Étude du facteur d'épissage CWC27 et de son lien avec les maladies génétiques et l'assemblage de l'EJC

In metazoans, most coding genes contain introns that are co-transcriptionally removed from pre-mRNAs by the spliceosome. In addition to excise introns, the splicing step dresses nascent mRNAs with RNA Binding Proteins (RBPs) that modulate the following steps of mRNA lifecycle. A major constituent of messenger ribonucleoprotein (mRNP) particles is the Exon Junction Complex (EJC), which is deposited onto mRNA during splicing. The EJC core is structurally organized around the RNA helicase eIF4A3 and serves as a binding platform for multiple peripheral factors. EIF4A3 is specifically recruited to the spliceosome by the splicing factor CWC22 before intron excision. EJCs plays a central role in mRNA destiny by contributing to the regulation of alternative splicing as well as mRNA localization, translation and decay. Multiple interrogations subsist regarding the mechanism by which EJCs are assembled by the spliceosome and the link existing between altered expression of EJC proteins and the related developmental defects and human pathological disorders. The aim of my PhD project was to characterize the splicing factor CWC27 whose precise function is unknown. The first part of my research work consisted in participating to the characterization of pathogenic mutations identified in the CWC27 human gene in collaboration with human geneticists. This study revealed that CWC27 truncating mutations are associated with syndromic and non-syndromic Retinis Pigmentosa (RP). I notably showed that the homozygous mutation c.599+1G>A of CWC27 carried by two siblings leads to the expression of two new alternative transcripts containing a premature termination codon (PTC).In a second part, my aim was to biochemically and functionally characterize CWC27. To efficiently purified CWC27, I first edited the genome of human HeLa cells by CRISPR–Cas9 method to fuse an affinity FLAG-tag to endogenous CWC27 proteins. Precipitation of FLAG-CWC27 coupled to quantitative label-free mass spectrometry identified both CWC22 and eIF4A3 as major partners of CWC27. By expressing full-length and truncated versions of CWC22 and CWC27, we showed that the C-terminal domain of CWC27 mediates the interaction with both CWC22 and eIF4A3. Interestingly, CWC27 truncations recapitulating mutations found in patients lead to the loss of interactions with both CWC22 and eIF4A3. Using recombinant human proteins, we reconstituted in vitro the CWC27/CWC22/eIF4A3 trimeric complex and showed that CWC27 stabilizes the binding of eIF4A3 to CWC22. In collaboration with structural biologists, we solved the 3D structure of the CWC27/CWC22/eIF4A3 core complex showing how CWC27 contacts CWC22 at the atomic level and illuminating the early contacts of eIF4A3 with splicing factors. Finally, we used mRNA sequencing to question the impact of CWC27 knock-down (KD) on human cell transcriptome in an immortalized retinal pigment epithelial cell line. CWC27 KD leads to important gene expression changes, including splicing defects, and many are common to changes associated to CWC22 KD. Strikingly, numerous genes encoding factors involved in inflammation are strongly up-regulated under CWC27 or CWC22 down regulation, which could explain the Retinis Pigmentosa phenotype. Our results reveal that CWC27 is involved in the early stages of EJC assembly and establish an unexpected link between core splicing factors mutations with inflammation activation in retinal epithelium. This study paves the way for understanding the molecular causes of retinal degeneration associated to human spliceosomopathies.Chez les métazoaires, la majorité des gènes codant des protéines contiennent des introns qui sont excisés par la machinerie d’épissage ou « spliceosome » pendant leur transcription. En plus d’enlever les introns, l'étape d'épissage habille les ARNm naissants avec des protéines de liaison à l'ARN (RBPs) qui modulent les étapes suivantes du cycle de vie des ARNm. Le complexe EJC (Exon Junction Complex) déposé par le spliceosome sur les ARNm est un composant majeur de l’ensemble des protéines couvrant les ARNm (particules mRNP). L'EJC est structurellement organisé autour de l'ARN hélicase eIF4A3 qui sert de plate-forme de liaison pour de multiples facteurs annexes. EIF4A3 est spécifiquement recrutée par la machinerie d’épissage par le facteur d’épissage CWC22 avant l'excision des introns. L’EJC joue un rôle central dans le devenir des ARNm en contribuant à la régulation de l’épissage alternatif ainsi que de la localization, la traduction et la dégradation des ARNm. De multiples interrogations subsistent quant au mécanisme par lequel les EJCs sont assemblés par le spliceosome ainsi que le lien existant entre l'expression altérée de protéines de l’EJC et des défauts de développement ou des pathologies humaines. L’objectif de mon doctorat est de caractériser le facteur d'épissage CWC27 dont la fonction précise est inconnue. La première partie de mon travail de recherche consiste en la caractérisation de mutations pathogènes identifiées dans le gène humain CWC27 en collaboration avec des généticiens humains. Cette étude a révélé que des mutations tronquantes de CWC27 sont associées à un phénotype de Rétinite pigmentaire (RP) avec parfois des défauts de développement. J'ai notamment montré que la mutation homozygote c.599+1G>A de CWC27 conduit à l'expression de deux nouveaux transcrits alternatifs contenant un codon de terminaison précoce (PTC). Dans un deuxième temps, j’ai caractérisé biochimiquement et fonctionnellement la protéine CWC27. Afin de purifier efficacement CWC27, j’ai fusionné la protéine endogène avec une étiquette d'affinité FLAG introduite dans le génome des cellules HeLa par la méthode CRISPR-Cas9. La précipitation de FLAG-CWC27 couplée à la spectrométrie de masse quantitative a identifié CWC22 et eIF4A3 comme des partenaires majeurs de CWC27. En exprimant des versions sauvages ou tronquées de CWC22 et CWC27, nous montrons que le domaine C-terminal de CWC27 est médiateur de l'interaction avec CWC22 et eIF4A3. Les mutations correspondant à celles observées chez les patients entraînent la perte des interactions avec CWC22 et eIF4A3. Puis, en utilisant des protéines recombinantes, nous avons reconstitué in vitro le complexe trimérique CWC27/CWC22/eIF4A3 et montré que CWC27 stabilise la liaison de eIF4A3 à CWC22. En collaboration avec des biologistes structuraux, nous avons résolu la structure 3D du complexe CWC27/CWC22/eIF4A3. Cette structure éclaire les premiers contacts de eIF4A3 avec les facteurs d’épissage. Puis, nous avons étudié par RNA-seq, l'impact de la perte de CWC27 sur le transcriptome de cellules épithéliales pigmentaires rétiniennes humaines immortalisées. Un ARN interférent anti-CWC27 entraîne d'importants changements dans l'expression des gènes, y compris des défauts d'épissage, et beaucoup sont communs aux à ceux dus à la perte de CWC22. De nombreux gènes codant pour des facteurs impliqués dans l'inflammation sont fortement affectés par la perte de CWC27 ou CWC22, ce qui pourrait rendre compte du phénotype Rétinite pigmentaire. Nos résultats révèlent que CWC27 est impliquée dans les premières étapes de l'assemblage de l'EJC. Ils établissent un lien inattendu entre des mutations des facteurs d'épissage centraux et l'activation de l'inflammation dans l'épithélium rétinien. Cette étude ouvre la voie à la compréhension des causes moléculaires de la dégénérescence rétinienne associée aux spliceosomopathies humaines

Structural and functional insights into CWC27/CWC22 heterodimer linking the exon junction complex to spliceosomes

International audienceHuman CWC27 is an uncharacterized splicing factor and mutations in its gene are linked to retinal degeneration and other developmental defects. We identify the splicing factor CWC22 as the major CWC27 partner. Both CWC27 and CWC22 are present in published Bact spliceosome structures, but no interacting domains are visible. Here, the structure of a CWC27/CWC22 heterodimer bound to the exon junction complex (EJC) core component eIF4A3 is solved at 3Å-resolution. According to spliceosomal structures, the EJC is recruited in the C complex, once CWC27 has left. Our 3D structure of the eIF4A3/CWC22/CWC27 complex is compatible with the Bact spliceosome structure but not with that of the C complex, where a CWC27 loop would clash with the EJC core subunit Y14. A CWC27/CWC22 building block might thus form an intermediate landing platform for eIF4A3 onto the Bact complex prior to its conversion into C complex. Knock-down of either CWC27 or CWC22 in immortalized retinal pigment epithelial cells affects numerous common genes, indicating that these proteins cooperate, targeting the same pathways. As the most up-regulated genes encode factors involved in inflammation, our findings suggest a possible link to the retinal degeneration associated with CWC27 deficiencies

Efficacy of Different Bacillus of Calmette-Guérin (BCG) Strains on Recurrence Rates among Intermediate/High-Risk Non-Muscle Invasive Bladder Cancers (NMIBCs): Single-Arm Study Systematic Review, Cumulative and Network Meta-Analysis

Background: In an era of Bacillus of Calmette-Guérin (BCG) shortages, the comparative efficacy from different adjuvant intravesical BCG strains in non-muscle invasive bladder cancer (NMIBC) has not been clearly elucidated. We aim to compare, through a systematic review and meta-analysis, the cumulative BC recurrence rates and the best efficacy profile of worldwide available BCG strains over the last forty years. Methods: PubMed, Scopus, Web of Science, Embase, and Cochrane databases were searched from 1982 up to 2022. A meta-analysis of pooled BC recurrence rates was stratified for studies with ≤3-y vs. >3-y recurrence-free survival (RFS) endpoints and the strain of BCG. Sensitivity analysis, sub-group analysis, and meta-regression were implemented to investigate the contribution of moderators to heterogeneity. A random-effect network meta-analysis was performed to compare BCG strains on a multi-treatment level. Results: In total, n = 62 series with n = 15,412 patients in n = 100 study arms and n = 10 different BCG strains were reviewed. BCG Tokyo 172 exhibited the lowest pooled BC recurrence rate among studies with ≤3-y RFS (0.22 (95%CI 0.16–0.28). No clinically relevant difference was noted among strains at >3-y RFS outcomes. Sub-group and meta-regression analyses highlighted the influence of NMIBC risk-group classification and previous intravesical treated categories. Out of the n = 11 studies with n = 7 BCG strains included in the network, BCG RIVM, Tice, and Tokyo 172 presented with the best-predicted probability for efficacy, yet no single strain was significantly superior to another in preventing BC recurrence risk. Conclusion: We did not identify a BCG stain providing a clinically significant lower BC recurrence rate. While these findings might discourage investment in future head-to-head randomized comparison, we were, however, able to highlight some potential enhanced benefits from the genetically different BCG RIVM, Tice, and Tokyo 172. This evidence would support the use of such strains for future BCG trials in NMIBCs

On the Redox-Activity and Health-Effects of Atmospheric Primary and Secondary Aerosol: Phenomenology

The RHAPS (Redox-Activity And Health-Effects Of Atmospheric Primary And Secondary Aerosol) project was launched in 2019 with the major objective of identifying specific properties of the fine atmospheric aerosol from combustion sources that are responsible for toxicological effects and can be used as new metrics for health-related outdoor pollution studies. In this paper, we present the overall methodology of RHAPS and introduce the phenomenology and the first data observed. A comprehensive physico-chemical aerosol characterization has been achieved by means of high-time resolution measurements (e.g., number size distributions, refractory chemical components, elemental composition) and low-time resolution analyses (e.g., oxidative potential, toxicological assays, chemical composition). Preliminary results indicate that, at the real atmospheric conditions observed (i.e., daily PM1 from less than 4 to more than 50 ug m-3), high/low mass concentrations of PM1, as well as black carbon (BC) and water soluble Oxidative Potential (WSOP,) do not necessarily translate into high/low toxicity. Notably, these findings were observed during a variety of atmospheric conditions and aerosol properties and with different toxicological assessments. Findings suggest a higher complexity in the relations observed between atmospheric aerosol and toxicological endpoints that go beyond the currently used PM1 metrics. Finally, we provide an outlook to companion papers where data will be analyzed in more detail, with the focus on source apportionment of PM1 and the role of source emissions on aerosol toxicity, the OP as a predictive variable for PM1 toxicity, and the related role of SOA possessing redox-active capacity, exposure-response relationships for PM1, and air quality models to forecast PM1 toxicity

Urology in the time of coronavirus: reduced access to urgent and emergent urological care during the coronavirus disease 2019 outbreak in Italy

The coronavirus disease 2019 (COVID-19) pandemic has put a substantial burden on the Italian healthcare system, resulting in the restructuring of hospitals to care for COVID-19 patients. However, this has likely impacted access to care for patients experiencing other conditions. We aimed to quantify the impact of COVID-19 on access to care for patients with urgent/emergent urological conditions throughout Italy