Pseudomonas aeruginosa cleaves the decoding center of Caenorhabditis elegans ribosomes

Pathogens such as Pseudomonas aeruginosa advantageously modify animal host physiology, for example, by inhibiting host protein synthesis. Translational inhibition of insects and mammalian hosts by P. aeruginosa utilizes the well-known exotoxin A effector. However, for the infection of Caenorhabditis elegans by P. aeruginosa, the precise pathways and mechanism(s) of translational inhibition are not well understood. We found that upon exposure to P. aeruginosa PA14, C. elegans undergoes a rapid loss of intact ribosomes accompanied by the accumulation of ribosomes cleaved at helix 69 (H69) of the 26S ribosomal RNA (rRNA), a key part of ribosome decoding center. H69 cleavage is elicited by certain virulent P. aeruginosa isolates in a quorum sensing (QS)-dependent manner and independently of exotoxin A-mediated translational repression. H69 cleavage is antagonized by the 3 major host defense pathways defined by the pmk-1, fshr-1, and zip-2 genes. The level of H69 cleavage increases with the bacterial exposure time, and it is predominantly localized in the worm\u27s intestinal tissue. Genetic and genomic analysis suggests that H69 cleavage leads to the activation of the worm\u27s zip-2-mediated defense response pathway, consistent with translational inhibition. Taken together, our observations suggest that P. aeruginosa deploys a virulence mechanism to induce ribosome degradation and H69 cleavage of host ribosomes. In this manner, P. aeruginosa would impair host translation and block antibacterial responses

An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments

Deep sequencing of strand-specific cDNA libraries is now a ubiquitous tool for identifying and quantifying RNAs in diverse sample types. The accuracy of conclusions drawn from these analyses depends on precise and quantitative conversion of the RNA sample into a DNA library suitable for sequencing. Here, we describe an optimized method of preparing strand-specific RNA deep sequencing libraries from small RNAs and variably sized RNA fragments obtained from ribonucleoprotein particle footprinting experiments or fragmentation of long RNAs. Our approach works across a wide range of input amounts (400 pg to 200 ng), is easy to follow and produces a library in 2-3 days at relatively low reagent cost, all while giving the user complete control over every step. Because all enzymatic reactions were optimized and driven to apparent completion, sequence diversity and species abundance in the input sample are well preserved

Lentiviral RNAs can use different mechanisms for translation initiation

Abstract The full-length genomic RNA of lentiviruses can be translated to produce proteins and incorporated as genomic RNA in the viral particle. Interestingly, both functions are driven by the genomic 5 -UTR (5 -untranslated region), which harbours structural RNA motifs for the replication cycle of the virus. Recent work has shown that this RNA architecture also functions as an IRES (internal ribosome entry site) in HIV-1 and -2, and in SIV (simian immunodeficiency virus). In addition, the IRES extends to the gag coding region for all these viruses and this leads to the synthesis of shorter isoforms of the Gag polyprotein from downstream initiation codons. In the present study, we have investigated how different members of the lentivirus family (namely HIV-1 and -2, and SIV) can initiate protein synthesis by distinct mechanisms. For this, we have used the competitive reticulocyte lysate that we have recently described. Our results show that HIV-1 is able to drive the synthesis of the Gag polyprotein both by a classical cap-dependent mechanism and an IRES, whereas HIV-2 and SIV appear to use exclusively an IRES mechanism

Translation-dependent and independent mRNA decay occur through mutually exclusive pathways that are defined by ribosome density during T Cell activation [preprint]

mRNA translation and degradation are strongly interconnected processes that participate in the fine tuning of gene expression. Particularly, targeting mRNAs to translation-dependent degradation (TDD) could attenuate protein expression by making any increase in mRNA translation self-limiting. However, the extent to which TDD is a general mechanism for limiting protein expression is currently unknown. Here we describe a comprehensive analysis of basal and signal-induced TDD in mouse primary CD4 T cells. Our data indicate that most cellular transcripts are decayed to some extent in a translation-dependent manner, both in resting and activated cells. Our analysis further identifies the length of untranslated regions, the density of ribosomes and the GC content of the coding region as major determinants of TDD magnitude. Consistent with this, all transcripts that undergo changes in ribosome density upon T cell activation display a corresponding change in their TDD level. Surprisingly, the amplitude of translation-independent mRNA decay (TID) appears as a mirror image of TDD. Moreover, TID also responds to changes in ribosome density upon T cell activation but in the opposite direction from the one observed for TDD. Our data demonstrate a strong interconnection between mRNA translation and decay in mammalian cells. Furthermore, they indicate that ribosome density is a major determinant of the pathway by which transcripts are degraded within cells

Site Specific Knock-In Genome Editing in Human HSCs Using Baboon Envelope gp Pseudotypedviral Derived “Nanoblades” Loaded with Cas9/sgRNA Combined with Donor Encoding AAV-6

Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. Here, we have designed ?Nanoblades?, a new technology that will deliver a genomic cleaving agent into cells. These are genetically modified Murine Leukemia Virus (MLV) or HIV derived virus like particle (VLP), in which the viral structural protein Gag has been fused to the Cas9. These VLPs are thus loaded with Cas9 protein together with the guide RNAs. Thus, nanoblades are devoid of any viral-derived genetic material. Highly efficient gene editing was obtained in cell lines, IPS cells and primary mouse and human cells (Mangeot et al. Nature Communication, 2019). However, their delivery into target cells can be technically challenging when working with primary immune cells. Now we showed that nanoblades were remarkably efficient for entry into human T, B and hematopoietic stem cells thanks to their surface co-pseudotyping with baboon retroviral and VSVG envelope glycoproteins. We were able to induce efficient, transient and very rapidlygenome-editing in human induced pluripotent stem cells reaching up to 70% in the empty spiracles homeobox 1 (EMX1) and muscular dystrophy (MD) gene locus. A brief nanoblade incubation of primary human T and B cells resulted in 40% and 20% editing of the Wiskott-Aldrich syndrome (WAS) gene locus, while hematopoietic stem cells treated for 18 h with nanoblades allowed 30-40% gene editing in the WAS gene locus and up to 80% for the Myd88 genomic target. Moreover, for the HIV- and MLV-derived nanoblades no cell toxicity and low to undetectable off-target effects were demonstrated.Finally, we also treated hHSCs with nanoblades in combination with an AAV-6 donor encoding vector resulting in over 20% of stable expression cassette knock-in into the WAS gene locus. Currently, we are evaluating these gene modified HSCs for their long-term reconstitution of NOD/SCIDgC-/- mice.Summarizing, this new technology is simple to implement in any laboratory, shows high flexibility for different targets including primary immune cells of murine and human origin, is relatively inexpensive and therefore have important prospects for basic and clinical translation in the area of gene therapy.Fil: Gutierrez, Alejandra. Inserm; FranciaFil: Abrey Recalde, Maria Jimena. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; Argentina. Inserm; FranciaFil: Mangeot, Philippe E.. Inserm; FranciaFil: Costa, Caroline. Inserm; FranciaFil: Bernandin, Ornellie. Inserm; FranciaFil: Fusil, Floriane. Inserm; FranciaFil: Froment, Gisèle. Inserm; FranciaFil: Martin, Francisco. Inserm; FranciaFil: Bellabdelah, Karim. Universidad de Granada; EspañaFil: Ricci, Emiliano P.. Inserm; FranciaFil: Ayuso, Eduard. Universite de Nantes; FranciaFil: Cosset, François loic. Inserm; FranciaFil: Verhoeyen, Els. Inserm; FranciaAmerican Society of Cell and Gene Therapy 22nd Annual MettingWashingtonEstados UnidosAmerican Society of Cell and Gene Therap

System-wide profiling of RNA-binding proteins uncovers key regulators of virus infection

International audienceGraphical Abstract Highlights d A quarter of the RBPome changes upon SINV infection d Alterations in RBP activity are largely explained by changes in RNA availability d Altered RBPs are crucial for viral infection efficacy d GEMIN5 binds to the 5 0 end of SINV RNAs and regulates viral gene expressio

Le profilage ribosomique

L’explosion du nombre de techniques basées sur le séquençage massif parallèle est actuellement en train de révolutionner l’étude des systèmes biologiques en permettant à l’expérimentateur d’avoir une vision globale des processus se déroulant à l’échelle moléculaire. Parmi ces nouvelles approches, le profilage ribosomique est un outil particulièrement puissant pour l’étude de la traduction à un niveau de détail jamais égalé auparavant. Cette technique permet notamment de cartographier très précisément la position des ribosomes sur l’ensemble des ARN messagers en cours de traduction dans la cellule à un moment donné. Dans le cas d’une infection virale, il est ainsi possible d’étudier les mécanismes souvent très complexes et encore mal compris qui sont mis en place par les virus pour assurer la production des protéines nécessaires à leur multiplication. Cette synthèse a pour but de discuter la manière dont le profilage ribosomique peut nous permettre de mieux comprendre le cycle de réplication virale, mais aussi de montrer les biais liés à la technique à prendre en compte lors de l’analyse des résultats