Strand‐specific, high‐resolution mapping of modified RNA polymerase II

Reversible modification of the RNAPII C-terminal domain links transcription with RNA processing and surveillance activities. To better understand this, we mapped the location of RNAPII carrying the five types of CTD phosphorylation on the RNA transcript, providing strand-specific, nucleotide-resolution information, and we used a machine learning-based approach to define RNAPII states. This revealed enrichment of Ser5P, and depletion of Tyr1P, Ser2P, Thr4P, and Ser7P in the transcription start site (TSS) proximal ~150 nt of most genes, with depletion of all modifications close to the poly(A) site. The TSS region also showed elevated RNAPII relative to regions further 3′, with high recruitment of RNA surveillance and termination factors, and correlated with the previously mapped 3′ ends of short, unstable ncRNA transcripts. A hidden Markov model identified distinct modification states associated with initiating, early elongating and later elongating RNAPII. The initiation state was enriched near the TSS of protein-coding genes and persisted throughout exon 1 of intron-containing genes. Notably, unstable ncRNAs apparently failed to transition into the elongation states seen on protein-coding genes

Transcriptome-wide analysis of alternative routes for RNA substrates into the exosome complex

<div><p>The RNA exosome complex functions in both the accurate processing and rapid degradation of many classes of RNA. Functional and structural analyses indicate that RNA can either be threaded through the central channel of the exosome or more directly access the active sites of the ribonucleases Rrp44 and Rrp6, but it was unclear how many substrates follow each pathway <i>in vivo</i>. We used CRAC (UV crosslinking and analysis of cDNA) in growing cells to identify transcriptome-wide interactions of RNAs with the major nuclear exosome-cofactor Mtr4 and with individual exosome subunits (Rrp6, Csl4, Rrp41 and Rrp44) along the threaded RNA path. We compared exosome complexes lacking Rrp44 exonuclease activity, carrying a mutation in the Rrp44 S1 RNA-binding domain predicted to disfavor direct access, or with multiple mutations in Rrp41 reported to impede RNA access to the central channel <i>in vitro</i>. Preferential use of channel-threading was seen for mRNAs, 5S rRNA, scR1 (SRP) and aborted tRNAs transcripts. Conversely, pre-tRNAs preferentially accessed Rrp44 directly. Both routes participated in degradation and maturation of RNAPI transcripts, with hand-over during processing. Rrp41 mutations blocked substrate passage through the channel to Rrp44 only for cytoplasmic mRNAs, supporting the predicted widening of the lumen in the Rrp6-associated, nuclear complex. Many exosome substrates exhibited clear preferences for a specific path to Rrp44. Other targets showed redundancy, possibly allowing the efficient handling of highly diverse RNA-protein complexes and RNA structures. Both threading and direct access routes involve the RNA helicase Mtr4. mRNAs that are predominately nuclear or cytoplasmic exosome substrates can be distinguished <i>in vivo</i>.</p></div

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

Recent Advances in Peptidoglycan Synthesis and Regulation in Bacteria

International audienceBacteria must synthesize their cell wall and membrane during their cell cycle, with peptidoglycan being the primary component of the cell wall in most bacteria. Peptidoglycan is a three-dimensional polymer that enables bacteria to resist cytoplasmic osmotic pressure, maintain their cell shape and protect themselves from environmental threats. Numerous antibiotics that are currently used target enzymes involved in the synthesis of the cell wall, particularly peptidoglycan synthases. In this review, we highlight recent progress in our understanding of peptidoglycan synthesis, remodeling, repair, and regulation in two model bacteria: the Gram-negative Escherichia coli and the Gram-positive Bacillus subtilis. By summarizing the latest findings in this field, we hope to provide a comprehensive overview of peptidoglycan biology, which is critical for our understanding of bacterial adaptation and antibiotic resistance