Caractérisation de la RNase E et du dégradosome d'ARN de la bactérie marine adaptée au froid Pseudoalteromonas haloplanktis

La dégradation des ARNm est l'un des processus biologiques essentiels qui implique chez les bactéries, l'intervention d'un complexe multiprotéique appelé « dégradosome d'ARN ». Chez E.coli, la formation de ce complexe nécessite l'endoribonucléase essentielle « RNase E », qui porte dans sa région non conservée, des îlots de 15 à 40 acides aminés appelés « microdomaines ». Ces microdomaines sont responsables des interactions entre la RNase E et trois protéines : l'hélicase à ARN RhlB, l'enzyme de la glycolyse énolase et l'exoribonucléase PNPase. Chez d'autres bactéries, plusieurs dégradosomes d’ARN assemblés sur la RNase E ont été identifiés et ils présentent une composition protéique différente du dégradosome d'E.coli. Il y a ainsi une plasticité des interactions RNase E-protéines qui serait due à la divergence de la séquence des microdomaines et leur évolution. Cependant, au début de ce travail de thèse, seuls les microdomaines de la RNase E d'E.coli ont été caractérisés. Les travaux de thèse présentés dans ce manuscrit se sont focalisés sur la bactérie marine adaptée au froid et relativement distante d'E.coli, Pseudoalteromonas haloplanktis, pour caractériser le dégradosome d'ARN et les microdomaines responsables des interactions RNase E-protéines. Le dégradosome d'ARN adapté au froid de P.haloplanktis est composé de la RNase E, de l'exoribonucléase PNPase et de l'hélicase à ARN RhlB. Chez E.coli, la RNase E de P.haloplanktis est capable de restaurer la viabilité d'E.coli dépourvue de RNase E sans conférer un avantage de croissance à E.coli à basse température. De plus, la RNase E de P.haloplanktis est capable d'interagir avec la protéine RhlB d'E.coli. Les microdomaines d'interaction entre la RNase E et les protéines RhlB et PNPase ont été caractérisés ce qui a permis de mieux comprendre la conservation de ces deux types d'interaction retrouvés au sein du dégradosome d'ARN d'E.coli, malgré la distance évolutive séparant P.haloplanktis et E.coli. L'interaction RNase E-PNPase de P.haloplanktis implique un microdomaine de séquence non conservée en comparaison avec celui de l'interaction RNase E-PNPase d'E.coli. Ceci suggère une conservation structurale de l'interaction RNase E-PNPase. Quant à l'interaction RNase E-RhlB, le microdomaine d'interaction présente des acides aminés conservés et similaires au microdomaine d'interaction RNase E-RhlB d'E.coli. L'analyse de ces microdomaines est élargie de manière in silico à d'autres RNase E des gamma-protéobactéries pour avoir une vue d'ensemble de leur distribution. Enfin, une étude phylogénétique de la protéine RhlB a été initiée dans le but de voir une corrélation potentielle entre la présence du microdomaine de fixation de RhlB et la présence de RhlB chez les gamma-protéobactéries.RNA degradation is an essential biological process which implies in bacteria a multienzyme complex called RNA « degradosome ». In E. coli, this complex contains the RNase E, the DEAD-box RNA helicase RhlB, the glycolytic enzyme enolase and the exoribonuclease PNPase. The C-terminal natively unstructured region of the RNase E serves as the scaffold for binding other components of the RNA degradosome. In this region, microdomains from 15 to 40 amino acids are necessary for protein-protein interactions with other components of the RNA degradosome. In other bacteria, several RNase E-based complexes have been identified but the composition of the associated proteins is variable. Thus, there is a plasticity of the RNase E-protein interactions that would be due to the sequence divergence of the microdomains and their evolution. However, at the beginning of this work, only the microdomains of the RNase E of E. coli have been characterized. In this work, the RNA degradosome of the marine bacterium Pseudoalteromonas haloplanktis distantly related to E. coli, has been characterized. The RNase E of P. haloplanktis associates with RhlB and PNPase but not enolase. In E. coli, the RNase E of P. haloplanktis can restore the viability of E. coli lacking RNase E without conferring a growth advantage to E. coli at low temperature. In addition, the RNase E of P. haloplanktis can make a heterologous interaction only with RhlB of E. coli. Microdomains corresponding to the RNase E binding sites for RhlB and PNPase were mapped. The sequence of the RhlB binding site of the RNase E of P. haloplanktis is related to the sequence of the RhlB binding site of the RNase E of E. coli. For the PNPase binding site of the RNase E of P. haloplanktis, the sequence is not conserved compared to the PNPase binding site of the RNase E of E. coli. The characterization of these two microdomains in the RNase E of P. haloplankits shows the conservation of these interactions found in the RNA degradosome of E. coli over a large evolutionary distance separating P. haloplanktis and E. coli. This suggests that the structural motif of these interactions is conserved. The analysis of these microdomains is expanded in silico to others RNase E of the gamma-proteobacteria, to have an overview of their distribution. A phylogenetic study of the protein RhlB was initiated to see a potential correlation between the presence of microdomain binding site of RhlB and the presence of RhlB in gamma-proteobacteria

Hematopoietic progenitors express embryonic stem cell and germ layer genes

Cell therapy for tissue regeneration requires cells with high self-renewal potential and with the capacity to differentiate into multiple differentiated cell lineages, like embryonic stem cells (ESCs) and adult somatic cells induced to pluripotency (iPSCs) by genetic manipulation. Here we report that normal adult mammalian bone marrow contains cells, expressing the cell surface antigen CD34, that naturally express the genes that are characteristic of ESCs and that are required to generate iPSCs. In addition, these CD34+ cells spontaneously express, without genetic manipulation, genes characteristic of the three embryonic germ layers ectoderm, mesoderm and endoderm. In addition to the neural lineage genes we previously reported in these CD34+ cells, we found that they express genes of the mesodermal cardiac muscle lineage and of the endodermal pancreatic lineage as well as intestinal lineage genes. Thus, these normal cells in the adult spontaneously exhibit the characteristics of embryonic-like stem cells

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Competitive folding of RNA structures at a termination-antitermination site

Antitermination is a regulatory process based on the competitive folding of terminator antiterminator structures that can form in the leader region of nascent transcripts. In the case of the Bacillus subtilis licS gene involved in D-glucosides utilization, the binding of the antitermination protein Lid to a short RNA hairpin (RAT) prevents the formation of an overlapping terminator and thereby allows transcription to proceed. Here, we monitored in vitro the competition between termination and antitermination by combining bulk and single-molecule fluorescence-based assays using labeled RNA oligonucleotide constructs of increasing length that mimic the progressive transcription of the terminator invading the antiterminator hairpin. Although high affinity binding is abolished as soon as the antiterminator basal stem is disrupted by the invading terminator, Lid-can still bind and promote closing of the partially unfolded RAT hairpin. However, binding no longer occurs once the antiterminator structure has been disrupted by the full-length terminator. Based on these findings, we propose a kinetic competition model for the sequential events taking place at the termination antitermination site, where LicT needs to capture its RAT target before completion of the terminator to remain tightly bound during RNAP pausing, before finally dissociating irreversibly from the elongated licS transcript

Intestinal inhibition of <em>Atg7</em> prevents tumour initiation through a microbiome-influenced immune response and suppresses tumour growth

Here, we show that autophagy is activated in the intestinal epithelium in murine and human colorectal cancer and that the conditional inactivation of Atg7 in intestinal epithelial cells inhibits the formation of pre-cancerous lesions in Apc(+/-) mice by enhancing anti-tumour responses. The antibody-mediated depletion of CD8(+) T cells showed that these cells are essential for the anti-tumoral responses mediated by the inhibition of autophagy. We show that Atg7 deficiency leads to intestinal dysbiosis and that the microbiota is required for anticancer responses. In addition, Atg7 deficiency resulted in a stress response accompanied by metabolic defects, AMPK activation and p53-mediated cell-cycle arrest in tumour cells but not in normal tissue. This study reveals that the inhibition of autophagy within the epithelium may prevent the development and progression of colorectal cancer in genetically predisposed patients