Microarray detection of novel nuclear RNA substrates for the exosome

Microarray analyses were performed on yeast strains mutant for the nuclear-specific exosome components Rrp6p and Rrp47p/Lrp1p or the core component Rrp41p/Ski6p, at permissive temperature and following transfer to 37 degrees C. 339 mRNAs showed clearly altered expression levels, with an unexpectedly high degree of heterogeneity in the different exosome mutants. In contrast, no clear alterations were seen in strains lacking the cytoplasmic exosome component Ski7p. 27 mRNAs that were overexpressed in each strain defective in the nuclear exosome are good candidates for regulation by nuclear turnover. These included the mRNA for the autoregulated RNA-binding protein Nrd1p. Northern and primer extension analyses confirmed the elevated NRD1 mRNA levels in exosome mutants, and revealed the accumulation of truncated 5' fragments of the mRNA. These contain a predicted Nrd1p-binding site, potentially sequestering the protein and disrupting its autoregulation. Several genes located immediately downstream of independently transcribed snoRNA genes were overexpressed in exosome mutants, presumably due to stabilization of the products of transcription termination read-through. Further analyses indicated that many snoRNA and snRNA genes are inefficiently terminated, but read-through transcripts into downstream ORFs are normally rapidly degraded by the exosome. Copyright (c) 2006 John Wiley & Sons, Ltd

Unphosphorylated SR-Like Protein Npl3 Stimulates RNA Polymerase II Elongation

The production of a functional mRNA is regulated at every step of transcription. An area not well-understood is the transition of RNA polymerase II from elongation to termination. The S. cerevisiae SR-like protein Npl3 functions to negatively regulate transcription termination by antagonizing the binding of polyA/termination proteins to the mRNA. In this study, Npl3 is shown to interact with the CTD and have a direct stimulatory effect on the elongation activity of the polymerase. The interaction is inhibited by phosphorylation of Npl3. In addition, Casein Kinase 2 was found to be required for the phosphorylation of Npl3 and affect its ability to compete against Rna15 (Cleavage Factor I) for binding to polyA signals. Our results suggest that phosphorylation of Npl3 promotes its dissociation from the mRNA/RNAP II, and contributes to the association of the polyA/termination factor Rna15. This work defines a novel role for Npl3 in elongation and its regulation by phosphorylation

Multiple roles of RNAs

MasterIn this practical course, we propose to explore in detail the substrates and mechanisms of a major translation-dependent RNA degradation pathway, the Nonsense Mediated mRNA Decay (NMD). NMD degrades transcripts with premature stop codons through poorly understood mechanism that are conserved from yeast to humans. The lectures will be delivered by RNA specialists who will ficus on the multiple roles of RNA and specific technologies related to RNA studies. The aim of the practical course is to learn classical and innovative RNA-related techniques that include transcriptome analysis using RNA-seq, Northern blots and RT-qPCR. Student study different RNA populations that associate with the NMD machinery. We will compare transcripts from a wild-type and from mutants yeast strains in which essential NMD factor or cofactor genes have been deleted. Genome wide analysis of the resultswill integrate phenotypic data (changes in RNA abundance) to illustrate the diversity of NMD targets. see more at https://research.pasteur.fr/fr/course/multiple-roles-of-rnas-2/Dans ce cours pratique, nous proposons d'explorer en détail les substrats et les mécanismes d'une voie majeure de dégradation de l'ARN dépendant de la traduction, le Nonsense Mediated mRNA Decay (NMD). Le NMD dégrade les transcrits contenant des codons stop prématurés par le biais de mécanismes mal compris qui sont conservés de la levure à l'homme. Les conférences seront données par des spécialistes de l'ARN qui parleront des multiples rôles de l'ARN et des technologies spécifiques liées à l'étude de l'ARN. L'objectif du cours pratique est d'apprendre les techniques classiques et innovantes liées à l'ARN qui comprennent l'analyse du transcriptome en utilisant le RNA-seq, les Northern blots et la RT-qPCR. Les étudiants étudient différentes populations d'ARN qui s'associent à la machinerie NMD. Nous comparerons les transcrits d'un type sauvage et de souches de levure mutantes dans lesquelles des gènes de facteurs ou de cofacteurs NMD essentiels ont été supprimés. L'analyse des résultats à l'échelle du génome intégrera des données phénotypiques (changements dans l'abondance de l'ARN) pour illustrer la diversité des cibles de la NMD. Pour en savoir plus : https://research.pasteur.fr/fr/course/multiple-roles-of-rnas-2

ETUDE DU ROLE PARTICULIER JOUE PAR LE GENE RRP5 DANS LA MATURATION DES ARN RIBOSOMAUX CHEZ LA LEVURE SACCHAROMYCES CEREVISIAE

ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Le Viroïde Avocado sunblotch (étude de sa réplication dans la levure Saccharomyces cerevisiae et de sa structure)

Les viroïdes sont les plus petits agents pathogènes connus (246 à 401 nt). Ce sont des ARN nus, simple-brin, circulaires et non-codants dont les deux séquences complémentaires nommées (+) et (-) co-existent dans les cellules. Il existe deux familles : les Pospiviroidae et les Avsunviroidae. Ces derniers ont dans la séquence de chaque polarité un ribozyme en tête de marteau, indispensable à leur réplication ARN dépendante. À ce jour, tous les viroïdes ont été identifiés chez des végétaux supérieurs. Le premier objectif de ma thèse a été de tester la réplication d un Avsunviroidae, le viroïde Avocado sunblotch (ASBVd), dans un système modèle non-photosynthétique, la levure Saccharomyces cerevisiae. J ai démontré que l ASBVd est capable de se répliquer et de se maintenir pendant au moins 25 générations dans la levure. De plus, l ASBVd est sensible à la dégradation des ARN nucléaire et cytoplasmique de S. cerevisiae. Les interactions des viroïdes avec les facteurs cellulaires semblent intimement liés à leurs caractéristiques structurales et catalytiques. Un très haut degré de complémentarité entre les différentes régions de ces ARN leur permet d adopter des structures complexes. Le deuxième objectif de ma thèse a été d étudier le comportement cinétique et structural des brins (+) et (-). J ai mis en évidence des différences de propriétés biophysiques entre les deux brins et une plus grande efficacité d auto-clivage de l ASBVd (-). La structure de l ASBVd est déterminée par une technique biochimique innovante et haut débit, le SHAPE (sélective 2 -hydroxyl acylation analysed by primer extension), pour préciser et localiser les différences structurales dans deux polaritésPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

A Nuclear Surveillance Pathway for mRNAs with Defective Polyadenylation

The pap1-5 mutation in poly(A) polymerase causes rapid depletion of mRNAs at restrictive temperatures. Residual mRNAs are polyadenylated, indicating that Pap1-5p retains at least partial activity. In pap1-5 strains lacking Rrp6p, a nucleus-specific component of the exosome complex of 3′-5′ exonucleases, accumulation of poly(A)(+) mRNA was largely restored and growth was improved. The catalytically inactive mutant Rrp6-1p did not increase growth of the pap1-5 strain and conferred much less mRNA stabilization than rrp6Δ. This may indicate that the major function of Rrp6p is in RNA surveillance. Inactivation of core exosome components, Rrp41p and Mtr3p, or the nuclear RNA helicase Mtr4p gave different phenotypes, with accumulation of deadenylated and 3′-truncated mRNAs. We speculate that slowed mRNA polyadenylation in the pap1-5 strain is detected by a surveillance activity of Rrp6p, triggering rapid deadenylation and exosome-mediated degradation. In wild-type strains, assembly of the cleavage and polyadenylation complex might be suboptimal at cryptic polyadenylation sites, causing slowed polyadenylation

In vitro selection of adenine-dependent ribozyme against Tpl2/Cot oncogene.

International audienceHairpin ribozymes possess the properties of RNA sequence-specific recognition and site-specific cleavage. These properties make them a powerful extension of the antisense approach for the inhibition of gene expression. From a randomized RNA pool of hairpin ribozymes, using the systematic evolution of ligands by exponential enrichment, we have obtained an adenine-dependent hairpin ribozyme, Tpl2/Cot (tumour progression locus 2) ribozyme, which cleaves the Tpl2/Cot kinase mRNA sequence at nucleotides A225/G226 relative to the start codon of translation. This serine/threonine kinase activates the mitogen-activated protein kinase pathway implicated in cell proliferation in cancer. The selected 'Tpl2/Cot-YL ribozyme' efficiently cleaves its target sequence in cis and in trans; furthermore, the ribozyme efficiently cleaves a longer target sequence of 54 nucleotides in trans, as well as the full-length mRNA

Additional Layer of Regulation via Convergent Gene Orientation in Yeasts

International audienc