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

Etude structurale et fonctionnelle de motifs en "K-turn" présents dans les ARN, rôle dans l'assemblage et le mécanisme d'action de particules ribonucléoprotéiques impliquées dans la maturation des ARN

La snoRNP U3 est une particule ribonucléoprotéique nucléolaire qui est requise pour la synthèse de l'ARN ribosomique de la petite sous-unité, l'ARNr 18S. Le snoRNA U3 contient 2 domaines (5' et 3'). Nous avons étudié le rôle d'interactions formées entre le domaine 5' et le pré-ARNr dans la production de l'ARNr 18S. Le snoRNA U3 est associé à des protéines. Deux sites d'ancrage des protéines sont contenus dans le domaine 3' (les motifs C'/D et B/C). Ils adoptent une structure particulière en "K-turn" et fixent chacun la protéine Snu13 qui permet le recrutement des protéines Nop1, Nop56 et Nop58 sur le motif C'/D et Rrp9 sur le motif B/C. Nous avons identifié les déterminants de l'ARN requis pour la fixation de Snu13p et identifié les éléments de chaque motif permettant la fixation des protéines distinctes, en particulier Rrp9p sur le motif B/C. Le motif de reconnaissance de Snu13p ayant des homologies avec ceux des protéines L7Ae d'archaea, et SBP2 de vertébrés, nous avons comparé les spécificités d'interaction de ces 3 protéines avec l'ARN. Enfin, nous avons étudié des changements de conformations du pré-ARN ribosomique lors de sa maturation.In eukarya, the nucleolar U3 snoRNP plays a crucial role in 18S rRNA maturation. U3 snoRNA contains 2 domains (5' and 3'). We studied the role for 18S rRNA production of base-pair interactions formed between its 5' domain and the pre-rRNA. U3 snoRNA binds several proteins. Its 3' domain contains two protein anchoring sites (the C'/D and B/C motifs). They adopt a peculiar K-turn structure and each K-turn binds protein Snu13. Binding of this protein is required for recruitment of proteins Nop1, Nop56 and Nop58 on the C'/D motif and of protein Rrp9 on the B/C motif. We determined the RNA determinants, required for the specific recruitment of these proteins, in particular Rrp9p. The RNA binding domain of protein Snu13p has strong homologies with those of the archaeal L7Ae and vertebrate SBP2 proteins. We compared the RNA binding specificity of these 3 proteins. Finally, we studied RNA conformational changes in pre-ribosomal RNAs during the maturation process.NANCY1-SCD Sciences & Techniques (545782101) / SudocSudocFranceF

Synergy between NMR measurements and MD simulations of protein/RNA complexes: Application to the RRMs, the most common RNA Recognition Motifs

RNA Recognition Motif (RRM) proteins represent an abundant and variable class of proteins playing key roles in RNA biology. We present a joint atomistic molecular dynamics (MD) and experimental study of two RRM-containing proteins bound with their single-stranded target RNAs, namely the Fox-1 and SRSF1 complexes. The simulations are used in conjunction with NMR spectroscopy to interpret and expand the available structural data. We accumulate more than 50 µs of simulations and show that the MD method is robust enough to reliably describe the structural dynamics of the RRM-RNA complexes. The simulations predict unanticipated specific participation of Arg142 at the protein-RNA interface of the SRFS1 complex, which is subsequently confirmed by NMR and ITC measurements with mutant protein. We also predict that several segments of the protein-RNA interface may involve competition between dynamical local substates rather than firmly formed interactions, which is indirectly consistent with the primary NMR data. We demonstrate that the simulations can reveal important information not obtainable from the NMR data alone, can be used to interpret the NMR atomistic models, and can provide qualified predictions. Finally, we propose a protocol for “MD-adapted structure ensemble” as a way to integrate the simulation predictions and expand upon the deposited NMR structures. Unbiased µs-scale atomistic MD could become a technique routinely complementing the NMR measurements of protein-RNA complexe

Tandem hnRNP A1 RNA recognition motifs act in concert to repress the splicing of survival motor neuron exon 7

HnRNP A1 regulates many alternative splicing events by the recognition of splicing silencer elements. Here, we provide the solution structures of its two RNA recognition motifs (RRMs) in complex with short RNA. In addition, we show by NMR that both RRMs of hnRNP A1 can bind simultaneously to a single bipartite motif of the human intronic splicing silencer ISS-N1, which controls survival of motor neuron exon 7 splicing. RRM2 binds to the upstream motif and RRM1 to the downstream motif. Combining the insights from the structure with in cell splicing assays we show that the architecture and organization of the two RRMs is essential to hnRNP A1 function. The disruption of the inter-RRM interaction or the loss of RNA binding capacity of either RRM impairs splicing repression by hnRNP A1. Furthermore, both binding sites within the ISS-N1 are important for splicing repression and their contributions are cumulative rather than synergistic.ISSN:2050-084

Structural flexibility enables alternative maturation, ARGONAUTE sorting and activities of miR168, a global gene silencing regulator in plants

In eukaryotes, the RNase-III Dicer often produces length/sequence microRNA (miRNA) variants, called "isomiRs", owing to intrinsic structural/sequence determinants of the miRNA precursors (pre-miRNAs). In this study, we combined biophysics, genetics and biochemistry approaches to study Arabidopsis miR168, the key feedback regulator of central plant silencing effector protein ARGONAUTE1 (AGO1). We identified a motif conserved among plant pre-miR168 orthologs, which enables flexible internal base-pairing underlying at least three metastable structural configurations. These configurations promote alternative, accurate Dicer cleavage events generating length and structural isomiR168 variants with distinctive AGO sorting properties and modes of action. Among these isomiR168s, a duplex with a 22-nt guide strand exhibits strikingly preferential affinity for AGO10, the closest AGO1 paralog. The 22-nt miR168-AGO10 complex antagonizes AGO1 accumulation in part via "transitive RNAi", a silencing-amplification process, to maintain appropriate AGO1 cellular homeostasis. Furthermore, we found that the tombusviral P19 silencing-suppressor protein displays markedly weaker affinity for the 22-nt form among its isomiR168 cargoes, thereby promoting AGO10-directed suppression of AGO1-mediated antiviral silencing. Taken together, these findings indicate that structural flexibility, a previously overlooked property of pre-miRNAs, considerably increases the versatility and regulatory potential of individual MIRNA genes, and that some pathogens might have evolved the capacity or mechanisms to usurp this property

Exon-independent recruitment of SRSF1 is mediated by U1 snRNP stem-loop 3

SRSF1 protein and U1 snRNPs are closely connected splicing factors. They both stimulate exon inclusion, SRSF1 by binding to exonic splicing enhancer sequences (ESEs) and U1 snRNPs by binding to the downstream 5 ' splice site (SS), and both factors affect 5 ' SS selection. The binding of U1 snRNPs initiates spliceosome assembly, but SR proteins such as SRSF1 can in some cases substitute for it. The mechanistic basis of this relationship is poorly understood. We show here by single-molecule methods that a single molecule of SRSF1 can be recruited by a U1 snRNP. This reaction is independent of exon sequences and separate from the U1-independent process of binding to an ESE. Structural analysis and cross-linking data show that SRSF1 contacts U1 snRNA stem-loop 3, which is required for splicing. We suggest that the recruitment of SRSF1 to a U1 snRNP at a 5 ' SS is the basis for exon definition by U1 snRNP and might be one of the principal functions of U1 snRNPs in the core reactions of splicing in mammals.ISSN:0261-4189ISSN:1460-207

Exon-independent recruitment of SRSF1 is mediated by U1 snRNP stem-loop 3

SRSF1 protein and U1 snRNPs are closely connected splicing factors. They both stimulate exon inclusion, SRSF1 by binding to exonic splicing enhancer sequences (ESEs) and U1 snRNPs by binding to the downstream 5′ splice site (SS), and both factors affect 5′ SS selection. The binding of U1 snRNPs initiates spliceosome assembly, but SR proteins such as SRSF1 can in some cases substitute for it. The mechanistic basis of this relationship is poorly understood. We show here by single-molecule methods that a single molecule of SRSF1 can be recruited by a U1 snRNP. This reaction is independent of exon sequences and separate from the U1-independent process of binding to an ESE. Structural analysis and cross-linking data show that SRSF1 contacts U1 snRNA stem-loop 3, which is required for splicing. We suggest that the recruitment of SRSF1 to a U1 snRNP at a 5′SS is the basis for exon definition by U1 snRNP and might be one of the principal functions of U1 snRNPs in the core reactions of splicing in mammals

A home-visiting intervention targeting determinants of infant mental health: the study protocol for the CAPEDP randomized controlled trial in France

<p>Abstract</p> <p>Background</p> <p>Several studies suggest that the number of risk factors rather than their nature is key to mental health disorders in childhood.</p> <p>Method and design</p> <p>The objective of this multicentre randomized controlled parallel trial (PROBE methodology) is to assess the impact in a multi-risk French urban sample of a home-visiting program targeting child mental health and its major determinants. This paper describes the protocol of this study. In the study, pregnant women were eligible if they were: living in the intervention area; able to speak French, less than 26 years old; having their first child; less than 27 weeks of amenorrhea; and if at least one of the following criteria were true: less than twelve years of education, intending to bring up their child without the presence of the child’s father, and 3) low income. Participants were randomized into either the intervention or the control group. All had access to usual care in mother-child centres and community mental health services free of charge in every neighbourhood. Psychologists conducted all home visits, which were planned on a weekly basis from the 7^th month of pregnancy and progressively decreasing in frequency until the child’s second birthday. Principle outcome measures included child mental health at 24 months and two major mediating variables for infant mental health: postnatal maternal depression and the quality of the caring environment. A total of 440 families were recruited, of which a subsample of 120 families received specific attachment and caregiver behaviour assessment. Assessment was conducted by an independent assessment team during home visits and, for the attachment study, in a specifically created Attachment Assessment laboratory.</p> <p>Discussion</p> <p>The CAPEDP study is the first large-scale randomised, controlled infant mental health promotion programme to take place in France. A major specificity of the program was that all home visits were conducted by specifically trained, supervised psychologists rather than nurses. Significant challenges included designing a mental health promotion programme targeting vulnerable families within one of the most generous but little assessed health and social care systems in the Western World.</p> <p>Trial registration</p> <p>Current Clinical trial number is NCT00392847.</p