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A TALE OF TWO METHYLATION MODIFICATIONS IN ARCHAEAL RNAsChatterjee, Kunal 01 May 2014 (has links)
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.
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STRUCTURAL AND FUNCTIONAL STUDIES OF ARCHAEAL BOX C/D GUIDE RNA AND ROLE OF A PUTATIVE HUMAN PSEUDOURIDINE SYNTHASE, PUS10 IN APOPTOSISJana, Sujata 01 May 2017 (has links)
RNAs undergo different posttranscriptional chemical modifications, which affect their structural stability and functional diversity. RNA methylation is a very common type of post-transcriptional modification and is present in all domains of life: Archaea, Eukaryotes and Bacteria. Some of these methylations are catalyzed either by a RNA-protein complex or by stand-alone enzymes. The RNA-protein complex (Ribonucleoprotein complex) is comprised of a small RNA known as the guide RNA (Box C/D RNA) and core proteins (L7Ae, Nop5, and Fibrillarin). Box C/D RNAs contain conserved regions, called box C and box D near their 5’ and 3’ termini, respectively, and their imperfect copies called box C’ and box D’, internally. A short stretch of sequence between these Boxes are known as the guide/spacer regions, as the guide region helps in recruiting and positioning a specific target RNA for modification. Both in Archaea and Eukarya, box C and box D, as well as box C’ and box D’ together can form a structure called a Kink-turn (K-turn) that is characterized by a canonical Watson-Crick base-paired stem on one side, and a non-canonical stem on the other, separated by a 3-nucleotide loop. In Archaea box C’ and D’ can also form a K-loop, where the canonical stem of K-turn is replaced by a loop. Archaeal L7Ae binds first to the K-turn or K-loop and allows the recruitment of other proteins to form the complex. The presence of a unique box C/D RNA of Haloferax volcanii, called sR-tMet has been reported previously to guide the 2’-O-methylation of C34 in elongator pre-tRNAMet. Here we tried to characterize the structure-function relationship of this guide RNA under in vivo conditions. This RNA lacks a conventional K-turn or K-loop at its C’/D’ motif. We have created an H. volcanii strain that has a genomic deletion of sR-tMet. The sR-tMet gene is not essential for H. volcanii but this sR-tMet deleted strain lacks the 2’-O-methylation of C34 of its elongator tRNAMet. Unlike the close sR-tMet homologs (sR8 from Methanocaldococcus jannaschii and sR49 from Pyrococcus abyssi), the Box C’/D’ motif of sR-tMet is neither a K-turn nor a K-loop. The introduction of proper K-loop in the Box C’/D’ motif (sR-tMet with K-loop) abolished its Cm34 modification function in ΔsR-tMet strain. Direct interaction between L7Ae and the K-loop is not an absolute requirement for its function. However, disruption of the G/A and A/G pairing in Box C/D motif and Box D’ suggests the importance of these non-Watson crick base pairings in respect to sR-tMet’s function. Several other mutational studies have revealed that peculiar sR-tMet guide RNA from H. volcanii, behaves more like a Eukaryotic Box C/D RNA (where the K-loop is not required and presence of longer spacer length) than regular Archaeal one. Pseudouridine synthase 10 (Pus10) is the most recently identified Ψ synthase, found only in higher eukaryotes and Archaea. Archaeal Pus10 produces either tRNA Ψ55 or both tRNA Ψ54 and Ψ55 modifications. In Human, its Ψ synthase activity is not yet confirmed and interestingly it has been implicated in apoptosis. Herein for the first time we revealed that this putative RNA Ψ synthase protein, Human Pus10 (HuP10), translocates from the nucleus to the cytoplasm in TRAIL induced apoptosis. This nucleo-cytoplasmic movement of HuP10 occurs through the CRM1 mediated nuclear export pathway and Caspase 3 influences this movement. HuP10 also mediates crosstalk between the extrinsic and intrinsic pathways during TRAIL-induced apoptosis. Other than its involvement in apoptosis, we have also uncovered that HuP10 is involved in regulation of cell proliferation. Depletion (knockdown) of this protein in different cancer cell lines, promotes cell migration and anchorage-independent cell growth in the absence of any apoptotic stimulation.
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STRUCTURAL AND FUNCTIONAL STUDIES OF H. VOLCANII BOX C/D PROTEINS AND ROLES FOR HUMAN PUS10 BEYOND PSEUDORURIDINE SYNTHESISBosmeny, Michael Stephen 01 May 2022 (has links) (PDF)
RNA in all forms of life contain a myriad of post-transcriptional modifications. These modifications are important for processing and structural reasons, and includes 2’-O-methylation and pseudouridylation. Some of these modifications are the product of stand-alone proteins and others are the product of RNA-protein complexes.In eukaryotic and archaeal cells, the Box C/D ribonucleoprotein complex is one of the complexes responsible for 2’-O-methylation activity. In Archaea, this complex consists of the enzymatic protein, Fibrillarin, plus two other structural proteins, Nop5 and L7Ae, along with a guide RNA, which all come together to modify a specific target RNA sequence. A methyl group is added to the 2’ hydroxyl of the nucleotide’s ribose sugar. These modifications are found both in ribosomal RNA and tRNAs. This work focuses on the interactions between Nop5, Fibrillarin, and the guide RNA used in this complex. The objective was to identify important amino acid sequences in these proteins which are essential for the operation of the complex. The size of the archaeal Box C/D complex is also investigated using size chromatography.Similarly, Pus10 is one protein responsible for pseudouridylation in Archaea and eukaryotes. Pseudouridylation is the isomerization of uridine (U) to pseudouridine (Ψ). Pus10 is known to produce Ψ54 and Ψ55 in the TΨC loop of some archaeal and eukaryotic tRNAs. However, current research suggests it could have additional jobs in the cellular lifecycle, such as roles in apoptosis and the regulation of eukaryotic cell cycle. During mammalian cell apoptosis, Pus10 translocates from the nucleus to the cytoplasm and is believed to be involved in cytochrome c release. This is suspected to be related to Caspase-3’s role in apoptosis. Caspase-3 is activated in both the extrinsic and intrinsic apoptotic pathways. It has been shown that activation of the extrinsic apoptotic pathway via TRAIL affects the localization of Pus10. Here we test Pus10’s actions in cells under the effect of intrinsic apoptotic pathway compounds.Pus10 has also shown to have an effect on cell proliferation. Cells in which Pus10 has been depleted show increased growth rates. Here we investigate expression levels of proteins involved in cell cycle regulation, in both wild-type and Pus10-depleted cells, and attempt to compile a model of how Pus10 could be interacting with this system, using RNA-Seq, qPCR, and ChIP.SARS-CoV-2, also known as COVID-19, is a coronavirus that quickly became a pandemic in late 2019, early 2020. In the two years since then, the virus has undergone many mutations. By tracking the spread of these mutations at a national or regional level, by studying the spread pattern, we can make predictions, and possibly even prevent the next pandemic.
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Maturation of tRNA in Haloferax volcaniiNist, Richard Neil 06 September 2011 (has links)
No description available.
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Caracterização funcional das proteínas Nop17p e Rsa1p de Saccharomyces cerevisiae / Functional characterization of the Saccharomyces cerevisiae proteins Nop17p and Rsa1pPrieto, Marcela Bach 19 September 2014 (has links)
Nop17p e Rsa1p são proteínas nucleolares em Saccharomyces cerevisiae, as quais foram identificadas pela sua associação a dois complexos celulares: os snoRNPs de box C/D, através de interação com as subunidades Nop58p e Snu13p, respectivamente, e o R2TP/Hsp90p. Nop17p parece ser responsável por direcionar a chaperona Hsp90p durante a montagem dos snoRNPs, e a associação de Rsa1p a estes complexos ainda não tem uma função estabelecida. Neste trabalho, nós mostramos que a ausência de ambas as proteínas afetam a estabilidade da proteína Nop58p dos snoRNPs e afetam a localização do snoRNA U3. Em relação à ordem de interação das proteínas do core de snoRNps de box C/D, Nop17p associa-se de maneira transiente a Nop1p/Snu13p, seguida da ligação de Nop58p ao complexo. Quanto à rede de interação do R2TP, obtivemos o mutante Nop17(N307S), que não mais interage com Tah1p. Este mutante interage com a subunidade Rvb1p do R2TP, mas não se associa com outras proteínas parceiras de Nop17p(WT). Apesar da importância da interação Nop17p-Tah1p, sua interrupção não afeta o crescimento celular, o que sugere a possibilidade de outro fator estar envolvido na associação entre Nop17p e Hsp90p. / Nop17p and Rsa1p are Saccharomyces cerevisiae nucleolar proteins, which were identified for its association with two cellular complexes: box C/D snoRNPs, through interaction with the core subunits Nop58p and Snu13p respectively, and the R2TP/Hsp90p. Nop17p seems to be responsible for directing Hsp90p to the assembly of snoRNPs. The Rsa1p association to these complexes still have no defined function. In this work, we showed that both proteins absence affect Nop58p stability and causes a mislocalization of the U3 snoRNA. Relativel to the order of assembly of the box C/D snoRNPs core proteins, Nop17p associates transiently with Nop1p/Snu13p, followed by the Nop58p joining to the complex. To study in more detail the protein interactions within the R2TP complex, we obtained the Nop17(N307S) mutant, which no longer interacts withTah1p, but still interacts withRvb1p, another R2TP subunit. Nop17(N307S) does not interact with other Nop17p(WT) partners. Despite the importance of the Nop17p-Tah1p association, the disruption of this interaction does not affect cell growth, suggesting the involvement of a second factor on the Nop17p and Hsp90p association.
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Caracterização funcional das proteínas Nop17p e Rsa1p de Saccharomyces cerevisiae / Functional characterization of the Saccharomyces cerevisiae proteins Nop17p and Rsa1pMarcela Bach Prieto 19 September 2014 (has links)
Nop17p e Rsa1p são proteínas nucleolares em Saccharomyces cerevisiae, as quais foram identificadas pela sua associação a dois complexos celulares: os snoRNPs de box C/D, através de interação com as subunidades Nop58p e Snu13p, respectivamente, e o R2TP/Hsp90p. Nop17p parece ser responsável por direcionar a chaperona Hsp90p durante a montagem dos snoRNPs, e a associação de Rsa1p a estes complexos ainda não tem uma função estabelecida. Neste trabalho, nós mostramos que a ausência de ambas as proteínas afetam a estabilidade da proteína Nop58p dos snoRNPs e afetam a localização do snoRNA U3. Em relação à ordem de interação das proteínas do core de snoRNps de box C/D, Nop17p associa-se de maneira transiente a Nop1p/Snu13p, seguida da ligação de Nop58p ao complexo. Quanto à rede de interação do R2TP, obtivemos o mutante Nop17(N307S), que não mais interage com Tah1p. Este mutante interage com a subunidade Rvb1p do R2TP, mas não se associa com outras proteínas parceiras de Nop17p(WT). Apesar da importância da interação Nop17p-Tah1p, sua interrupção não afeta o crescimento celular, o que sugere a possibilidade de outro fator estar envolvido na associação entre Nop17p e Hsp90p. / Nop17p and Rsa1p are Saccharomyces cerevisiae nucleolar proteins, which were identified for its association with two cellular complexes: box C/D snoRNPs, through interaction with the core subunits Nop58p and Snu13p respectively, and the R2TP/Hsp90p. Nop17p seems to be responsible for directing Hsp90p to the assembly of snoRNPs. The Rsa1p association to these complexes still have no defined function. In this work, we showed that both proteins absence affect Nop58p stability and causes a mislocalization of the U3 snoRNA. Relativel to the order of assembly of the box C/D snoRNPs core proteins, Nop17p associates transiently with Nop1p/Snu13p, followed by the Nop58p joining to the complex. To study in more detail the protein interactions within the R2TP complex, we obtained the Nop17(N307S) mutant, which no longer interacts withTah1p, but still interacts withRvb1p, another R2TP subunit. Nop17(N307S) does not interact with other Nop17p(WT) partners. Despite the importance of the Nop17p-Tah1p association, the disruption of this interaction does not affect cell growth, suggesting the involvement of a second factor on the Nop17p and Hsp90p association.
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Structure / Function Relationship of Archaeal Box C/D and H/ACA ProteinsBosmeny, Michael 01 May 2016 (has links)
Ribonucleoprotein complexes are responsible for some of the post-transcriptional modifications of RNA that occur within the cell, including 2'-O-methylation and pseudouridylation. These modifications contribute, among other things, to RNA folding, inhibition of degradation, and general cellular viability. In this study, we identify residues within the proteins of these complexes that are important to the functioning of the Box C/D and Box H/ACA complexes. Candidates were selected based on previous work and mutant versions of the proteins were introduced in-vivo. Assays were done to determine the functionality of the mutant complex. This work is divided into three parts, focused on the three proteins investigated. The first part is concerned with Nop5, a protein in the Box C/D RNP complex. Nop5 is known to interact with all other proteins and RNAs in the complex, and is believed to serve a primarily structural role, aligning the other components. Mutagenesis study of suspected significant amino acids in this protein showed that it is difficult to disrupt the operation of Nop5 with single changes, but is possible with more extensive mutation. The second part concerns Fibrillarin, the catalytic protein of the Box C/D ribonucleoprotein complex. Previous mutagenesis work identified several important amino acids involved with AdoMet transfer and complex formation. The methylation ability of these mutant complexes were further examined in this work by confirming that the same modification, or lack thereof, occurred at a second rRNA position. The final part of this work is about Nop10, part of the Box H/ACA complex. This work is only preliminary, but begins the process of testing suspected essential amino acids in the structure.
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Analyse fonctionnelle des protéines Hit1 et Bcd1 impliquées dans la biogenèse des snoRNP à boîtes C/D eucaryotes / Functional analysis of the Hit1p and Bcd1p proteins involved in eukaryotic box C/D snoRNP biogenesisTiotiu, Decebal 10 October 2016 (has links)
Chez les eucaryotes, la biogenèse des ribosomes débute dans le nucléole par la maturation et la modification des ARN ribosomiques (ARNr), et fait intervenir des centaines de particules ribonucléoprotéiques (RNP) distinctes, comme les petites RNP nucléolaires (snoRNP) à boîtes C/D, qui portent une activité méthyl transférase ciblée sur la position 2’-OH des riboses. Leur biogenèse nécessite l’intervention transitoire de facteurs protéiques constituant une machinerie d’assemblage spécifique. Mon travail de thèse a visé à étudier le rôle fonctionnel de deux de ces facteurs chez la levure S. cerevisiae les protéines Hit1 et Bcd1. Hit1p avait été trouvée au laboratoire être impliquée dans la biogenèse des snoRNP à boîtes C/D, et il était connu que l’expression de Bcd1p est essentielle à la viabilité cellulaire et pour la stabilité des snoRNA à boîtes C/D. Lors de ce travail, nous avons retrouvé le domaine fonctionnel de Hit1p et identifié les acides aminés impliqués dans l’interaction avec Rsa1p, un autre facteur d’assemblage. Par une approche similaire, nous avons recherché les domaines nécessaires à la fonctionnalité de Bcd1p. Le mécanisme par lequel Bcd1p influence spécifiquement les taux de snoRNA à boîtes C/D reste inconnu, mais au cours de ce travail j’ai identifié un nouveau partenaire potentiel pour cette protéine - la chaperonne d’histone Rtt106p. La dernière partie de mon travail a visé à rechercher le lien fonctionnel entre Rtt106p et l’expression des snoRNA à boîtes C/D / In eukaryotes, ribosome biogenesis begins in the nucleolus, by maturation and modification of ribosomal RNAs (rRNA) and involves hundreds of distinct ribonucleoprotein particles, like box C/D small nucleolar RNPs (snoRNPs). Their assembly requires the transient intervention of protein factors constituting a specific assembly machinery. My PhD work aimed to investigate the functional role of two such factors, Bcd1p and Hit1p, in the yeast S. cerevisiae. Hit1p involvement in box C/D snoRNP biogenesis was revealed in our lab, and it was known that Bcd1p expression is essential to cell viability and box C/D snoRNA stability. During this work, we identified the functional domain of Hit1p, and the aminoacids involved in its interaction with Rsa1, another assembly factor. By a similar approach we identified the functional domains of Bcd1p. The mechanism by which Bcd1p specifically influences box C/D snoRNA levels is unknown. However, I identified a potentially new partner for this protein – the Rtt106p histone chaperone. The last part of my work aimed to search for a functional link between this histone chaperone and box C/D snoRNA expression
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Rôle de la protéine Bcd1p/BCD1 dans les étapes précoces de la biogenèse des snoRNP à boîtes C/D eucaryotes / Functions of Bcd1p/BCD1 in the early steps of box C/D snoRNP biogenesisPaul, Arnaud 27 September 2018 (has links)
La biogenèse des ribosomes matures et fonctionnels est notamment dépendante de petites particules ribonucléoprotéiques composées d’ARN et de protéines, les snoRNP (small nucleolar RiboNucleoProteins). Celles-ci sont subdivisées en deux familles : les snoRNP à boîtes C/D et les snoRNP à boîtes H/ACA. Ces deux classes de snoRNP catalysent des modifications chimiques, respectivement de 2’-O-méthylation et de pseudouridylation, sur des positions spécifiques des ARN ribosomiques (ARNr), ou sont impliquées dans des clivages du long ARNr précurseur. Les snoRNP à boîtes C/D sont composées d’un snoARN à boîtes C/D servant de guide pour cibler la position à modifier, et d’un jeu invariant de quatre protéines : Snu13p/SNU13, Nop1p/Fibrillarine, Nop56p/NOP56 et Nop58p/NOP58 (levure/Homme). Ces snoRNP sont produites par la cellule grâce à la présence de plusieurs complexes protéiques constituant une machinerie pour leur assemblage. Outre plusieurs facteurs protéiques déjà connus dans la biogenèse de snoRNP à boîtes C/D comme les protéines Rsa1p/NUFIP, Hit1p/ZNHIT3 et les protéines du complexe R2TP, d’autres protéines pourraient compléter cette machinerie. Parmi ces facteurs additionnels, la protéine Bcd1p/ZNHIT6, pour Box C/D snoRNA protein 1, est essentielle pour maintenir spécifiquement la stabilité in vivo des snoARN à boîtes C/D, et des associations ont pu être identifiées entre Bcd1p/ZNHIT6 avec différents partenaires protéiques de la machinerie d’assemblage de ces particules. Toutefois, l’étape d’assemblage où Bcd1p/ZNHIT6 intervient et la fonction qu’elle y accomplit demeurent inconnues. L’utilisation d’outils in vivo et in vitro chez la levure S. cerevisiae et chez les mammifères nous ont permis de progresser dans la compréhension de la fonction de Bcd1p/ZNHIT6 dans l’assemblage des snoRNP à boîtes C/D. Bcd1p est un facteur d’assemblage recruté de manière co-transcriptionnelle sur les loci codant les snoARN à boîtes C/D et est requis pour le recrutement des complexes d’assemblage sur les snoARN en cours de transcription. Plus spécifiquement, Bcd1p affecte l’interaction de Nop58p avec le facteur d’assemblage Rsa1p, suggérant une fonction dans le recrutement de Nop58p dans une pré-snoRNP en cours d’assemblage. Ce travail a permis d’apporter des informations importantes permettant d’expliquer le caractère essentiel de Bcd1p dans la fonction et la biogenèse des snoRNP à boîtes C/D / Ribosome biogenesis is especially dependent on the action of small RNA/proteins complexes called small nucleolar ribonucleoproteins (snoRNPs). They are divided into two main families: the so-called box C/D snoRNPs and box H/ACA snoRNPs. Each category performs specific enzymatic processes, 2’-O-methylation and pseudouridylation, respectively, and induces target-specific chemical modification on rRNAs. Few snoRNPs are also essential for pre-rRNA processing. The box C/D snoRNPs are formed by the association of a box C/D snoRNA with a set of four invariant proteins: Snu13p/SNU13, Nop1p/Fibrillarine, Nop56p/NOP56 and Nop58p/NOP58 (yeast/Human). Biogenesis of these RNPs relies on the action of several proteins complexes which constitute a dedicated assembly machinery. Rsa1p/NUFIP, Hit1p/ZNHIT3, and components of the R2TP complex are the best characterized protein actors of this machinery. Additional protein factors probably participate in box C/D snoRNP biogenesis; Bcd1p/ZNHIT6 (Box C/D snoRNA protein 1) is such a candidate as it is essential for the in vivo stability of box C/D snoRNAs, and it was found associated with proteins involved in this machinery in yeast and Human. However, the mechanism governing the recruitment of this protein towards the biogenesis of box C/D snoRNP, and the step of the assembly process relying on the presence of Bcd1p are still unknown. In S. cerevisiae and Human, in vivo and in vitro tools allowed us to improve the understanding of the functions of Bcd1p/ZNHIT6 in box C/D snoRNP assembly. Bcd1p is an assembly factor that is recruited co-transcriptionally on box C/D snoRNA loci, and is required for the recruitment of assembly complexes on nascent snoRNAs. Bcd1p is important for Nop58p association with the assembly factor Rsa1p, which suggests that its primary function is to recruit Nop58p to nascent pre-snoRNPs. This work evidenced important information on the essential role of Bcd1p in C/D snoRNP biogenesis and function
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Étude des interactions protéine-protéine entre le complexe de Survie des MotoNeurones (SMN) et les facteurs d'assemblage des RNP à boîtes C/D et H/ACA / Study of the protein-protein interactions between the SMN complex and the factors required for box C/D and H/ACA RNP assemblyHuttin, Alexandra 11 December 2012 (has links)
Les particules ribonucléoprotéiques (RNP) à boîtes C/D et H/ACA sont impliquées dans la maturation des UsnRNA et des précurseurs des ARNr. L'assemblage de ces RNP dans les cellules est un processus complexe faisant intervenir de nombreux facteurs cellulaires dont NUFIP, commun aux deux RNP, et NAF1, spécifique aux RNP à boîtes H/ACA. Le complexe de Survie des Motoneurones (SMN) est essentiel à la survie cellulaire et est nécessaire à l'assemblage d'une autre RNP, les UsnRNP, composants des spliceosomes. Un déficit en protéine SMN conduit à une pathologie grave, l'amyotrophie spinale. Plusieurs études suggèrent que le complexe SMN puisse également jouer un rôle dans l'assemblage des RNP à boîtes C/D et H/ACA. Dans le but d'obtenir de plus amples informations, nous avons testé si des interactions existent entre les constituants du complexe SMN et i) les protéines associées aux RNP matures, ainsi que ii) les autres facteurs d'assemblage déjà connus. Ainsi, par une approche de double hybride chez la levure, nous avons observé des interactions fortes entre NAF1 et les protéines Gemin3 et Gemin8 du complexe SMN. Comme la protéine coeur GAR1 des RNP à boîte H/ACA interagit avec la protéine SMN, ces données suggèrent que le complexe SMN participe à l'échange de NAF1 par GAR1, qui est une étape clé de la biogenèse des RNP à boîtes H/ACA. De plus, nous avons mis en évidence des interactions entre Gemin3/NUFIP, Gemin4/NUFIP et Gemin6/NUFIP. L'étude de cette dernière interaction a été approfondie. Nous avons montré que l'interaction est directe, qu'elle existe dans les cellules de mammifères à la fois dans le cytoplasme et le noyau, et nous avons défini les domaines de chaque protéine nécessaires à l'interaction, en collaboration avec l'équipe d'E. Bertrand (IGM Montpellier). Ces résultats ouvrent de larges perspectives quant à un lien fonctionnel entre le complexe SMN et NUFIP dans l'assemblage des RNP à boîtes C/D et H/ACA, mais aussi dans l'assemblage de la snRNP U4 et dans le mécanisme de traduction localisée dans les cellules / Box C/D and H/ACA ribonucleoparticles (RNPs) are required for UsnRNA and ribosomal RNA maturation. Their assembly in cells is a complex process, which implicates numerous cellular factors, such as NUFIP, a common assembly factor, and NAF1, which is a specific factor for H/ACA box RNP assembly. The Survival of Motoneurons (SMN) complex is essential for cell survival and is required for the assembly of another class of RNPs, the UsnRNPs, which are essential components of the splicing machinery. Decreased levels of the SMN protein lead to a severe disease, the spinal muscular atrophy. Several studies led to the proposal that the SMN complex also plays a role in the assembly of box C/D and H/ACA RNPs. In order to obtain more information, we analyzed whether some interactions may exist between components of the SMN complex and i) core proteins of mature RNPs, or ii) factors already known to be involved in the assembly. Using a yeast two-hybrid approach, we observed strong interactions between NAF1 and the SMN complex components, Gemin3 and Gemin8. Since the core H/ACA protein GAR1 interacts with the SMN protein, our data suggest that the SMN complex participates to the exchange of NAF1 by GAR1, which is a crucial step of H/ACA box RNP biogenesis. Furthermore, we discovered strong interactions between Gemin3/NUFIP, Gemin4/NUFIP and Gemin6/NUFIP. Concerning the Gemin6/NUFIP interaction, we showed that is direct, that it exists in both compartments in mammalian cells and we defined domains of both proteins necessary for the interaction in collaboration with the E. Bertrand team (IGM Montpellier). These results open new perspectives concerning functional links between the SMN complex and NUFIP in box H/ACA and C/D RNP assembly, but also in U4 snRNP assembly and in the mechanism of localized translation
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