The Hsp90 chaperone controls the biogenesis of L7Ae RNPs through conserved machinery

RNA-binding proteins of the L7Ae family are at the heart of many essential ribonucleoproteins (RNPs), including box C/D and H/ACA small nucleolar RNPs, U4 small nuclear RNP, telomerase, and messenger RNPs coding for selenoproteins. In this study, we show that Nufip and its yeast homologue Rsa1 are key components of the machinery that assembles these RNPs. We observed that Rsa1 and Nufip bind several L7Ae proteins and tether them to other core proteins in the immature particles. Surprisingly, Rsa1 and Nufip also link assembling RNPs with the AAA + adenosine triphosphatases hRvb1 and hRvb2 and with the Hsp90 chaperone through two conserved adaptors, Tah1/hSpagh and Pih1. Inhibition of Hsp90 in human cells prevents the accumulation of U3, U4, and telomerase RNAs and decreases the levels of newly synthesized hNop58, hNHP2, 15.5K, and SBP2. Thus, Hsp90 may control the folding of these proteins during the formation of new RNPs. This suggests that Hsp90 functions as a master regulator of cell proliferation by allowing simultaneous control of cell signaling and cell growth

A second base pair interaction between U3 small nucleolar RNA and the 5′-ETS region is required for early cleavage of the yeast pre-ribosomal RNA

In eukaryotes, U3 snoRNA is essential for pre-rRNA maturation. Its 5′-domain was found to form base pair interactions with the 18S and 5′-ETS parts of the pre-rRNA. In Xenopus laevis, two segments of U3 snoRNA form base-pair interactions with the 5′-ETS region and only one of them is essential to the maturation process. In Saccharomyces cerevisiae, two similar U3 snoRNA–5′ ETS interactions are possible; but, the functional importance of only one of them had been tested. Surprisingly, this interaction, which corresponds to the non-essential one in X. laevis, is essential for cell growth and pre-rRNA maturation in yeast. In parallel with [Dutca et al. (2011) The initial U3 snoRNA:pre-rRNA base pairing interaction required for pre-18S rRNA folding revealed by in vivo chemical probing. Nucleic Acids Research, 39, 5164–5180], here we show, that the second possible 11-bp long interaction between the 5′ domain of S. cerevisiae U3 snoRNA and the pre-rRNA 5′-ETS region (helix VI) is also essential for pre-rRNA processing and cell growth. Compensatory mutations in one-half of helix VI fully restored cell growth. Only a partial restoration of growth was obtained upon extension of compensatory mutations to the entire helix VI, suggesting sequence requirement for binding of specific proteins. Accordingly, we got strong evidences for a role of segment VI in the association of proteins Mpp10, Imp4 and Imp3

Etudes sur le mécanisme d'action du petit ARN nucléolaire U3 de la levure Saccharomyces cerevisiae et sur la protéine Snu13p/15,5 kD associée à cet ARN et son homologue d'archaea L7Ae

Le snoRNA U3 a un rôle essentiel dans la maturation du pré-ARN ribosomique d'eucaryotes. Son domaine 5' forme plusieurs hélices avec le pré-ARNr. Son domaine 3' contient deux sites d'ancrage des protéines de la snoRNP U3, dont Snu13p. Nous avons montré que, chez S. cerevisiae, les segments du domaine 5' du snoRNA U3 impliqués dans la formation des hélices hétérologues, ainsi que les séquences les liant, ont des rôles distincts dans le processus de maturation. Les études structurale, phylogénétique et fonctionnelle, que nous avons menées sur la fixation de Snu13p au domaine 3', mettent en évidence deux sites formant des structures particulières en K-turn. In vivo, des mutations dans le site B/C engendrent plus de défauts fonctionnels que dans le site C'/D. Nous avons comparé la spécificité de fixation à l'ARN de Snu13p à celle de son homologue d'archaea L7Ae, et mis en évidence un lien possible entre le snoRNA U3 et la protéine Rsa1, intervenant dans la maturation de la sous-unité 60S.In eukarya, U3 snoRNA plays a crucial role in ribosomal RNA maturation. Its 5' domain forms several base-pair interactions with the pre-ribosomal RNA and its 3' domain contains two anchoring sites for the U3 snoRNP proteins, including Snu13p. We showed, in yeast, that the segments of the U3 snoRNA 5' domain, which are involved in the intermolecular base-pair interactions and the sequences linking these segments, have differential functional roles in the maturation process. Our structural, phylogenetic, and functional study of Snu13p binding on U3 snoRNA demonstrated the presence of two binding sites that form peculiar K-turn structure. In vivo experiments showed that mutations in the B/C motif are more deleterious for U3 snoRNA function than mutations in the C'/D motif. We compared the RNA binding specificity of Snu13p and its archaeal homolog L7Ae. Finally, we found a possible link between protein Rsa1, involved in the late steps of 60S subunit maturation, and U3 snoRNA.NANCY1-SCD Sciences & Techniques (545782101) / SudocSudocFranceF

Synergistic defects in pre-rRNA processing from mutations in the U3-specific protein Rrp9 and U3 snoRNA

International audienceU3 snoRNA and the associated Rrp9/U3-55K protein are essential for 18S rRNA production by the SSU-processome complex. U3 and Rrp9 are required for early pre-rRNA cleavages at sites A0, A1 and A2, but the mechanism remains unclear. Substitution of Arg 289 in Rrp9 to Ala (R289A) specifically reduced cleavage at sites A1 and A2. Surprisingly, R289 is located on the surface of the Rrp9 β-propeller structure opposite to U3 snoRNA. To understand this, we first characterized the protein-protein interaction network of Rrp9 within the SSU-processome. This identified a direct interaction between the Rrp9 β-propeller domain and Rrp36, the strength of which was reduced by the R289A substitution, implicating this interaction in the observed processing phenotype. The Rrp9 R289A mutation also showed strong synergistic negative interactions with mutations in U3 that destabilize the U3/pre-rRNA base-pair interactions or reduce the length of their linking segments. We propose that the Rrp9 β-propeller and U3/pre-rRNA binding cooperate in the structure or stability of the SSU-processome. Additionally, our analysis of U3 variants gave insights into the function of individual segments of the 5'-terminal 72-nt sequence of U3. We interpret these data in the light of recently reported SSU-processome structures