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

The Kink Turn, a Key Architectural Element in RNA Structure

AbstractKink turns (k-turns) are widespread structural elements that introduce an axial bend into duplex RNA with an included angle of 50°. These mediate key tertiary interactions and bind specific proteins including members of the L7Ae family. The standard k-turn comprises a three-nucleotide bulge followed by G·A and A·G pairs. The RNA kinks by an association of the two minor grooves, stabilized by the formation of a number of key cross-strand hydrogen bonds mostly involving the adenine bases of the G·A and A·G pairs. The k-turns may be divided into two conformational classes, depending on the receptor for one of these hydrogen bonds. k-turns become folded by one of three different processes. Some, but not all, k-turns become folded in the presence of metal ions. Whether or not a given k-turn is folded under these conditions is determined by its sequence. We present a set of rules for the prediction of folding properties and the structure adopted on local sequence

The K-turn motif in riboswitches and other RNA species

AbstractThe kink turn is a widespread structure motif that introduces a tight bend into the axis of duplex RNA. This generally functions to mediate tertiary interactions, and to serve as a specific protein binding site. K-turns or closely related structures are found in at least seven different riboswitch structures, where they function as key architectural elements that help generate the ligand binding pocket. This article is part of a Special Issue entitled: Riboswitches

Non-coding RNA annotation of the genome of Trichoplax adhaerens

A detailed annotation of non-protein coding RNAs is typically missing in initial releases of newly sequenced genomes. Here we report on a comprehensive ncRNA annotation of the genome of Trichoplax adhaerens, the presumably most basal metazoan whose genome has been published to-date. Since blast identified only a small fraction of the best-conserved ncRNAs—in particular rRNAs, tRNAs and some snRNAs—we developed a semi-global dynamic programming tool, GotohScan, to increase the sensitivity of the homology search. It successfully identified the full complement of major and minor spliceosomal snRNAs, the genes for RNase P and MRP RNAs, the SRP RNA, as well as several small nucleolar RNAs. We did not find any microRNA candidates homologous to known eumetazoan sequences. Interestingly, most ncRNAs, including the pol-III transcripts, appear as single-copy genes or with very small copy numbers in the Trichoplax genome

UtpA and UtpB chaperone nascent pre-ribosomal RNA and U3 snoRNA to initiate eukaryotic ribosome assembly

Early eukaryotic ribosome biogenesis involves large multi-protein complexes, which co-transcriptionally associate with pre-ribosomal RNA to form the small subunit processome. The precise mechanisms by which two of the largest multi-protein complexes—UtpA and UtpB—interact with nascent pre-ribosomal RNA are poorly understood. Here, we combined biochemical and structural biology approaches with ensembles of RNA–protein cross-linking data to elucidate the essential functions of both complexes. We show that UtpA contains a large composite RNA-binding site and captures the 5′ end of pre-ribosomal RNA. UtpB forms an extended structure that binds early pre-ribosomal intermediates in close proximity to architectural sites such as an RNA duplex formed by the 5′ ETS and U3 snoRNA as well as the 3′ boundary of the 18S rRNA. Both complexes therefore act as vital RNA chaperones to initiate eukaryotic ribosome assembly

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