strategy associating RNA-immunoprecipitation (RIP) with deep-sequencing. In the final session of results, I describe the different steps of RIP experiment that we started to set up. The main finding arisen from the work presented in this thesis is that the EKC/KEOPS complex strongly impacts translation, accordingly with its direct role in tRNA modification, providing a novel twist to understanding its primary function and its impact on several essential cellular functions like transcription and telomere homeostasis
Il complesso EKC/KEOPS è costituito nel lievito da cinque subunità: tre piccole proteine (Pcc1, Pcc2 e Cgi121), di cui non sono note caratteristiche biochimiche rilevanti, Bud32, una protein chinasi atipica e Kae1, la cui funzione è sconosciuta. Fatta eccezione per Pcc2, presente solo nei Funghi, l’intero complesso EKC/KEOPS è conservato negli Archaea e negli Eucarioti, mentre Kae1, il membro più conservato e, probabilmente, più antico del complesso, presenta ortologhi anche nei Batteri. Sebbene il complesso sia implicato in diversi processi cellulari, tra cui trascrizione, omeostasi telomerica e mantenimento della stabilità genomica, il suo preciso meccanismo d’azione o la sua precisa funzione molecolare non sono mai stati descritti finora. Il mio lavoro di Ricerca svolto durante il Dottorato è inserito in una rete di collaborazioni volta ad elucidare i possibili meccanismi d’azione molecolare del complesso, adottando diversi approcci sperimentali e usando il lievito Saccharomyces cerevisiae come organismo modello. All’inizio del mio Dottorato, ho preso parte allo studio della relazione enzima/substrato che già diversi dati indicavano per Bud32 e Kae1. In particolare, ho provato a definire l’importanza e il ruolo fisiologico della fosforilazione della serina in posizione 367 di Kae1, identificata, grazie ad analisi di spettrometria di massa (MS), come uno dei residui di Kae1 fosforilati da Bud32. I risultati di un test di fosforilazione in vitro, condotto usando proteine ricombinanti, tra cui una forma di Kae1 mutagenizzata (e quindi non più fosforilabile) in posizione 367 come substrato di Bud32, assieme all’analisi del fenotipo di crescita di un ceppo di lievito mutante kae1-S367A, indicano che la serina367 non è il principale residuo di Kae1 ad essere fosforilato da Bud32. Per poter identificare e definire l’importanza di altri residui di Kae1 fosforilati da Bud32, saranno quindi necessarie ulteriori analisi. Parallelamente, l’analisi del trascrittoma di ceppi esprimenti subunità mutanti del complesso EKC/KEOPS (eseguite dal gruppo diretto da Frank Holstege), ha rivelato uno specifico profilo di geni up-regolati: fra i trascritti risultati particolarmente arricchiti sono stati identificati quelli corrispondenti ai geni bersaglio dell’attivatore trascrizionale Gcn4. L’espressione di GCN4, il principale regolatore del controllo generale degli amminoacidi, è regolata a livello traduzionale, attraverso un meccanismo di regolazione dell’iniziazione che dipende da 4 ORFs presenti nella regione 5’ UTR dell’mRNA di GCN4 (uORFs 1-4). Usando diversi reporter Gcn4-LacZ, abbiamo dimostrato che la traduzione di GCN4 è derepressa nei mutanti EKC/KEOPS, in seguito a una ridotta capacità di riconoscere le uORFs inibitorie. Altri collaboratori e anche due gruppi in modo indipendente hanno dimostrato il deficit della modificazione universalmente conservata t6A37 (una treonil-carbamoilazione del residuo di adenosina in posizione 37 di tutti i tRNA che decodificano codoni ANN, incluso il tRNA iniziatore tRNAiMet) nei mutanti EKC/KEOPS. Questa modificazione è necessaria per il corretto riconoscimento codone-anticodone e per la stabilità di questa interazione. Un difetto di t6A37 altera la traduzione a diversi livelli, in particolare la fase iniziale della traduzione, ed è la causa della derepressione di Gcn4 nei mutanti EKC/KEOPS. Ciò è inoltre supportato da dati che dimostrano l’esistenza di una forte interazione genetica tra i mutanti EKC/KEOPS e il fattore eIF5 implicato nell’inizio della traduzione, ma anche con geni coinvolti nella biosintesi della treonina o nel metabolismo dei tRNA. A questo punto, ci siamo chiesti se il complesso EKC/KEOPS sia in grado di modificare, mediante l’aggiunta di un gruppo treonil-carbamoilico all’adenosina, anche altri RNA oltre ai tRNA. Ho quindi tentato di riprodurre un esperimento di “primer extension” già pubblicato, in cui si correla l’assenza della modificazione t6A con la scomparsa/diminuzione di una banda d’arresto in corrispondenza del nucleotide A37, prodotta durante l’allungamento di un primer radiomarcato durante la retrotrascrizione di un tRNA che decodifica un codone ANN. Una volta messa a punto la tecnica usando un substrato già testato, avrei potuto analizzare qualsiasi altro tipo di RNA, scegliendo un appropriato oligonucleotide da estendere. Tuttavia, anche usando diverse condizioni sperimentali identiche o comparabili a quelle pubblicate, non sono mai riuscita ad ottenere lo stesso risultato. Allo scopo di identificare gli RNA legati dal compesso EKC/KEOPS, abbiamo scelto una strategia che associa l’immunoprecipitazione dell’RNA (RIP) al suo sequenziamento. Nella parte finale dei risultati descrivo le diverse tappe dell’esperimento RIP che ho iniziato a mettere a punto. Il risultato principale derivante dal lavoro presentato in questa tesi è la dimostrazione del coinvolgimento del complesso EKC/KEOPS nel processo di traduzione, come conseguenza del suo ruolo diretto nella modificazione t6A37 dei tRNAs. Questo risultato rappresenta una nuova svolta nella comprensione della funzione primaria del complesso e del suo impatto su diverse funzioni cellulari essenziali, tra cui la trascrizione e l’omeostasi dei telomeri

tRNA molecules undergo extensive post-transcriptional modifications to produce several variations of the four canonical nucleotides. Despite the huge number of nucleotide modifications that have been identified in tRNAs, to date their biological roles and regulations continue to be poorly understood. The uridines at the wobble position of the eukaryotic cytoplasmic tRNAs tK^UUU, tQ^UUG and tE^UUC are methoxycarbonylmethylated and thiolated to form mcm⁵s²U₃₄ by the ELP- and URM1-pathways, respectively. Several in vitro experiments have implicated these modifications in modulating wobbling capacity. Moreover, mutations in the ELP- and URM1-pathway genes have been associated with physiological defects in several organisms, including complex neurological disorders in humans. In this thesis we used a systems biology approach to study the in vivo functional relevance of mcm⁵s²U₃₄. A sensitive, robust and quantitative proteomics workflow was developed and applied to investigate differential proteome composition in budding yeast mutants deficient in U₃₄ modifications. We find that, in vivo and under normal conditions, mcm⁵s²U₃₄ fine tunes proteome composition by ensuring efficient translation of mRNAs biased for AAA, CAA and GAA codons. Importantly, our results connect these tRNA modifications with various cellular stress response pathways. Follow up analyses of yeast cells subjected to environmental stresses were conducted and led to the discovery that the biosynthesis of mcm⁵s²U₃₄ is dynamically regulated in response to growth conditions in a URM1-pathway dependent fashion. We propose that this regulation allows the cells to adjust their translational capacity during unfavourable growth conditions and contributes to the management of the environmental stress response. Overall, this thesis presents the first extensive investigation of the functional relevance of tRNA nucleotide modifications and reports one of the few known cases wherein cells regulate the levels of modified nucleotides to fine tune their metabolism in response to environmental cues. We expect that dynamic modulation of RNA modifications will prove to be a more general regulatory mechanism of cellular processes. The experimental and analytical approaches presented in this dissertation will provide a general framework for future studies in this field.

Doutoramento em Biomedicina
Protein synthesis is central to life and is being intensively studied at various levels. The exception is mRNA translational fidelity whose study has been hampered by technical difficulties in detecting amino acid misincorporations in proteins. Few genes have so far been associated to the control of protein synthesis fidelity and it is unclear how many genes control this biological process. We investigated the role of RNA modification by RNA modifying enzymes (RNAmods) in protein synthesis efficiency and accuracy. Our hypothesis was that RNAmods that modify tRNA nucleosides (tRNAmods) have a significant impact on protein synthesis through modulation of codonanticodon interactions. To address this issue, we focused our work on tRNAmods involved in the modification of tRNA anticodons. The biology of these enzymes is still poorly understood, but they are involved in RNA processing, stability and function and their deregulation is associated with cancer, neurodegenerative, metabolic and other diseases. We have set up a yeast genetic screen and used mass-spectrometry methods to determine the role of tRNAmods on proteome homeostasis. Our work identified a subgroup of yeast tRNAmods that play essential roles in protein synthesis fidelity and folding. The genes that encode insoluble proteins isolated from yeast cells lacking U34 modification were enriched in codon sites that are decoded by the hypomodified tRNAs. These aggregated proteins also participate in specific biological processes, suggesting that tRNAmods are linked to specific physiological pathways. Interestingly, we detected amino acid misincorporations at the codon sites decoded by the anticodons of the hypomodified tRNAs, demonstrating that tRNA U34 modifications control translational error rate.
A síntese proteica é central para a vida e tem sido extensivamente estudada a vários níveis. Contudo, o estudo da fidelidade da tradução do mRNA tem progredido lentamente devido a dificuldades técnicas na deteção de incorporações incorretas de aminoácidos nas proteínas. Poucos genes têm sido associados com o controlo da fidelidade da síntese proteica e não é evidente quais os genes que controlam este processo biológico. Nesta tese investigámos o papel da modificação dos nucleósidos do RNA na eficiência e precisão da síntese proteica. A nossa hipótese é que as enzimas que modificam nucleósidos do tRNA (tRNAmods) têm um impacto significativo na síntese proteica através da modulação das interações codão-anticodão. A biologia das tRNAmods e das modificações do tRNA são ainda pouco conhecidas, mas estão envolvidas na estabilidade e função do RNA e mutações nos seus genes causam doenças neurodegenerativas, metabólicas, cancro, entre outras. Neste projeto realizámos um rastreio genético em levedura com o objetivo de identificar tRNAmods que asseguram a homeostase do proteoma (proteostase) e usámos espectrometria de massa para clarificar o papel das tRNAmods na fidelidade da síntese proteica. Os resultados do estudo genético mostram que um sub-grupo de tRNAmods envolvidas na modificação de nucleósidos do anticodão do tRNA são essenciais para manter a estabilidade do proteoma. Outras tRNAmods estudadas não produziram impactos visíveis na proteostase. Os genes de proteínas agregadas que isolámos a partir de células de levedura com tRNAs hipomodificados são enriquecidos em codões descodificados por estes tRNAs. Os nossos dados mostram também que tais proteínas participam em processos biológicos específicos e têm níveis de aminoácidos errados mais elevados que as células wild-type. Estes dados mostram que certas modificações do tRNA são essenciais para a fisiologia celular, estabilidade do proteoma e fidelidade da síntese proteica.

Le séquençage à haut débit est une technique très utile pour l’étude des ARN. Pendant mon doctorat, nous l’avons utilisé pour la caractérisation des ARN extracellulaire (ARNex) du plasma humain. Les ARNex du plasma sont retrouvés soit à l’état soluble sous forme de complexes ribonucléoprotéiques (RNP) ou encapsulés au sein de vésicules extracellulaires (VE) de diverses origines (exosomes, microvesicles, …). Dans ce projet, j’ai démontré que le plasma contenait principalement des micro ARN, le fragment hY4 et des ARN ribosomiques dégradés. Par ailleurs, après chromatographie à exclusion de tailles ou par traitement consécutif protéinase K/RNase A, des VE hautement purifiées peuvent être obtenus. Nous ne retrouvons plus en majorité les micro ARN et l’ARN hY4 dans ces échantillons mais plutôt des ARN du microbiote humain, montrant une composition différente entre les ARNex solubles et ceux des vésicules purifiées. Par ailleurs, j’ai également effectué une étude comparative de kits commerciaux qui sont supposés purifier les exosomes par précipitation. La composition en ARN de ces fractions est très similaire au plasma humain total, montrant une forte contamination par les RNP solubles. Ainsi, nous sommes en mesure de proposer un protocole pour l’étude des ARNex dans le cadre de biopsies liquides avec des échantillons cliniques afin de découvrir de potentiels biomarqueurs de diagnostic. Au-delà de la caractérisation d’ARN, le séquençage à haut-débit peut être utilisé pour la détection et quantification des modifications post-transcriptionnelles. Pendant ma thèse, j’ai utilisé le séquençage pour l’analyse des 2’O-méthylation des ARN de transfert chez E. coli par RiboMethSeq. Sous plusieurs conditions de stress (manque de nutriments ou des concentrations non létales d’antibiotiques), certaines 2’O-méthylations montrent une réponse adaptative. Alors que plus de la moitié des 2’O-méthylations en position 18 (Gm18) sont augmentées dans toutes les conditions de stress étudiées, les positions Nm34 montrent un effet opposé avec une diminution dans certains stress (chloramphénicol et streptomycine). Chacun de ces deux comportements peut être relié à un phénomène de régulation cellulaire en réponse au stress : un changement au niveau de la wobble base pourrait être un moyen de réguler la traduction en modifiant l’usage des codons. En ce qui concerne Gm18, son rôle dans l’évasion du système immunitaire inné lors de l’invasion d’un hôte est en cours d’élucidation
For less than a decade, high-throughput sequencing became a very powerful, sensitive and precise technique for the study of ribonucleic acids. During my PhD thesis, I used this technology for in-depth characterization of the extracellular RNA (exRNA) content of human plasma. exRNA in plasma exists either in a “soluble state” as a component of ribonucleoprotein (RNP) complexes or encapsulated into extracellular vesicles (EV) of diverse origins (exosomes, microvesicles, …). In this project, I demonstrated that whole human plasma contains mostly micro RNA and the fragment of RNA hY4, as well as degraded ribosomal RNA. Moreover, using a rigorous strategy via size exclusion chromatography or consecutive proteinase K/RNase A treatments, highly purified EVs can be obtained. miRNAs and RNA hY4 fragments were not present in majority of samples, demonstrating a huge difference between soluble exRNA and exRNA from purified EVs. The RNA content of these EVs mainly reflects RNA composition of human microbiota. In addition, I also performed a comparative analysis of commercially available “exosome-enrichment” kits which are supposed to purify human exosomes by precipitation. Their RNA composition was found to be almost identical to human plasma, showing strong uncontrolled contamination by soluble RNPs. Based on this study, we were able to propose a protocol for studies in exRNA in the field of liquid biopsies with clinical sample in order to discover new diagnostic biomarkers. Apart from the characterization of RNA, high-throughput sequencing can be used for detection and quantification of RNA post-transcriptional modifications. During my PhD thesis I applied deep sequencing for analysis of transfer RNA (tRNA) 2’-O-methylations in model bacteria (E. coli) using RiboMethSeq. Under several stress conditions, such as starvation and non-lethal antibiotics concentrations, some 2’-O-methylated nucleotides show an adaptive response. While over than half of Gm18 show a global increase under all investigated stress conditions, ribomethylated residues at position 34 show an opposite effect for some antibiotic treatments (chloramphenicol and streptomycin). Each of these dynamic profiles can be linked to cell regulation in response to stress. Change at the tRNA wobble base (position 34) could be a way to regulate translation by modifying the codon usage. Concerning Gm18, its role in the escape from the human innate immune system during host invasion is currently elucidated

Les ARN de transfert (ARNt) sont des composants essentiels de la machinerie de traduction génétique. Pour être fonctionnels, ces ARNt subissent des modifications chimiques post-transcriptionnelles. Ces modifications permettent d’améliorer la reconnaissance entre l’ARNt et ses partenaires durant la traduction et assurent ainsi la fidélité et l’efficacité de la traduction. Le soufre est présent dans plusieurs nucléosides au sein de ces ARNt, comme dérivé de la thiouridine (s4U8, s2U34, m5s2U54), de la 2-thioadénosine (ms2i6A37, ms2t6A37) et la 2-thiocytidine (s2C32).Mon projet a consisté en l’étude structurale et fonctionnelle de la famille d’enzymes TtcA/TtuA, une superfamille dépendante d’un centre [FeS] impliquée dans la thiolation des ARNt.La thiolation de la méthyl-uridine universellement conservée à la position 54, catalysée par l’enzyme TtuA, stabilise les ARNt de bactéries thermophiles et d’archées hyperthermophiles et est nécessaire pour la croissance à haute température de ces organismes. La thiolation de l’uridine en position 34 dans la boucle de l’anticodon, qui est nécessaire pour une croissance normale et la résistance aux stress chez la levure, est effectuée par deux systèmes complètement différents : la protéine MmnA qui a été bien étudiéE et est présente chez les bactéries et les mitochondries des organismes eucaryotes et les protéines NcsA/NcsA/Ctu1 dans tous les autres organismes, dont le cytoplasme des eucaryotes.Des études spectroscopiques, cristallographique et des tests d’activité de TtuA et NcsA ont montré que : (i) le centre [4Fe-4S] est coordonné par seulement trois cystéines qui sont entièrement conservées, permettant au quatrième fer de fixer du sulfure exogène, qui agit probablement comme agent sulfurant ; (ii) le site de fixation de l’ATP est adjacent au centre [4Fe-4S]. Un nouveau mécanisme de sulfuration des ARNt a été proposé, dans lequel l’atome de fer non liant du centre [4Fe-4S] catalytique fonctionne comme transporteur de soufre, ouvrant ainsi de nouvelles perspectives sur la fonction des centres [Fe-S] en biologie
Transfer RNAs are essential components of cellular translation machinery. To achieve their function they possess several post-transcriptional chemical modifications. These modifications improve recognition between tRNA and its partners during translation and thus ensure translation fidelity and efficiency. Sulfur is present in several of these modified nucleosides: as thiouridine and its derivatives (s4U8, s2U34, m5s2U54), 2-thioadenosine derivatives (ms2i6A37, ms2t6A37) and 2-thiocytidine (s2C32).My project consisted in the structural and functional study of enzymes of the TtcA/TtuA family a [4Fe-4S]-dependent superfamily, involved in the thiolation of transfer RNAs (tRNAs).My aim was to show that enzymes that catalyze the simple non-redox substitution of the C2-uridine carbonyl oxygen by sulfur at position 54 (TtuA) and 34 (Ncs6/Ctu1/NcsA) in tRNAs use an iron-sulfur cluster cofactor and elucidate the biochemical and structural mechanisms of the TtuA and NcsA reactions.The thiolation of the universally conserved methyl-uridine at position 54, catalyzed by TtuA, stabilizes tRNAs from thermophilic bacteria and hyperthermophilic archaea and is required for growth at high temperature of these organisms. On the other hand, the thiolation of uridine 34 in the anticodon loop of tRNAs, which is required for normal growth and stress resistance in yeast, is carried out by two completely different systems: the well-studied MnmA protein (present in bacteria and in the eukaryotic mitochondrion) and the Nsc6/NcsA/Ctu1 proteins in all other organisms, including the eukaryotic cytoplasm.Spectroscopic and crystallographic analysis, together with activity tests enzymatic of TtuA and NcsA showed that: (i) the [4Fe-4S] cluster is ligated by three cysteines only that are fully conserved, allowing the fourth unique iron to bind an exogenous sulfide, which likely acts as the sulfurating agent; (ii) the ATP-binding site is adjacent to the cluster. A new mechanism for tRNA sulfuration was proposed, in which the unique iron of the catalytic [4Fe-4S] cluster functions as a sulfur carrier, opening new perspectives regarding functions of iron-sulfur cluster in biology

Studies of ribosomal reading frame maintenance are often based on frameshift mutation suppression experiments. In this thesis, suppression of a frameshift mutation in Salmonella enterica serovar Typhimurium by a tRNA and a ribosomal protein are described. The +1 frameshift mutation hisC3072 (that contains an extra G in a run of Gs) is corrected by mutations in the argU gene coding for the minor tRNAArgmnm5UCU. The altered tRNAArgmnm5UCU has a decreased stability and reduced aminoacylation due to changed secondary and/or tertiary structure. Protein sequencing revealed that during the translation of the GAA-AGA frameshifting site the altered tRNAArgmnm5UCU reads the AGA codon inefficiently. This induces a ribosomal pause, allowing the tRNAGlumnm5s2UUC residing in the ribosomal P-site to slip forward one nucleotide. The same frameshift mutation (hisC3072) was also suppressed by defects in the large ribosomal subunit protein L9. Single base substitutions, truncations, and absence of this protein induced ribosome slippage. Mutated ribosome could shift to the overlapping codon in the +1 frame, or bypass to a codon further downstream in the +1 frame. The signal for stimulation of slippage and function of L9 needs to be investigated. During the search for suppressors of the hisD3749 frameshift mutation, a spontaneous mutant was isolated in the iscU gene that contained greatly decreased levels of the thiolated tRNA modifications ms2io6A and s2C. The iscU gene belongs to the iscR-iscSUA-hscBA-fdx operon coding for proteins involved in the assembly of [Fe-S] clusters. As has been shown earlier, IscS influences the synthesis of all thiolated nucleosides in tRNA by mobilizing sulfur from cysteine. In this thesis, it is demonstrated that IscU, HscA, and Fdx proteins are required for the synthesis of the tRNA modifications ms2io6A and s2C but are dispensable for the synthesis of s4U and (c)mnm5s2U. Based on these results it is proposed that two distinct pathways exist in the formation of thiolated nucleosides in tRNA: one is an [Fe-S] cluster-dependent pathway for the synthesis of ms2io6A and s2C and the other is an [Fe-S] cluster-independent pathway for the synthesis of s4U and (c)mnm5s2U. MiaB is a [Fe-S] protein required for the introduction of sulfur in ms2io6A. TtcA is proposed to be involved in the synthesis of s2C. This protein contains a CXXC conserved motif essential for cytidine thiolation that, together with an additional CXXC motif in the C-terminus may serve as an [Fe-S] cluster ligation site.

La méthylation de l’uridine sur son carbone 5 est apparue au cours de l’évolution sous plusieurs formes. Tout d’abord, les thymidylate synthases permettent la synthèse de novo du dTMP, un précurseur essentiel de l’ADN des trois règnes du vivant. Deux familles de thymidylate synthases sont connues à ce jour : ThyA et la flavo-enzyme ThyX, codées par des gènes hétérologues et ayant des structures et mécanismes réactionnels radicalement différents. En outre, cette méthylation de l’uridine est apparue (probablement plus tard) sous forme de modifications post-transcriptionnelles des ARNt et ARNr. Cette thèse vise à questionner les contraintes évolutives ayant menés indépendament à ces quatres types de méthylation de l’uridine.Une première partie décrit l’identification d’une voie métabolique permetant la complémentation du phénotype d’auxotrophie pour la thymidine par des analogues nucléotidiques chez Escherichia coli. Une approche de biologie synthétique en vue d’établir une voie alternative de biosynthèse du thymidylate a aussi été mise en œuvre. Une technique de sélection de gènes de complémentation du phénotype d’auxotrophie pour la thymidine, issus de mutagénèse aléatoire, a pu être développée. Dans une seconde partie, des études biochimiques et sppectroscopiques ont été réalisées sur la méthyle-transférase flavine-dépendante TrmFO, responsable de la méthylation post-transciptionnelle de l’uridine 54 des ARNt de certains microorganismes.L’implication de certains résidus dans la fixation du substrat a pu être déterminée d’une part, et certains intermédiaires réactionnels potentiels ont été caractérisés spectralement d’autre part. Ces dernières observations s’appuient, en outre, sur des études en cours de spectroscopie résolue en temps et des simulations de dynamique moléculaire afin de mieux comprendre les flavoprotéines en général et les méthyle transférases flavine-dépendantes en particulier
Enzymes catalyzing the methylation of uridine at its carbon 5 position have appeared independently in different forms across evolution. Thymidylate synthases ThyA and the flavoprotein ThyX catalyze the de novo synthesis of dTMP, an essential DNA precursor in the three domains of life. They are encoded by heterologous genes and have drastically different structures and reaction mechanisms. On the other hand, this uridine methylation is also performed by tRNA and rRNA post-transcriptional modification enzymes.This thesis assesses the question of the evolutionary constraints that have led independently to four kinds of uridine methylation. The first part describes the identification of a metabolic pathway allowing the complementation of thymidine auxotrophy by non-natural nucleotide analogs in Escherichia coli. A synthetic biology approach, aiming to establish an alternative pathway for thymidylate biosynthesis, was also implemented and a selection strategy for thymidine auxotrophy-complementing genes, could be developed.In a second part, biochemical and spectral studies where realised on the flavin-dependent methyltransferase TrmFO, responsible for the post-transcriptional methylation of uridine at the invariant position 54 of tRNA in several microorganisms. The involvement of specific amino acid residues in substrate fixation and in stabilization of potential reaction intermediates was demonstrated. Their spectral characterization supports previously proposed reaction schemes for flavin-dependent thymidylate forming enzymes. These observations are currently being pursued by parallel approaches combining time-resolved spectroscopy and molecular dynamics simulations, aiming to further our understanding of how flavin mediates the transfer of carbon molecules from folate to uracil rings

Neisseria gonorrhoeae (Ng) encounters different microenvironments during its life-cycle. Some of these niches have different concentrations of oxygen, which influences the rate of Ng growth; as well as iron, an element essential for Ng survival. Differential expression of several proteins allows the bacteria to adapt to the diverse conditions it comes encounters. One protein affected by environmental changes during Ng growth is Gcp, a tRNA-modification enzyme essential for protein synthesis. To study the regulation of expression of Gcp, we first analyzed the sequence of its ORF, gcp. Orthologs of this gene are found in all kingdoms of life. In silico analysis shows that among Neisseria species, gcp ranges in homology from 76% to 99%, at the nucleotide level. Reverse transcription PCR indicates that gcp is expressed as part of an operon, together with three cytochrome-associated genes cyc4, resB and resC. Rapid amplification of complementary DNA ends determined the start of transcription of cyc4 (and possibly of the cyc4-gcp operon) at 95 nucleotides from the gene start codon. Transcriptional fusions determined that the promoter region upstream of cyc4 is the strongest promoter in the operon. However, the region directly upstream of gcp also has low level of promoter activity, suggesting that the gene may be expressed from two different promoters. Semi-quantitative determination of the concentration of gcp mRNA indicates that the transcription of the gene is significantly repressed when Ng is grown under low iron or low oxygen conditions. Analysis of an fnr mutant, grown under the same conditions as its parental wild type, indicates that the FNR transcriptional regulator is involved in the repression of gcp in low iron or low oxygen conditions. Contrary to expectation, the cyc4 promoter is upregulated when Ng is grown under low oxygen or low iron conditions. However, these results cannot be compared to the original promoter strength. Determination of which was performed on bacteria grown in liquid medium. Coregulation of gcp with cytochrome genes can guarantee low levels of protein synthesis when Ng encounters adverse microenvironments and needs its energy redirected to the expression of genes that would allow it to survive.

Transfer ribonucleic acids (tRNAs) are extensively modified, especially their anticodon loops. Modifications at position 34 (wobble base) and 37 in these loops affect the tRNAs’ decoding ability, while modifications outside the anticodon loops, e.g. m1A58 of tRNAMeti, may be crucial for tRNA structure or stability. A number of gene products are required for the formation of modified nucleosides, e.g. at least 26 proteins (including Elongator complex) are needed for U34 modifications in yeast, and methyl transferase activity of the Trm6/61p complex is needed to form m1A58. The aim of the studies which this thesis is based upon was to investigate the functional aspects of tRNA modifications and regulation of the modifying enzymes’ activity. First, the hypothesis that ncm5U34, mcm5U34, or mcm5s2U34 modifications may be essential for reading frame maintenance was investigated. The results show that mcm5 and s2 group of mcm5s2U play a vital role in reading frame maintenance. Subsequent experiments showed that the +1 frameshifting event at Lys AAA codon occurs via peptidyl-tRNA slippage due to a slow entry of the hypomodified tRNA-Lys. Moreover, the hypothesis that Elp1p N-terminal truncation may regulate Elongator activity was investigated. Cleavage of Elp1p was found to occur between residue 203 (Lys) and 204 (Ala) and to depend on the vacuolar protease Prb1p. However, including trichloroacetic acid (TCA) during protein extraction abolished the appearance of truncated Elp1p, showing that its truncation is a preparation artifact. Finally, in glioma cell line C6, PKCα was found to interact with TRM61. RNA silencing of TRM6/61 causes a growth defect that can be partially suppressed by tRNAMeti overexpression. PKCα overexpression reduces the nuclear level of TRM61, likely resulting in reduced level of TRM6/61 complex in the nucleus. Furthermore, lower expression of PKCα in the highly aggressive GBM (relative to its expression in less aggressive Grade II/III glioblastomas) is accompanied by increased expression of TRM6/61 mRNAs and tRNAMeti, highlighting the clinical relevance of the studies.

Enterococci were once thought to be harmless, commensal organisms that colonize the gastrointestinal tract of humans and other mammals. In the last 30 years, however, concern has grown in the clinical setting over two particular species, Enterococcus faecalis and Enterococcus faecium, which are frequently found to be the etiologic agents of nosocomial infections. Aminoacyl-phosphatidylglycerol synthases (aaPGSs) are integral membrane proteins that add amino acids to phosphatidylglycerol (PG) in the cellular envelope of bacteria. Addition of amino acids to PG confers resistance to various therapeutic antimicrobial agents, and contributes to evasion of the host immune response in a number of clinically relevant microorganisms. E. faecium possesses two distinct aaPGSs: aaPGS1 and aaPGS2. In addition, another gene coding for a putative hydrolase (pHyd) is located in the same operon as aaPGS2, and has no known function. To investigate the physiological relevance of aa-PG formation, and the function of aaPGS1, aaPGS2, and pHyd in E. faecium, we generated individual knockouts of these genes using a markerless deletion strategy. Deletion of aaPGS1 did not noticeably alter lipid aminoacylation, whereas deletion of aaPGS2 led to a loss of aa-PG synthesis. Deletion of pHyd also led to a loss of lipid aminoacylation; however, additional experiments are needed to verify that expression of aaPGS2 (which resides just downstream in the same operon) is unaffected in the pHyd-deletion strain. Development of the mutant strains described here will enable us to investigate additional phenotypes associated with these genes, and to determine whether aa-PG formation contributes to antibiotic resistance in E. faecium as in several other pathogenic microorganisms.
B.S.
Bachelors
Burnett School of Biomedical Sciences
Molecular Biology and Microbiology

Wyosine derivatives are highly complex modified ribonucleic acid (RNA) bases found in archaea and eukarya. They are a modification of a genetically encoded guanosine found at position 37 of phenylalanine encoding transfer ribonucleic acid (tRNA). The second step in the biosynthesis of all wyosine derivatives, in both archaea and eukarya, is the transformation of N-methylguanosine to 4-demethylwyosine by the radical S-adenosyl-l-methionine enzyme TYW1. When these studies were initiated, the substrate of TYW1 was unknown. Four possible substrates; acetyl CoA, acetyl phosphate, phosphoenolpyruvate, and pyruvate; were tested for activity. Only incubation with pyruvate led to production of 4-demethylwyosine. As only two new carbons are incorporated into the RNA base at this step, ¹³C isotopologues were used to identify the carbons that are transferred into 4-demethylwyosine. These experiments revealed that C2 and C3 of pyruvate are incorporated into 4-demethylwyosine, with C1 lost as an unknown byproduct. Utilizing pyruvate containing deuteriums in place of protons on the C3 carbon, the regiochemistry of the addition was determined. It was found that C3 forms the methyl group of 4-demethylwyosine and C2 becomes the bridging carbon in the imidazoline ring. The site of hydrogen atom abstraction by 5'-deoxyadenosyl radical was identified as the N-methylguanosine methyl group through the use of tRNA containing a deuterated methyl group. The putative mechanism for this transformation involved the formation of an enzyme substrate Schiff base through a conserved lysine residue. Utilizing sodium cyanoborohydride a Schiff base was trapped between TYW1 and pyruvate. The mass of the trapped adduct responded as expected when different isotopologues of pyruvate were used, demonstrating that it is due to pyruvate. Moreover, the fragment of TYW1 that contained the trapped adduct contained two lysine residues, one of which was shown to be required for activity both in vivo and in vitro. It was initially proposed that TYW1 contained two iron-sulfur clusters, and then subsequently shown to have two 4Fe-4S clusters. Site directed mutagenesis, along with iron and sulfide analysis identified the cysteines; as C26, C39, and C52; coordinating the second 4Fe-4S cluster. This study identified pyruvate as the substrate of TYW1, and provided evidence for key steps in the transformation of N-methylguanosine to 4-demethylwyosine.

Inosine is a guanosine analogue that when is found at the wobble position of the tRNAs (I34) expands its codon recognition capability. Inosine can wobble pair with cytosine, adenosine and uridine. Because inosine is not genomically encoded, essential enzymes are responsible for the hydrolytic deamination of adenosine to inosine, specifically at the wobble position of the tRNAs. In Bacteria, the modification is mostly found in tRNAArg, catalysed by the homodimeric tRNA adenosine deaminase A (TadA), with a conserved active site coordinated with an atom of Zn+2. In Eukarya, the modification is present in up to eight different tRNAs, catalysed by the heterodimeric enzyme ADAT (ADAT2-ADAT3), which originally evolved from TadA by duplication and divergence. ADAT2 is considered the catalytic subunit because it conserves the active site, whereas ADAT3, which lacks one of the essential catalytic residues, is thought to play a structural role. This substrate expansion, significantly influenced the evolution of eukaryotic genomes in terms of tRNA gene abundance and codon usage. However, the selection pressures driving this process remain unclear. In this thesis, we characterize the human transcriptome and proteome in terms of frequency and distribution of ADAT-related codons. Human codon usage indicates that I34 modified tRNAs are preferred for the translation of highly repetitive coding sequences, suggesting that I34 is an important modification for the synthesis of proteins of highly skewed amino acid composition. Persuaded by these results we extend the analysis to a series of eukaryotic and bacterial organisms, spanning the whole tree of life. We find that the preference for codons that are recognized by I34-modified tRNAs, in genes with highly biased codon composition, is universal among eukaryotes, and we report that, unexpectedly, the bacterial phylum of Firmicutes shows a similar preference. We experimentally demonstrate that the Firmicute Oenococcus oeni presents a functional expansion of I34 modification to other tRNAs other than tRNAArg, and that this process likely starts with the emergence of unmodified A34-containing tRNAs. Our findings also indicate that several ancestral bacterial groups lack both TadA and A34-tRNAs, suggesting that these species never developed the machinery to generate I34- modified tRNAs. On the other hand limited sets of bacterial species have either lost the system secondarily, or expanded it to additional tRNA substrates. In Eukaryotes, we show that a large variability in the use of I34 can be found in protists, while the modification becomes fixed in Metazoa, Fungi and Plant kingdoms.
La inosina és un anàleg de la guanosina, que quan es troba a la posició 34 dels tRNAs, expandeix el nombre de codons que aquests tRNA són capaços de reconèixer. La inosina pot emparellar-se mitjançant wobbling amb citosina, adenosina i uridina. Degut que la inosina no està codificada al genoma, existeixen enzims essencials encarregats de la deaminar la adenosina a inosina específicament a la posició 34 dels tRNAs. Als organismes bacterians, aquesta modificació es troba principalment a tRNAArg i és catalitzada per l’enzim homodimeric tRNA adenosina desaminasa A (TadA), que disposa d’un centre actiu conservat. Als organismes eucariòtics, aquesta modificació és present en fins a vuit tRNAs diferents, catalitzada per l’enzim heterodimeric ADAT (ADAT2-ADAT3). Aquest enzim ha evolucionat a partir de TadA per duplicació i divergència. ADAT2 és considerat la subunitat catalítica, ja que conserva el centre actiu mentre que ADAT3 n’ha perdut un dels residus essencials i es considera que té un paper en el reconeixement dels substrats. L’expansió en el reconeixement de substrats entre TadA i ADAT ha influenciat significativament en la composició dels genomes eucariotes, particularment en l’abundància de gens de tRNA i en el biaix de la composició de codons. Tanmateix, les pressions selectives que condueixen aquests processos romanen desconegudes. En aquesta tesi, hem caracteritzat el transcriptoma i el proteoma humà respecte la freqüència i distribució de codons relacionats amb ADAT. Els nostres resultats indiquen que la composició de codons del transcriptoma humà està esbiaixada promovent una dependència en l’ús de I34, especialment en regions altament repetitives. Persuadits per aquests resultats, hem estès les nostres anàlisis a un conjunt d’organismes eucariotes i bacterians per tal de representar tot l’arbre de la vida. Hem comprovat que aquesta preferència per codons que són reconeguts per tRNAs amb I34 és generalitzada només als eucariotes, tot i que sorprenentment, també és present al fílum bacterià dels Firmicutes. Els nostres resultats també indiquen que alguns grups bacterians ancestrals no disposen de tRNAs amb A34 ni de l’enzim TadA, cosa que suggereix que aquestes espècies mai han desenvolupat la maquinària per generar tRNAs amb I34. Altres conjunts de bactèries indiquen tant la pèrdua secundària d’aquest sistema, com l’expansió a d’altres tRNAs. Hem demostrat experimentalment que Oenococcus oeni, pertanyent als Firmicutes, presenta altres tRNAs amb I34 a part del tRNAArg i que també presenta tRNA amb A34 no modificats. Entre els organismes eucariotes, els protists presenten una gran variabilitat en l’ús de tRNA amb I34, mentre que en Metazoa, Fungi i Plantae, tots els tRNAs amb I34 són presents.

[EN] Post-transcriptional modification of the wobble uridine (U34) of a tRNA set is an evolutionary conserved process, produced by homologous proteins from the MnmA/MTU1, MnmE/GTPBP3 and MnmG/MTO1 families. Mutations in the human genes MTU1 and GTPBP3 or MTO1 produce acute infantile liver failure, and hypertrophic cardiomyopathy and lactic acidosis, respectively, which usually cause lethality in the first months of life. It is assumed that the primary cause of these diseases is the lack of the modifications introduced by the MTU1 protein in position 2 (tiol) and GTPBP3 and MTO1 proteins (taurinomethylation) in position 5 at U34 in a subgroup of mt-tRNAs. Nevertheless, the molecular mechanisms underlying these diseases (and other diseases associated with such modifications) are not clear. The reason why the typical defects of oxidative phosphorylation (due to impaired mitochondrial translation) produce such wide range of phenotypes is still unknown. Our hypothesis sustains that the mitochondria-nucleus retrograde signaling pathways triggered by the hypomodification at position 2 and 5 of U34 are different, and that each nuclear response is modulated by the genetic and epigenetic programs of cells and organisms. In this work, we have used the nematode Caenorhabditis elegans as a model organism to study the effects of inactivating the homologue proteins to MTU1, GTPBP3 and MTO1, which we have named as MTTU-1, MTCU-1 and MTCU-2, respectively. We have proved that these nuclear encoded proteins are located in mitochondria and are involved in U34 modification of mt-tRNAs. The mtcu-1 and mtcu-2 mutants show a reduction in fertility, while the mttu-1 mutant shows a reduction in fertility and a lengthening of the reproductive cycle (both phenotypes are thermosensitive). The phenotypes exhibited by the mttu-1, mtcu-1 and mtcu-2 mutants support our hypothesis, in which the mttu-1 single mutation, on the one hand, and the mtcu-1 and mtcu-2 single mutations, on the other hand, trigger different retrograde signaling pathways which produce specific nucear expression. Thus, a nuclear dependent phenotypic trait (as transcription or mt-tRNAs stability) and the expression of nuclear genes as ucp-4, hsp-6, hsp-60 and other genes involved in mitochondrial metabolism show a differential pattern in both group of mutants. hsp-6 and hsp-60 genes (UPRmt markers) are downregulated in mttu-1 single mutant, which could be related to fertility and reproductive cycle thermosensitivity. The three single mutants exhibit reduced expression of glycolysis and ß-oxidation genes (usually more drastic in the mttu-1 mutant), an induction of a glutaminolysis marker, and an induction of the ucp-4 gene, which encodes a transporter of the succinate to the mitochondria. Due to all three single mutants display a mild OXPHOS dysfunction, we propose that the observed changes in the expression of genes involved in the mitochondrial metabolism reveal a TCA cycle reprogramming aimed to compensate the reduction of acetil-CoA (coming from glycolysis and fatty acid oxidation) though the activation of anaplerotic pathways characterized by the succinate import to mitochondria by UCP-4 and the incorporation of 2-oxoglurate from glutaminolysis. We also analyze the effects of the simultanous suppression of modifications at positions 2 and 5 of U34 in C. elegans. The double mutant mtcu-2;mttu-1 displayed a severe OXPHOS dysfunction and a 5-fold higher AMP/ATP ratio, which was associated with embryonic lethality, developmental arrest in primary larval stages, penetrant sterility in adults and extended lifespan. This lifespan extension is modulated by signaling pathways which depend on AMPK (specifically on AAK-1 catalitic subunit) and steroid hormones, through DAF-9 and DAF-12 proteins. This work shows the important gene reprogramming related to mitochondrial metabolism in response to U34 hypomodification of mt-tRNAs, and shows new connexions between signaling pathways that extend lifespan.
[ES] La modificación post-transcripcional de la uridina de tambaleo (U34) de ciertos tRNAs es un proceso conservado evolutivamente, realizado por proteínas homólogas de las familias MnmA/MTU1, MnmE/GTPBP3 y MnmG/MTO1, y biológicamente relevante. De hecho, mutaciones en los genes humanos MTU1 y GTPBP3 o MTO1 causan fallo hepático infantil agudo y cardiomiopatía hipertrófica infantil, respectivamente, que producen letalidad durante los primeros meses de vida. Se asume que la causa primaria de estas enfermedades es la ausencia de las modificaciones introducidas por la proteína MTU1 en la posición 2 (tiol) y las proteínas GTPBP3 y MTO1 (taurinometil) en la posición 5 de la U34 en un grupo de mt-tRNAs. Se desconocen los mecanismos subyacentes y las razones por las que el déficit de OXPHOS resultante en todos los casos (atribuido a alteraciones de la traducción mitocondrial de proteínas) produce fenotipos tan diversos. Nuestra hipótesis es que la señalización retrógrada mitocondria-núcleo promovida por la hipomodificación de los mt-tRNAs en 2 ó 5 de la U34 es diferente y la respuesta nuclear viene modulada por el programa genético y epigenético de células y organismos. Hemos utilizado el nematodo C. elegans como modelo para estudiar los efectos producidos por la inactivación de las proteínas homólogas de MTU1, GTPBP3 y MTO1 a las que hemos denominado MTTU-1, MTCU-1 y MTCU-2. Hemos comprobado que estas proteínas, codificadas por el núcleo, son de localización mitocondrial y están implicadas en la modificación de la U34 de los mt-tRNAs. Los mutantes mtcu-1 y mtcu-2 presentan una reducción en su fertilidad y, en el caso del mutante simple mttu-1, fenotipos asociados a termosensibilidad. Los fenotipos exhibidos por los mutantes mttu-1, mtcu-1 y mtcu-2 sustentan la hipótesis de que la mutación mttu-1, y las mutaciones mtcu-1 y mtcu-2 promueven señales retrógradas diferentes que producen patrones de expresión nuclear específicos. Así, un rasgo fenotípico dependiente de genes nucleares (como lo es la transcripción y/o estabilidad de los mt-tRNAs) y la expresión de genes nucleares como ucp-4, hsp-6, hsp-60 y otros implicados en el metabolismo mitocondrial muestran un patrón diferente en los dos grupos de mutantes. Los genes hsp-6 y hsp-60 (marcadores de la UPRmt) están regulados a la baja en el mutante mttu-1. Los tres mutantes simples exhiben una reducción en la expresión de genes de la glicólisis y de la ß-oxidación de los ácidos grasos, una inducción en un marcador de glutaminolisis y una inducción en el gen ucp-4 (mayor en mttu-1) implicado en el transporte de succinato a la mitocondria. Dado que los tres mutantes simples presentan una disfunción OXPHOS relativamente suave, proponemos que los cambios de expresión en genes que modulan el metabolismo mitocondrial revelan una reprogramación del ciclo del TCA que compensa la disminución en el aporte de acetil-CoA procedente de glicólisis y oxidación de ácidos grasos con la activación de rutas anapleróticas del ciclo del TCA (importe de succinato a la mitocondria por UCP-4 y aporte de ¿-cetoglutarato procedente de la glutaminolisis). También analizamos los efectos de la anulación simultánea de las modificaciones en las posiciones 2 y 5 de la U34. El doble mutante mttu-1;mtcu-2 presenta una disfunción OXPHOS severa, con una ratio AMP/ATP 5 veces superior al control, que resulta en letalidad embrionaria, detención del desarrollo en estadios larvarios tempranos y esterilidad completa en los adultos que presentan, por otra parte, una longevidad unas dos veces superior a la cepa control. Este incremento de la longevidad está modulado por rutas de señalización que dependen de la subunidad catalítica AAK-1 (AMPK), y de hormonas esteroideas (proteínas DAF-9 y DAF-12). El trabajo muestra la importante reprogramación de genes relacionados con el metabolismo mitocondrial en respuesta a la hipomodificación de la U34 de los mt-tRNAs y
[CAT] La modificació post-transcripcional de la uridina de balanceig (U34) de certs tRNAs és un procés conservat evolutivament realitzat per proteïnes homòlogues a les de les famílies MnmA/MTU1, MnmE/GTPBP3 i MnmG/MTO1 i biològicament relevant. De fet, mutacions en els gens humans MTU1 i GTPBP3 o MTO1 causen fallada hepàtica infantil aguda i cardiomiopatia hipertròfica infantil amb acidosis làctica, respectivament, que produïxen letalitat durant els primers mesos de vida. S'assumix que la causa primària d'aquestes malalties és l'absència de les modificacions introduïdes per la proteïna MTU1 a la posició 2 (tiol) i per les proteïnes GTPBP3 i MTO1 (taurinometil) a la posició 5 de la U34 en un grup de mt-tRNAs. Es desconeixen els mecanismes subjacents en estes malalties i les raons per les quals el dèficit de la OXPHOS resultant en tots els casos (atribuït a alteracions de la traducció mitocondrial de proteïnes) produïx fenotips tan diversos. La nostra hipòtesi és que la senyalització retrògrada mitocondria-nucli promoguda per la hipomodificació dels mt-tRNAs en 2 o 5 de la U34 és diferent i la resposta nuclear en cada cas es dependent del programa genètic i epigenètic de cèl¿lules i organismes. Hem utilitzat el nematode C. elegans com a organisme model per a estudiar els efectes produïts per la inactivació de les proteïnes homòlogues de MTU1, GTPBP3 i MTO1 a les que hem denominat MTTU-1, MTCU-1 i MTCU-2. Hem comprovat que aquestes proteïnes, codificades pel nucli, són de localització mitocondrial i estan implicades en la modificació de la U34 dels mt-tRNAs. Els mutants mtcu-1 i mtcu-2 presenten una reducció en la seua fertilitat i, en el cas del mutant mttu-1, fenotipus associats a termosensibilitat. Els fenotipus exhibits pels mutants mttu-1, mtcu-1 i mtcu-2 sustenten la hipòtesi que la mutació mttu-1, i les mutacions mtcu-1 i mtcu-2 promouen senyals retrògrads diferents que produïxen patrons d'expressió nuclears específics. Així, un tret fenotípic dependent de gens nuclears (com ho és la transcripció i/o l'estabilitat dels mt-tRNAs) i l'expressió de gens nuclears com ucp-4, hsp-6, hsp-60 i altres implicats en el metabolisme mitocondrial mostren un patró diferent en els dos grups de mutants. Els gens hsp-6 i hsp-60 (marcadors de la UPRmt) estan regulats a la baixa en el mutant mttu-1. Els tres mutants simples exhibixen una reducció en l'expressió de gens de la glicòlisi i de la ß-oxidació dels àcids grassos, una inducció en un marcador de glutaminolisi i una inducció en el gen ucp-4 (major en el mutant mttu-1) implicat en el transport de succinat a la mitocondria. Atés que els tres mutants simples presenten una disfunció OXPHOS relativament suau, proposem que els canvis d'expressió en gens que modulen el metabolisme mitocondrial revelen una reprogramació del cicle del TCA que compensa la disminució en l'aportació d'acetil-CoA procedent de la glicòlisi i de l'oxidació d'àcids grassos amb l'activació de rutes anaplerótiques del cicle del TCA (importació de succinat a la mitocondria per UCP-4 i aportació de ¿-cetoglutarat de la glutaminolisi). També s'analitzen els efectes de l'anul¿lació simultània de les modificacions en 2 i 5 de la U34. El doble mutant mttu-1;mtcu-2 presenta una disfunció OXPHOS severa, amb una ràtio AMP/ATP 5 vegades superior al control, que resulta en letalitat embrionària, detenció del desenvolupament en estadis larvaris primerencs, esterilitat completa en els adults i una longevitat unes 2 vegades superior al control. Aquest increment de la longevitat està modulat per rutes de senyalització que depenen de la subunitat catalítica AAK-1 (AMPK), i d'hormones esteroidees (a través de les proteïnes DAF-9 i DAF-12). En resum, aquest treball mostra per primera vegada a nivell d'un animal model la important reprogramació de gens relacionats amb el metabolisme mitocondrial en resposta a la hipomodificació de la U34 dels mt-tRNAs i
Navarro González, MDC. (2016). Caenorhabditis elegans as a research tool to study mitochondrial diseases associated with defects in tRNA modification [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/61978
TESIS

Modified bases in RNA molecules are common, with over one-hundred individual modifications reported to date. In transfer RNA (tRNA), these modifications help hold the molecule in its proper conformation, allow the aminoacyl-synthetases to attach the amino acid to the correct tRNA for transfer to the ribosome, and in the correct matching of the codon-anticodon pair at the ribosome. Two positions on the tRNA molecule are significant in this last action; positions 34 and 37. Position 34 is the first base in the anticodon, and pairs with the last base in the codon during translation; modifications at this point allow the anticodon to pair with multiple codons. Modifications at position 37 help stabilise the anticodon loop of the tRNA, and help the efficiency of the translation. The threonylcarbamyoladenosine (t⁶A) modification at position 37 is required for the efficient translation of ANN codons. At least four enzymes, TsaBCDE, are involved in the formation of this modification, and are essential for bacterial viability. In this study, the interactions between the TsaB, TsaC, TsaD and TsaE have been investigated. Protein complex formation has been identified both in-vitro and in-vivo between TsaB and TsaD. The proteins interact in a 1:1 ratio, and the results indicate that this interaction stabilises the TsaD enzyme. The interaction appears to be unchanged over a physiological range of temperatures. No stable complex was identified between TsaC or TsaE with any of the other enzymes, however calorimetry suggests that there is an interaction between TsaE and both TsaB and TsaD. TsaC is believed to synthesise the threonylcarbamoyladenylate intermediate from threonine, bicarbonate and ATP; the activity of the enzyme complex (as measured by ATPase activity) in a media containing these compounds is increased in the presence of TsaC. If the cell is prevented from maintaining a source of t⁶A, it mounts a number of transcriptional responses. The transcription of certain amino acid biosynthetic genes is increased, corresponding to the tRNAs requiring the t⁶A modification, and in the transport and conversion of sulphates. This reflects the cellular response to starvation of an amino acid; the inability to incorporate the amino acids into the polypeptide chain due to the hypomodified tRNA is interpreted as the absence of that particular amino acid. Other transcriptional effects can be interpreted as a consequence of translational stalling where the t⁶A modification is required, leading to alterations in transcriptional promotion. TsaB and TsaD have also been shown to bind a small number of compounds in a high-throughput fragment-based drug discovery assay. Some of these have been shown to potentially affect the ATPase activity of the enzymes.

La méthylation de l'adénine en position 58 des ARNt (m1A58) est présente dans les trois domaines de vie et joue un rôle crucial chez plusieurs organismes. Sa formation est catalysée par la méthyltransférase SAM-dépendante TrmI. Alors que chez les eucaryotes et les bactéries, TrmI est site-spécifique pour l'adénine en position 58, chez l'archée Pyrococcus abyssi, TrmI est région-spécifique puisqu'elle catalyse également la méthylation de l'adénine en position 57. Nous nous sommes intéressés à cette enzyme, PabTrmI, pour comprendre cette différence de spécificité par rapport à ses homologues eucaryotes et bactériens.La structure cristallographique de l'enzyme, en complexe avec son cofacteur SAM ainsi qu'avec le produit de la réaction, la SAH, nous a conduit à construire différents mutants de la protéine et de son substrat ARNt. Nous avons ainsi montré que His78, située à l'entrée du site actif, est mobile et est importante pour l'efficacité catalytique de PabTrmI. L'analyse des positions de méthylation par spectrométrie de masse, simple et en tandem, montre qu'une partie de la région-spécificité de l'enzyme pour certains ARNt de P. abyssi, est liée à la présence de trois adénines consécutives, PabTrmI ne méthylant que la première adénine d'une séquence AA.En vue d'étudier les cinétiques rapides du mécanisme de retournement de la base de l'ARNt par l'enzyme région-spécifique PabTrmI et l'enzyme bactérienne site-spécifique TthTrmI de T. thermophilus, nous avons vérifié dans un premier temps, par spectrométrie de masse, qu'un mini-ARNt, constitué de la tige acceptrice et de la tige-boucle T, est substrat de TrmI.

N6-threonylcarbamoyl adenosine (t6A) is a universally conserved tRNA modification found at position 37 of tRNAs which decode ANN codons. Structural studies have implicated its presence as a requirement for the disruption of a U-turn motif in certain tRNAs, leading to the formation of properly structured anticodon stem loop. This structure is proposed to enhance the base pairing between U36 of tRNA and A1 of the codon which aids in translational frame maintenance. Despite significant effort since its discovery in the 1970s the enzymes involved in its biosynthesis remained undiscovered. Bioinformatic analysis identified two proteins as likely candidates for t6A synthesis, YrdC and YgjD. Subsequent gene knockout experiments in yeast were consistent with their involvement in t6A biosynthesis in vivo. Furthermore, clustering between the bacterial genes ygjD, yeaZ and yjeE as well as the identification of a protein interaction network between YgjD, YeaZ, and YjeE suggested that YeaZ and YjeE might be involved in t6A biosynthesis. The genes encoding ygjD, yeaZ, yrdC and yjeE were cloned from E. coli and the recombinant protein was purified. Experiments analyzing the incorporation of [U-14C]-L-threonine and [14C]-bicarbonate (substrates previously indicated in its biosynthesis) into tRNA in the presence of these four proteins demonstrated the first reconstitution of the t6A pathway in vitro. LC-MS analysis verified the formation of t6A, and these proteins were renamed TsaD (YgjD), TsaB (YeaZ), TsaC (YrdC), and TsaE (YjeE). Biochemical characterization of this pathway suggested that the formation of t6A proceeds through an unstable threonylcarbamoyl adenosine monophosphate (TC-AMP) intermediate, which is produced by TsaC from its substrates CO2, L-threonine and ATP. To investigate this reaction in more detail a coupled assay was developed, enabling sensitive detection of turn over. TsaC is a promiscuous enzyme which readily accepts several amino acids as substrates. The formation of t6A from TC-AMP is catalyzed by TsaD, TsaB, and TsaE. Of these three proteins only TsaD is universally conserved suggesting it is the protein catalyzing the transfer of the threonylcarbamoyl moiety to A37 of tRNA. This transfer is not promiscuous as only TC-AMP serves as an efficient substrate for t6A formation. Structural investigation of these proteins are consistent with the formation of a single protein complex potentially alleviating issues with the reactivity and instability of TC-AMP.