Добірка наукової літератури з теми "Box C/D small nucleolar RNA"

ABSTRACT The processing and methylation of precursor rRNA is mediated by the box C/D small nucleolar RNAs (snoRNAs). These snoRNAs differ from most cellular RNAs in that they are not exported to the cytoplasm. Instead, these RNAs are actively retained in the nucleus where they assemble with proteins into mature small nucleolar ribonucleoprotein particles and are targeted to their intranuclear site of action, the nucleolus. In this study, we have identified the cis-acting sequences responsible for the nuclear retention of U3 box C/D snoRNA by analyzing the nucleocytoplasmic distributions of an extensive panel of U3 RNA variants after injection of the RNAs into Xenopus oocyte nuclei. Our data indicate the importance of two conserved sequence motifs in retaining U3 RNA in the nucleus. The first motif is comprised of the conserved box C′ and box D sequences that characterize the box C/D family. The second motif contains conserved box sequences B and C. Either motif is sufficient for nuclear retention, but disruption of both motifs leads to mislocalization of the RNAs to the cytoplasm. Variant RNAs that are not retained also lack 5′ cap hypermethylation and fail to associate with fibrillarin. Furthermore, our results indicate that nuclear retention of U3 RNA does not simply reflect its nucleolar localization. A fragment of U3 containing the box B/C motif is not localized to nucleoli but retained in coiled bodies. Thus, nuclear retention and nucleolar localization are distinct processes with differing sequence requirements.

The Nucleolar Localization Elements (NoLEs) of Xenopus laevis U3 small nucleolar RNA (snoRNA) have been defined. Fluorescein-labeled wild-type U3 snoRNA injected intoXenopus oocyte nuclei localized specifically to nucleoli as shown by fluorescence microscopy. Injection of mutated U3 snoRNA revealed that the 5′ region containing Boxes A and A′, known to be important for rRNA processing, is not essential for nucleolar localization. Nucleolar localization of U3 snoRNA was independent of the presence and nature of the 5′ cap and the terminal stem. In contrast, Boxes C and D, common to the Box C/D snoRNA family, are critical elements for U3 localization. Mutation of the hinge region, Box B, or Box C′ led to reduced U3 nucleolar localization. Results of competition experiments suggested that Boxes C and D act in a cooperative manner. It is proposed that Box B facilitates U3 snoRNA nucleolar localization by the primary NoLEs (Boxes C and D), with the hinge region of U3 subsequently base pairing to the external transcribed spacer of pre-rRNA, thus positioning U3 snoRNA for its roles in rRNA processing.

Small nucleolar RNAs (snoRNAs) are a large family of eukaryotic RNAs that function within the nucleolus in the biogenesis of ribosomes. One major class of snoRNAs is the box C/D snoRNAs named for their conserved box C and box D sequence elements. We have investigated the involvement of cis-acting sequences and intranuclear structures in the localization of box C/D snoRNAs to the nucleolus by assaying the intranuclear distribution of fluorescently labeled U3, U8, and U14 snoRNAs injected into Xenopus oocyte nuclei. Analysis of an extensive panel of U3 RNA variants showed that the box C/D motif, comprised of box C′, box D, and the 3′ terminal stem of U3, is necessary and sufficient for the nucleolar localization of U3 snoRNA. Disruption of the elements of the box C/D motif of U8 and U14 snoRNAs also prevented nucleolar localization, indicating that all box C/D snoRNAs use a common nucleolar-targeting mechanism. Finally, we found that wild-type box C/D snoRNAs transiently associate with coiled bodies before they localize to nucleoli and that variant RNAs that lack an intact box C/D motif are detained within coiled bodies. These results suggest that coiled bodies play a role in the biogenesis and/or intranuclear transport of box C/D snoRNAs.

We have found that intron 11 of the nucleolin gene in humans and rodents encodes a previously unidentified small nucleolar RNA, termed U20. The single-copy U20 sequence is located on the same DNA strand as the nucleolin mRNA. U20 RNA, which does not possess a trimethyl cap, appears to result from intronic RNA processing and not from transcription of an independent gene. In mammals, U20 RNA is an 80-nucleotide-long, metabolically stable species, present at about 7 x 10(3) molecules per exponentially growing HeLa cell. It has a nucleolar localization, as indicated by fluorescence microscopy following in situ hybridization with digoxigenin-labeled oligonucleotides. U20 RNA contains the box C and box D sequence motifs, hallmarks of most small nucleolar RNAs reported to date, and is immunoprecipitated by antifibrillarin antibodies. It also exhibits a 5'-3' terminal stem bracketing the box C-box D motifs like U14, U15, U16, or Y RNA. A U20 homolog of similar size has been detected in all vertebrate classes by Northern (RNA) hybridization with mammalian oligonucleotide probes. U20 RNA contains an extended region (21 nucleotides) of perfect complementarity with a phylogenetically conserved sequence in 18S rRNA. This complementarity is strongly preserved among distant vertebrates, suggesting that U20 RNA may be involved in the formation of the small ribosomal subunit like nucleolin, the product of its host gene.

We have found that intron 11 of the nucleolin gene in humans and rodents encodes a previously unidentified small nucleolar RNA, termed U20. The single-copy U20 sequence is located on the same DNA strand as the nucleolin mRNA. U20 RNA, which does not possess a trimethyl cap, appears to result from intronic RNA processing and not from transcription of an independent gene. In mammals, U20 RNA is an 80-nucleotide-long, metabolically stable species, present at about 7 x 10(3) molecules per exponentially growing HeLa cell. It has a nucleolar localization, as indicated by fluorescence microscopy following in situ hybridization with digoxigenin-labeled oligonucleotides. U20 RNA contains the box C and box D sequence motifs, hallmarks of most small nucleolar RNAs reported to date, and is immunoprecipitated by antifibrillarin antibodies. It also exhibits a 5'-3' terminal stem bracketing the box C-box D motifs like U14, U15, U16, or Y RNA. A U20 homolog of similar size has been detected in all vertebrate classes by Northern (RNA) hybridization with mammalian oligonucleotide probes. U20 RNA contains an extended region (21 nucleotides) of perfect complementarity with a phylogenetically conserved sequence in 18S rRNA. This complementarity is strongly preserved among distant vertebrates, suggesting that U20 RNA may be involved in the formation of the small ribosomal subunit like nucleolin, the product of its host gene.

The nucleolar localization elements (NoLEs) of U17 small nucleolar RNA (snoRNA), which is essential for rRNA processing and belongs to the box H/ACA snoRNA family, were analyzed by fluorescence microscopy. Injection of mutant U17 transcripts into Xenopus laevisoocyte nuclei revealed that deletion of stems 1, 2, and 4 of U17 snoRNA reduced but did not prevent nucleolar localization. The deletion of stem 3 had no adverse effect. Therefore, the hairpins of the hairpin–hinge–hairpin–tail structure formed by these stems are not absolutely critical for nucleolar localization of U17, nor are sequences within stems 1, 3, and 4, which may tether U17 to the rRNA precursor by base pairing. In contrast, box H and box ACA are major NoLEs; their combined substitution or deletion abolished nucleolar localization of U17 snoRNA. Mutation of just box H or just the box ACA region alone did not fully abolish the nucleolar localization of U17. This indicates that the NoLEs of the box H/ACA snoRNA family function differently from the bipartite NoLEs (conserved boxes C and D) of box C/D snoRNAs, where mutation of either box alone prevents nucleolar localization.

We have previously developed a novel technique for isolation of cDNAs encoding M phase phosphoproteins (MPPs). In the work described herein, we further characterize MPP10, one of 10 novel proteins that we identified, with regard to its potential nucleolar function. We show that by cell fractionation, almost all MPP10 was found in isolated nucleoli. By immunofluorescence, MPP10 colocalized with nucleolar fibrillarin and other known nucleolar proteins in interphase cells but was not detected in the coiled bodies stained for either fibrillarin or p80 coilin, a protein found only in the coiled body. When nucleoli were separated into fibrillar and granular domains by treatment with actinomycin D, almost all the MPP10 was found in the fibrillar caps, which contain proteins involved in rRNA processing. In early to middle M phase of the cell cycle, MPP10 colocalized with fibrillarin to chromosome surfaces. At telophase, MPP10 was found in cellular structures that resembled nucleolus-derived bodies and prenucleolar bodies. Some of these bodies lacked fibrillarin, a previously described component of nucleolus-derived bodies and prenucleolar bodies, however, and the bulk of MPP10 arrived at the nucleolus later than fibrillarin. To further examine the properties of MPP10, we immunoprecipitated it from cell sonicates. The resulting precipitates contained U3 small nucleolar RNA (snoRNA) but no significant amounts of other box C/D snoRNAs. This association of MPP10 with U3 snoRNA was stable to 400 mM salt and suggested that MPP10 is a component of the human U3 small nucleolar ribonucleoprotein.

Le nucléole des mammifères contient des centaines de petits ARN nucléolaires à boîte C/D (SNORD) dont la grande majorité guide une 2'-O-ribose méthylation sur les précurseurs des ARN ribosomiques (pré-ARNr). Certains SNORD facilitent aussi les clivages que subissent le pré-ARNr ou modifient le petit ARN nucléaire U6. Des travaux récents laissent également entrevoir que certains SNORD interagissent avec des ARNm. C'est le cas par exemple pour SNORD115 qui est au cœur de mon travail de thèse. SNORD115 est exprimé uniquement dans le cerveau à partir de nombreux gènes répétés en tandem situés au locus SNURF-SNRPN dont l'expression est contrôlée par l'empreinte génomique parentale. Des défauts génétiques associés à ce locus chromosomique sont associés à une maladie rare: le syndrome de Prader-Willi (SPW). SNORD115 est remarquable car il possède une longue complémentarité conservée avec l'ARNm codant un récepteur à la sérotonine, le variant 5-HT2C. Certains travaux proposent que SNORD115 régule la voie 5-HT2C en modulant l'épissage alternatif ou l'édition A vers I du pré-ARNm 5-HT2C. Un défaut dans l'activité du 5-HT2C pourrait être à l'origine de l'hyperphagie et/ou des anomalies comportementales qui caractérisent le SPW. Mon projet de thèse principal consistait à éprouver cette hypothèse grâce à un nouveau modèle murin CRISPR/Cas9 invalidé pour SNORD115. Mes résultats montrent que la perte d'expression de SNORD115 ne perturbe pas la régulation post-transcriptionnelle du pré-ARNm 5-HT2C in vivo. D'autre part, des études réalisées dans l'équipe n'ont pas permis de révéler des anomalies marquées dans les phénotypes anxio-dépressifs, ni dans le comportement alimentaire. Ma thèse soulève donc des questions importantes quant au rôle régulateur de SNORD115 dans le cerveau et de sa contribution potentielle dans l'étiologie du SPW. En parallèle, j'ai aussi abordé le répertoire des 2'-O-méthylations de l'ARNr dans des tissus murins, notamment le cerveau. Ce travail s'inscrivait dans la thématique émergente de la théorie du "ribosome spécialisé" qui propose qu'une hétérogénéité structurale des composants du ribosome puisse se traduire par des changements dans les capacités fonctionnelles du ribosome. Mes résultats montrent des variations dans la méthylation pour un nombre très limité de sites, et ce principalement au cours du développement. Aussi, les ribosomes des tissus développementaux sont globalement moins méthylés que ceux des tissus adultes. Nous avons concentré nos efforts sur LSU-G4593 dont la méthylation guidée par SNORD78 est retrouvée uniquement au cours du développement. Nous proposons que des évènements d'épissage alternatif du gène-hôte de SNORD78 modulent la production de SNORD78, et de fait le niveau de méthylation LSU-Gm4593. Grâce à l'étude d'une lignée cellulaire humaine (HEK293) invalidée pour SNORD78, j'ai recherché les implications fonctionnelles de LSU-Gm4593. A ce jour, mes travaux ne montrent pas un rôle marqué dans la prolifération cellulaire, ni dans la fidélité de la traduction. La fonction précise de LSU-Gm4593 demeure donc incomprise
The nucleolus of mammalian cells contains hundreds of box C/D small nucleolar RNAs (SNORDs). Majority of them, guide sequence-specific 2'-O ribose methylations into ribosomal RNA (rRNA). Some of them facilitate RNA folding and cleavages of ribosomal RNA precursors or guide ribose methylations into spliceosomal small nuclear RNA U6. Recent studies propose that some SNORD could target other transcripts, possibly messenger RNA as suggested by the brain-specific SNORD115. SNORD115 is processed from tandemly repeated genes embedded in the imprinted SNURF-SNRPN domain. Defects in gene expression at this domain are causally linked to rare disease: the Prader-Willi Syndrome (PWS). Excitingly, SNORD115 displays an extensive region of complementary to a brain-specific mRNA encoding the serotonin receptor 5-HT2C. SNORD115 could influence 5-HT2C signaling by fine-tuning alternative splicing or A to I RNA editing of 5-HT2C pre-mRNA. Reduced 5-HT2C receptor activity could contribute to impaired emotional response and/or compulsive overeating that characterized the syndrome. My work was to test this hypothesis using a CRISPR/Cas9-mediated SNORD115 knockout mouse model. My results show that loss of SNORD115 expression, in vivo, does not alter the post-transcriptional regulation of 5-HT2C pre-mRNA processing. Others results from the team do not reveal any defects in anxio-depressive phenotypes and eating behaviour. Our study questions the regulatory roles of SNORD115 in brain functions and behavioural disturbance associated with PWS. On other hand, I have studied ribose methylation sites in rRNA from mouse tissues. This work was included in emerging field of the specialized ribosome hypothesis which suggests heterogeneity in ribosomes may impact activity of ribosomes. Our results show significant changes at few discrete set of sites, especially in rRNA from developing tissues. Also, rRNA from developing tissues is globally less methylated than rRNA from adult tissues. We focus on LSU-Gm4593 site because this position is specifically methylated only during development and hardly ever detected in adult tissues. Methylation at LSU-G4593 is guided by SNORD78. We propose that the expression levels of SNORD78 during development appeared to be regulated by alternative splicing of the host-gene and to correlate with the methylation level of its target site at LSU-G4593. We've used a human cell line (HEK293T) inactivated for the SNORD78 gene in order to understand the functionally role of the corresponding ribose methylation. Our work did not demonstrate any overt cellular phenotypes, even though translation fidelity and the precise function of LSU-Gm4593 remains unknown

Les cellules contiennent de nombreux ARNs non-codants qui jouent un rôle essentiel dans toutes les étapes de l'expression génique. Nos travaux concernent la caractérisation de possibles nouvelles fonctions associés à deux classes d'ARNs non-codants : l'ARN U1 et les scaARNs. Le petit ARN nucléaire U1 forme une ribonucléoparticule (snRNP U1) qui joue un rôle clé dans l'épissage des ARN pré-messagers. De plus, l'ARN U1 semble intervenir dans d'autres étapes de la synthèse des ARNm fonctionnels. Nos travaux ont démontré qu'une fraction de l'ARN U1 interagit spécifiquement avec la protéine TAF15, dans un complexe qui diffère dans sa composition protéique de la particule d'épissage canonique Sm snRNP U1. Nous avons constaté qu'à la différence de la particule d'épissage, la snRNP U1-TAF15 est fortement attachée à la chromatine. De plus, suite à l'arrêt de la transcription par l'ARN polymérase II, la particule U1-TAF15 se relocalise dans la coiffe périnucléolaire, à l'opposé de la particule Sm snRNP U1 qui s'accumule alors dans les "speckles". Une étude protéomique de la particule U1-TAF15 nous a permis d'établir des hypothèses quant à sa fonction qui sont actuellement à l'étude dans le laboratoire. Récemment, une protéine essentielle à l'adressage des scaARNs (ARNs spécifiques des Corps de Cajal) dans les Corps de Cajal a été identifiée (Tycowski et al. , 2009; Venteicher et al. , 2009). Une analyse par séquençage à haut-débit de l'ensemble des petits ARNs associés à WDR79 a permis d'identifier de nombreux nouveaux scaARNs putatifs. Parmi eux, l'analyse de deux ARNs à boite C/D a défini des structures essentielles pour leur localisation dans les corps de Cajal. Des études plus approfondies devraient nous permettre de déterminer des éléments spécifiques nécessaires à l'adressage des scaARNs à boite C/D vers les corps de Cajal. Par ailleurs, nous avons commencé la caractérisation d'un nouveau scaARN qui ciblerait la modification d'un ARN de transfert. Alors que les ARN C/D connus chez les Eucaryotes participent à la biogenèse des ARN ribosomiques et des petits ARN nucléaires, nous avons identifié pour la première fois chez les Eucaryotes un ARN C/D qui ciblerait un ARN de transfert
Noncoding regulatory RNAs (ncRNAs) are in the focus of current research, since they participate in nearly all cellular processes. To get further insights into the functional and structural complexity of ncRNAs, we studied human ncRNAs belonging to two classes of ncRNAs, the nucleoplasmic spliceosomal snRNAs and the nucleolar and Cajal body-specific box C/D 2'-O-methylation guide RNAs. The U1 snRNP is an evolutionarily conserved, abundant nucleoplasmic snRNP that plays a central role in pre-mRNA splicing. According to a recently emerging view, besides its constitutive role in splicing, the U1 snRNP has important regulatory functions in different steps of pre-mRNA production. We demonstrated that a fraction of the human U1 snRNA specifically associates with the nuclear RNA-binding protein TAF15 that is known to interact with a subpopulation of TFIID and RNA polymerase II complexes. The U1-TAF15 snRNP is structurally and functionally distinct from the well-characterized U1 spliceosomal snRNP and it tightly associates with chromatin. The function of U1-TAF15 snRNP remains unknown; it might contribute to the coupling of transcription and splicing. WDR79 (also called WRAP53) has been recently identified as an essential factor for targeting a subclass of box C/D and H/ACA modification guide RNAs, as well as telomerase H/ACA RNA, into the Cajal bodies. Accumulation of box C/D and H/ACA RNPs in Cajal bodies is essential for the biogenesis of functional spliceosomal snRNPs and telomere synthesis. Co-immunopurification of WDR79-associated human RNAs, followed by cDNA synthesis and deep sequencing identified a large number of novel Cajal body-specific RNAs. We are currently dissecting the cis-acting RNA element responsible for WDR79-binding and for targeting box C/D 2'-O-methylation guide RNPs into Cajal bodies. We have also identified a novel Cajal body-specific 2'-O-methylation guide RNA that is predicted to direct methylation of cytidine 34 at the Wobble position of tRNA-Met-CAT elongator. Interestingly, tRNA modification is a novel function for vertebrate box C/D scaRNPs

Eukaryotic cells contain scores of small nucleolar RNAs (snoRNAs), which are required for maturation of pre-rRNA. Two large snoRNA families exist defined by vital box C/D and box H/ACA motifs. The goal of the present study was to gain new insights into the structure and biogenesis of the box C/D snoRNAs; the knowledge developed from this effort was then recruited for practical applications. The investigation was conducted with the phylogenetically conserved U14 and U3 box C/D snoRNAs, from the yeast Saccharomyces cerevisiae. The specific aims included: (1) identification of cis-elements sufficient for biogenesis of the U14 snoRNA; (2) development of a functional map for the U3 snoRNA, and; (3) development of a U3-based model ribozyme system for in vivo studies. Conclusions derived from the U14 biogenesis studies are: (1) production of U14 involves ordered folding of the precursor RNA, and this step is required for formation of the vital box C/D structure motif, and; (2) the active box C/D motif, which is now predicted to consist solely of the box C and D elements, is necessary and sufficient for both accumulation and targeting RNA to the nucleolus. A general model for box C/D snoRNA biogenesis is proposed. Functional mapping of U3 revealed that: (1) boxes C$\sp\prime$ and D and flanking helices are critical for U3 accumulation; (2) boxes B and C are not essential for U3 production, but are important for function, due most likely to binding of a trans-acting factor(s); (3) the 5$\sp\prime$ portion of U3 is required for function, but not stability, and; (4) the non-conserved hairpins, which account for 50% of the molecule, are not required for accumulation or function. Based on the knowledge obtained with U14 and U3, a model ribozyme system featuring chimeric U3:ribozyme RNAs, or "snorbozymes", was developed and tested in vivo. Remarkably, the cleavage efficiency by a hammerhead ribozyme, both in cis- and in trans-configurations, appears quantitative! Other advantages of the system are: (1) a final product is stable, and; (2) authentic in vivo cleavage can be easily distinguished from artifactual cleavages. Snorbozymes are predicted to be useful for targeting natural transcripts in any eukaryotes, for fundamental research or practical applications.