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

Cytoplasmic processing of mRNAs is affected by the Rrp41-channel mutation.

<p>(A) Distributions of RNA classes in mapped reads recovered with Rrp44 in strains expressing wildtype Rrp41 (left columns) or the Rrp41-channel mutant that is predicted to partially occlude the central channel of the exosome (-channel, right columns). Two biological repeats are shown for each strain. (B-C) Metagene analysis of binding across mRNA genes for Rrp44 in strains expressing wild type Rrp41 (blue) or the Rrp41-channel mutant (green) aligned to the transcription start site (TSS), for all reads (B) or only reads that include non-encoded 3’ oligo(A) tails (C), normalized per millions mapped reads. Data from two biological repeats were averaged for each analysis. (D-F) RPKMs for each RNA species were averaged between two replicates of either Rrp44 with wild type Rrp41 or the Rrp41-channel mutant construct and displayed on a 2D scatter plot for top 200 mRNAs (D), top 200 CUTs (E) or top 200 SUTs (F). Species above the diagonal line are predicted to be strongly subject to nuclear degradation. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006699#pgen.1006699.s010" target="_blank">S5</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006699#pgen.1006699.s011" target="_blank">S6</a> Tables. (G) Mtr4 binding (RPKM) across mRNAs in function of ratio of Rrp44 binding between strains expressing wild type Rrp41 and the Rrp41-channel mutant. Mtr4 preferentially binds mRNAs not affected by channel mutation, consistent with nuclear degradation. (H-I) Distribution of reads recovered with Mtr4 or Rrp44 (with wild type Rrp41 or the Rrp41-channel mutant) across the <i>TDH3</i> gene (H), targeted less in Rrp41-channel strains, and <i>RPS14B</i>, which is not sensitive to channel mutation, normalized to millions of mapped reads. Scale is linear.</p

Exosome structure model and interactions.

<p>(A) Overview of the structures of the TRAMP nuclear cofactor complex and the exosome. The main components of the exosome are schematically represented: the cap in red, the PH-ring in green, forming a contiguous channel (in lighter color) through which the RNA can be threaded. Active sites are indicated in Rrp6 (orange; exonuclease) and Rrp44 (dark blue; endonuclease (endo) and 3’ ->5’ exonuclease (exo)). The Rrp44 S1 RNA binding domain is represented in yellow and the channel to access the Rrp44 exonuclease site in light blue. Two conformations are illustrated: “channel-threading" of the substrate in which the exosome barrel channel is connected to the Rrp44 channel (left panel). A structural rearrangement can disconnect both channels to allow “direct-access” of substrates to the Rrp44 exonuclase site (right panel). Proteins analyzed by CRAC are in bold color. (B) Domain structure of the Rrp44-HTP fusion. From N-terminus to C-terminus, the following domains are indicated: PIN (PilT N terminus) domain harboring endonuclease activity, CSD (Cold-Shock Domain) RNA binding domain, RNB (RNase II ribonuclease) domain harboring exonuclease activity, S1 RNA binding domain and the HTP-tag (His6, TEV protease cleavage site, protein A). (C) Distribution of reads mapped to different RNA substrate classes recovered in CRAC datasets. Two biological replicates are shown for each protein.</p

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

RNAPII transcripts show differences in threading through the channel and direct access to Rrp44.

<p>(A) Clustering based on reads per kilobase per million total mapped reads (RPKM) for each transcript for top 1000 mRNAs, top 200 SUTs, top 200 CUTs, 75 snoRNAs and 4 snRNAs. Hits were averaged between two replicates of either Rrp44-exo (column 2) or Rrp44-exo-S1 (column 3) constructs and arranged by k-medians clustering (k = 4, column 1). Location of mRNAs (grey), snRNAs (green), snoRNA (dark red), CUTs (blue) and SUTs (orange) were indicated in column 4. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006699#pgen.1006699.s009" target="_blank">S4 Table</a>. (B) Association of all RNAs from each cluster with Mtr4, Rrp6, Csl4, Rrp41, Rrp44-exo and Rrp44-exo-S1 in total RPKM. Averages between two independent experiments are shown with standard deviation, except for Rrp41 where fewer reads were recovered and only the largest dataset is shown.</p

Targeting of the pre-rRNA 5’ external transcribed spacer 5’ ETS (RNAPI transcript) involves both channel threading and channel-independent pathways to access Rrp44.

<p>(A) Northern analysis of RNAs coprecipitated with immunoaffinity purified (IP) active Rrp44-HTP (WT), Rrp44-exo-HTP (exo) or Rrp44-exo-S1-HTP (exo-S1), along with 2% input RNA. RNA species are detected with a probe hybridizing near the TSS of the 5’ ETS (+49–67, see panel B for location of the probe). Sybr safe staining for 5S rRNA is shown as loading control. (B) Distribution of reads across the 5’ ETS, recovered with Mtr4, Rrp6, Csl4, Rrp41 in an Rrp44-exo background, and Rrp44-exo and Rrp44-exo-S1, normalized to millions of mapped reads. Scale is linear. A diagram of the 5’ ETS and the 18S rRNA is also shown. (C) Model for 5’ ETS degradation. Following cotranscriptional cleavage of the pre-rRNA, the 5’ ETS is oligo-adenylated by TRAMP and targeted to Rrp44 through the channel. The 5’ ETS is subsequently released from the channel (possibly aided by Mtr4 activity) and subjected to new oligo-adenylation by TRAMP, before being targeted to Rrp44 through direct access.</p

RNAPIII transcripts show differences in threading through the channel.

<p>(A) RPMs (reads per million mapped reads) for each RNA species were averaged between two replicates of either Rrp44-exo (column 2) or Rrp44-exo-S1 (column 3) constructs and arranged by k-medians clustering (k = 4, column 1). Distributions of pre-tRNAs (blue), tRNAs (purple), 5S rRNA (yellow) and other non-coding RNAPIII transcripts (green) are indicated in column 4. Intron-containing pre-tRNAs are indicated in green in column 5. Transcripts discussed in the text are indicated in column 6. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006699#pgen.1006699.s008" target="_blank">S3 Table</a>. (B) Relative protein association of all RNAPIII transcripts from each cluster was calculated for Mtr4, Rrp6, Csl4, Rrp41, Rrp44-exo and Rrp44-exo-S1 in total RPM. Averages between two independent experiments are shown with standard deviation, except for Rrp41 where fewer reads were recovered and only the largest dataset is shown. (C) 2D scatter-plot comparing RPM across pre-tRNAs and tRNAs recovered with Rrp44-exo and Rrp44-exo-S1. (D-E) Northern analysis of RNAs coprecipitated with Rrp44-HTP (WT), Rrp44-exo-HTP (exo) or Rrp44-exo-S1-HTP (exo-S1), along with 2% (D) or 1% (E) input RNA, probed for RNAPIII transcripts: U6 snRNA, scR1, 5S rRNA, RPR1 (D) or tRNAPro(UGG) (E). Sybr safe staining for 5S rRNA is shown as loading control. Asterisks indicate previously reported truncation products that are known exosome substrates.</p