Caractérisation de la fonction moléculaire du petit ARN nucléolaire H/ACA, snR30, dans la biogenèse des ribosomes chez Saccharomyces cerevisiae

Les petits ARN nucléolaires (snoARN) à boîtes H/ACA représentent une classe d'ARN non-codant très abondante, très conservée mais aussi extrêmement diversifiée. Tandis que de nombreux ARN à boîtes H/ACA sont responsables de la pseudo-uridylation des ARN ribosomiques et des snARN de la machinerie de l'épissage, le groupe grandissant des ARN "orphelins" participe aux clivages nucléolytiques des précurseurs ribosomiques (pré-ARNr), à la synthèse des télomères et très certainement à d'autres processus nucléaires. Appartenant à ce dernier groupe, le petit ARN nucléolaire snR30 est connu depuis longtemps comme étant essentiel à l'accumulation de l'ARNr 18S chez S. cerevisiae. En revanche, les mécanismes moléculaires à la base de cette fonction restent encore inconnus. Grâce à une approche à la fois génétique et biochimique, nous avons démontré que durant la maturation des pré-ARNr, deux séquences conservées présentes dans la tige-boucle à l'extrémité 3' de snR30 s'hybrident avec deux courtes séquences du pré-ARNr localisées au niveau d'une région interne de l'ARNr 18S, uniquement présente chez les Eucaryotes. Cette nouvelle interaction entre snR30 et l'ARNr 18S est essentielle à la production de ce dernier et ils forment une structure snoARN-ARN cible totalement nouvelle pour les snoARN à boîtes H/ACA. D'autre part, nous avons aussi mis en évidence qu'outre les motifs de reconnaissance de l'ARNr 18S, la partie distale de la tige-boucle à l'extrémité 3' de snR30 contient des séquences additionnelles essentielles pour la maturation de l'ARNr 18S qui pourraient probablement être le lieu de fixation de protéines indispensables à la maturation du pré-ARNr. De précédentes expériences ont révélé la présence de trois protéines spécifiques à la ribonucléoparticule de snR30 en complément des quatre protéines communes à tous les snoARN à boîtes H/ACA. Dans un dernier temps, nous avons donc mis au point une nouvelle technique de purification par affinité qui, à terme, pourrait être un bon outil pour identifier les protéines spécifiques de la ribonucléoparticule de snR30 et ainsi pourrait permettre d'approfondir le mécanisme d'action de ce snoARN à boîtes H/ACA dans les étapes de clivages nucléolytiques du pre-ARNr.Ribosome synthesis takes place in the nucleolus where many box H/ACA snoRNAs direct pseudouridylation of rRNAs. The U17/snR30 box H/ACA snoRNA, instead of directing rRNA modification, functions in the nucleolytic processing of the precursor rRNA (pre-rRNA) and is required for accumulation of mature 18S rRNA. We have identified two short sequence motifs in the 18S rRNA that can base-pair with two evolutionarily conserved sequences elements, m1 and m2 motifs, of the yeast Saccharomyces cerevisiae snR30. Mutations in the 18S sequences disrupting the base-pairing with the m1 or m2 motifs of snR30 inhibit the accumulation of the mature 18S rRNA. However, compensatory mutations introduced into m1 or m2 motifs of snR30 restore 18S rRNA processing. The m1 and m2 motifs that constitute the opposite strands of an internal loop of U17/snR30 base-pair with short 18S sequences preceding and following a conserved stem-loop structure in the middle of the 18S rRNA. Further functional mapping of yeast snR30 indicated that its 3'-terminal hairpin contains additional essential elements. Thus I developed a novel tandem affinity purification method in order to identify the putative snR30-specific snoRNP proteins

18S rRNA processing requires base pairings of snR30 H/ACA snoRNA to eukaryote-specific 18S sequences

The H/ACA RNAs represent an abundant, evolutionarily conserved and functionally diverse class of non-coding RNAs. Many H/ACA RNAs direct pseudouridylation of rRNAs and snRNAs, while members of the rapidly growing group of ‘orphan' H/ACA RNAs participate in pre-rRNA processing, telomere synthesis and probably, in other nuclear processes. The yeast snR30 ‘orphan' H/ACA snoRNA has long been known to function in the nucleolytic processing of 18S rRNA, but its molecular role remained unknown. Here, we provide biochemical and genetic evidence demonstrating that during pre-rRNA processing, two evolutionarily conserved sequence elements in the 3′-hairpin of snR30 base-pair with short pre-rRNA sequences located in the eukaryote-specific internal region of 18S rRNA. The newly discovered snR30-18S base-pairing interactions are essential for 18S rRNA production and they constitute a complex snoRNA target RNA transient structure that is novel to H/ACA RNAs. We also demonstrate that besides the 18S recognition motifs, the distal part of the 3′-hairpin of snR30 contains an additional snoRNA element that is essential for 18S rRNA processing and that functions most likely as a snoRNP protein-binding site

Identification of novel proteins associated with yeast snR30 small nucleolar RNA

H/ACA small nucleolar RNPs (snoRNPs) that guide pseudouridylation reactions are comprised of one small nucleolar RNA (snoRNA) and four common proteins (Cbf5, Gar1, Nhp2 and Nop10). Unlike other H/ACA snoRNPs, snR30 is essential for the early processing reactions that lead to the production of 18S ribosomal RNA in the yeast Saccharomyces cerevisiae. To determine whether snR30 RNP contains specific proteins that contribute to its unique functional properties, we devised an affinity purification strategy using TAP-tagged Gar1 and an RNA aptamer inserted in snR30 snoRNA to selectively purify the RNP. Northern blotting and pCp labeling experiments showed that S1-tagged snR30 snoRNA can be selectively purified with streptavidin beads. Protein analysis revealed that aptamer-tagged snR30 RNA was associated with the four H/ACA proteins and a number of additional proteins: Nop6, ribosomal proteins S9 and S18 and histones H2B and H4. Using antibodies raised against Nop6 we show that endogenous Nop6 localizes to the nucleolus and that it cosediments with snR30 snoRNA in sucrose density gradients. We demonstrate through primer extension experiments that snR30 snoRNA is required for cleavages at site A0, A1 and A2, and that the absence of Nop6 decreases the efficiency of cleavage at site A2. Finally, electron microscopy analyses of chromatin spreads from cells depleted of snR30 snoRNA show that it is required for SSU processome assembly

Exploiting Oxytricha trifallax nanochromosomes to screen for non-coding RNA genes

We took advantage of the unusual genomic organization of the ciliate Oxytricha trifallax to screen for eukaryotic non-coding RNA (ncRNA) genes. Ciliates have two types of nuclei: a germ line micronucleus that is usually transcriptionally inactive, and a somatic macronucleus that contains a reduced, fragmented and rearranged genome that expresses all genes required for growth and asexual reproduction. In some ciliates including Oxytricha, the macronuclear genome is particularly extreme, consisting of thousands of tiny ‘nanochromosomes’, each of which usually contains only a single gene. Because the organism itself identifies and isolates most of its genes on single-gene nanochromosomes, nanochromosome structure could facilitate the discovery of unusual genes or gene classes, such as ncRNA genes. Using a draft Oxytricha genome assembly and a custom-written protein-coding genefinding program, we identified a subset of nanochromosomes that lack any detectable protein-coding gene, thereby strongly enriching for nanochromosomes that carry ncRNA genes. We found only a small proportion of non-coding nanochromosomes, suggesting that Oxytricha has few independent ncRNA genes besides homologs of already known RNAs. Other than new members of known ncRNA classes including C/D and H/ACA snoRNAs, our screen identified one new family of small RNA genes, named the Arisong RNAs, which share some of the features of small nuclear RNAs

Functional dichotomy of ribosomal proteins during the synthesis of mammalian 40S ribosomal subunits

Subsets of 40S ribosomal subunits are required for initiating rRNA processing, rRNA maturation, and nuclear export

Caractérisation de la fonction moléculaire du petit ARN nucléolaire H-ACA, snR30, dans la biogenèse des ribosomes chez Saccharomyces cerevisiae

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Interrelationships between Yeast Ribosomal Protein Assembly Events and Transient Ribosome Biogenesis Factors Interactions in Early Pre-Ribosomes

Early steps of eukaryotic ribosome biogenesis require a large set of ribosome biogenesis factors which transiently interact with nascent rRNA precursors (pre-rRNA). Most likely, concomitant with that initial contacts between ribosomal proteins (r-proteins) and ribosome precursors (pre-ribosomes) are established which are converted into robust interactions between pre-rRNA and r-proteins during the course of ribosome maturation. Here we analysed the interrelationship between r-protein assembly events and the transient interactions of ribosome biogenesis factors with early pre-ribosomal intermediates termed 90S pre-ribosomes or small ribosomal subunit (SSU) processome in yeast cells. We observed that components of the SSU processome UTP-A and UTP-B sub-modules were recruited to early pre-ribosomes independently of all tested r-proteins. On the other hand, groups of SSU processome components were identified whose association with early pre-ribosomes was affected by specific r-protein assembly events in the head-platform interface of the SSU. One of these components, Noc4p, appeared to be itself required for robust incorporation of r-proteins into the SSU head domain. Altogether, the data reveal an emerging network of specific interrelationships between local r-protein assembly events and the functional interactions of SSU processome components with early pre-ribosomes. They point towards some of these components being transient primary pre-rRNA in vivo binders and towards a role for others in coordinating the assembly of major SSU domains