Deciphering the modulation of cellular alternative splicing during reovirus infection

L’épissage alternatif (ÉA) est un processus de maturation des ARNm permettant de diversifier le potentiel codant provenant d’un même gène. L’ÉA modifie la séquence codante des ARNm en retirant des exons ou des parties d’exons, ou même en conservant des introns au sein de l’ARNm mature. L’ÉA est crucial pour la cellule, car il permet la production d’isoformes protéiques ayant des localisations cellulaires, des stabilités ou des régulations différentes. Il a été récemment démontré que plusieurs virus peuvent modifier l’ÉA des transcrits cellulaires. Cependant, notre compréhension du rôle de ce phénomène dans les interactions virus-hôte est encore limitée. Nous avons investigué si réovirus, un virus à ARN double-brin prometteur comme traitement contre le cancer, module l’ÉA lors de l’infection, le mécanisme de cette modulation ainsi que l’impact possible dans les interactions virus-hôtes. Nous avons identifié 240 évènements d’ÉA altérés durant l’infection avec réovirus, qui se situent majoritairement dans des transcrits codant pour des protéines impliquées dans la régulation de l’expression génique ainsi que le métabolisme des ARN. Nous avons aussi identifié la protéine virale μ2 comme étant le principal déterminant des changements d’ÉA causé par réovirus, et démontré que la protéine μ2 par elle-même peut influencer l’ÉA. De plus, nous avons établi que des composantes du splicéosome, et plus particulièrement EFTUD2, PRPF8 et SNRNP200 du complexe U5, interagissent avec μ2 et sont requises pour que réovirus module l’ÉA cellulaire. Finalement, les niveaux de ces protéines sont réduits durant l’infection par la protéine μ2. Nous avons par la suite déterminé les rôles respectifs d’EFTUD2, de PRPF8 et de SNRNP200 dans la réplication de réovirus et découvert des rôles distincts et qui se recoupent partiellement sur la survie cellulaire, l’apoptose, la nécroptose, l’induction de la réponse interféron, et la réplication du virus. Ces résultats démontrent l’importance de l’ÉA dans les interactions virus-hôte ainsi que l’implication des protéines du complexe U5 dans la réplication de réovirus.Abstract : Alternative splicing (AS) is an RNA maturation process allowing enhanced diversity in mature messenger RNA originating from the same gene. AS modifies the mRNA’s coding potential by removing exons or parts of exon, or even retaining introns in the mature RNA. AS is crucial in cells as it enables the production of proteins isoforms with different cellular localization, stability, and regulation. Recent evidence suggests that viruses alter cellular AS during infection, although our understanding of the role of these changes remains limited. We investigated if mammalian orthoreovirus (MRV), a double-stranded RNA virus from the Reoviridae family presenting a promising activity as an oncolytic virus, alters the host cell AS during infection, the mechanism of this modulation, and the possible impacts of these virus-host interactions on MRV replication. We identified 240 altered AS events upon MRV infection which belong to transcripts involved in the regulation of gene expression and RNA metabolism. We identified the viral protein μ2 as the main determinant of MRV’s alterations in cellular AS, and we showed that μ2, by itself, could impact cellular AS. Moreover, we discovered that central components of the U5 snRNP of the spliceosome EFTUD2, PRPF8, and SNRNP200 interacts with μ2, and are required for MRV modulation of AS. Finally, these U5 snRNP components are reduced at the protein level during MRV infection by the μ2 protein. We then deciphered the respective impact of EFTUD2, PRPF8, and SNRNP200 on MRV replication. We discovered distinct and partially overlapping novel roles for EFTUD2, PRPF8, and SNRNP200 in cell survival, apoptosis, necroptosis, and the induction of the interferon response pathway during MRV infection. Moreover, we demonstrated that EFTUD2 restricts viral replication of MRV, both in a single cycle and multiple cycles of viral replication. These findings provide additional insights into the importance of AS in the complexity of virus-host interactions and the implications of U5 snRNP proteins in MRV replication

Reovirus μ2 protein modulates host cell alternative splicing by reducing protein levels of U5 snRNP core components

Abstract : Mammalian orthoreovirus (MRV) is a doublestranded RNA virus from the Reoviridae family presenting a promising activity as an oncolytic virus. Recent studies have underlined MRV’s ability to alter cellular alternative splicing (AS) during infection, with a limited understanding of the mechanisms at play. In this study, we investigated how MRV modulates AS. Using a combination of cell biology and reverse genetics experiments, we demonstrated that theM1 gene segment, encoding the μ2 protein, is the primary determinant of MRV’s ability to alter AS, and that the amino acid at position 208 in μ2 is critical to induce these changes. Moreover, we showed that the expression of μ2 by itself is sufficient to trigger AS changes, and its ability to enter the nucleus is not required for all these changes. Moreover, we identified core components of the U5 snRNP (i.e. EFTUD2, PRPF8, and SNRNP200) as interactors of μ2 that are required for MRV modulation of AS. Finally, these U5 snRNP components are reduced at the protein level by both MRV infection and μ2 expression. Our findings identify the reduction of U5 snRNP components levels as a new mechanism by which viruses alter cellular AS

Global profiling of alternative RNA splicing events provides insights into molecular differences between various types of hepatocellular carcinoma

Protein families encoded by transcripts that are differentially spliced in various types of HCC. Table S2. Bioinformatical prediction of functional changes caused by some of ASEs identified. Table S3. List of tumor suppressors for which AS is dysregulated in various types of HCC. Table S4. List of oncogenes for which AS is dysregulated in various types of HCC. Table S5. List of kinases for which AS is dysregulated in various types of HCC. Table S6. List of transcription factors for which AS is dysregulated in various types of HCC. Table S7. List of genes for which AS is dysregulated in all types of HCC. Table S8. List of genes uniquely dysregulated in HBV-associated HCC. Table S9. List of genes uniquely dysregulated in HCV-associated HCC. Table S10. List of genes uniquely dysregulated in HBV&HCV-associated HCC. Table S11. List of genes uniquely dysregulated in virus-free HCC. Figure S1. Characterization of splicing mysregulation in HCC. Figure S2. Characterization of ASEs that are modified in HBV- and HCV-associated HCC. Figure S3. AS modifications in transcripts encoded by kinases and transcriptions factores in HBV- and HCV-associated HCC. Figure S4. Global profiling of ASE modifications in both HBV&HCV-associated HCC and virus-free-associated HCC. Figure S5. RNA splicing factors in HCC. Figure S6. Modifications to AS of 96 transcripts in response to knockdown of splicing factors with specific siRNAs (PDF 6675 kb

Inverse dynamical population synthesis: Constraining the initial conditions of young stellar clusters by studying their binary populations

Binary populations in young star clusters show multiplicity fractions both lower and up to twice as high as those observed in the Galactic field. We follow the evolution of a population of binary stars in dense and loose star clusters starting with an invariant initial binary population and a formal multiplicity fraction of unity, and demonstrate that these models can explain the observed binary properties in Taurus, Rho-Ophiuchus, Chamaeleon, Orion, IC 348, Upper Scorpius A, Praesepe, and the Pleiades. The model needs to consider solely different birth densities for these regions. The evolved theoretical orbital-parameter distributions are highly probable parent distributions for the observed ones. We constrain the birth conditions (stellar mass, M_ecl, and half-mass radius, r_h) for the derived progenitors of the star clusters and the overall present-day binary fractions allowed by the present model. The results compare very well with properties of molecular cloud clumps on the verge of star formation. Combining these with previously and independently obtained constraints on the birth densities of globular clusters, we identify a weak stellar mass -- half-mass radius correlation for cluster-forming cloud clumps, r_h / pc ~ (M_ecl / M_sun)^(0.13+-0.04). The ability of the model to reproduce the binary properties in all the investigated young objects, covering present-day densities from 1-10 stars pc^-3 (Taurus) to 2x10^4 stars pc^-3 (Orion), suggests that environment-dependent dynamical evolution plays an important role in shaping the present-day properties of binary populations in star clusters, and that the initial binary properties may not vary dramatically between different environments.Comment: accepted for publication in A&A, 14 pages, 7 figure

Effets des propriétés physiques et chimiques des substrats sur la croissance et le développement de plants d'épinette blanche en récipient après un saison de culture

Au Québec, chaque année, des millions de plants forestiers produits en récipient sont rejetés à cause de leur système racinaire déficient. Afin de vérifier l'effet du substrat sur cette problématique, le premier objectif était de caractériser les propriétés physiques et chimiques des substrats utilisés par huit pépinières forestières. La masse volumique apparente (MVA), la granulométrie des particules, la teneur en air (thêtaa), la conductivité hydraulique saturée (Ks), le coefficient d'efficacité des pores (γ) et un indice de diffusivité relative des gaz (DsD₀⁻¹) ont été mesurés. Le pH, la conductivité électrique (CE), et la capacité d'échange cationique effective (CECeff) des substrats, ainsi que leurs contenus en éléments nutritifs (N-NH₄, N-NO₂₋₃, P, K, Ca, Mg) ont été mesurés. De plus, les variables de croissance des plants ont été évaluées après une saison de croissance. Les pépinières ont présenté d'importantes variations au niveau de la croissance des plants et des propriétés du substrat. Thêtaa et Ks mesurées à la fin de la saison de croissance étaient corrélées négativement avec (i) la masse sèche de la partie aérienne des plants et (ii) le diamètre au collet. La MVA était toutefois corrélée positivement avec la masse racinaire. L'aération des substrats n'a pas limité la croissance des plants malgré des valeurs de DsD₀⁻¹ faibles pour certains substrats (<0.005). Le second objectif visait à vérifier, au cours de deux années de production (2008 et 2009), les effets des propriétés physiques et chimiques sur la croissance et le développement des plants d'épinette blanche (1+0) cultivés dans plusieurs substrats composés de tourbe grossière et fine (<0.5 mm), de perlite et de vermiculite. Les substrats ont affecté les variables de croissance. En 2008, l'augmentation de la porosité d'air (thêtaa) a eu un effet négatif sur la croissance alors qu'en 2009, la croissance était affectée par une réduction du coefficient d'efficacité des pores (γ) dans les substrats. Selon les résultats de cette étude, la valeur optimale de DsD₀⁻¹ recommandée pour la culture de l'épinette blanche en récipient serait comprise entre 0.003 et 0.016 et thêtaa située entre 0.07 et 0.14 cm³ cm⁻³

U5 snRNP Core Proteins Are Key Components of the Defense Response against Viral Infection through Their Roles in Programmed Cell Death and Interferon Induction

The spliceosome is a massive ribonucleoprotein structure composed of five small nuclear ribonucleoprotein (snRNP) complexes that catalyze the removal of introns from pre-mature RNA during constitutive and alternative splicing. EFTUD2, PRPF8, and SNRNP200 are core components of the U5 snRNP, which is crucial for spliceosome function as it coordinates and performs the last steps of the splicing reaction. Several studies have demonstrated U5 snRNP proteins as targeted during viral infection, with a limited understanding of their involvement in virus&ndash;host interactions. In the present study, we deciphered the respective impact of EFTUD2, PRPF8, and SNRNP200 on viral replication using mammalian reovirus as a model. Using a combination of RNA silencing, real-time cell analysis, cell death and viral replication assays, we discovered distinct and partially overlapping novel roles for EFTUD2, PRPF8, and SNRNP200 in cell survival, apoptosis, necroptosis, and the induction of the interferon response pathway. For instance, we demonstrated that EFTUD2 and SNRNP200 are required for both apoptosis and necroptosis, whereas EFTUD2 and PRPF8 are required for optimal interferon response against viral infection. Moreover, we demonstrated that EFTUD2 restricts viral replication, both in a single cycle and multiple cycles of viral replication. Altogether, these results establish U5 snRNP core components as key elements of the cellular antiviral response

How Many Mammalian Reovirus Proteins are involved in the Control of the Interferon Response?

As with most viruses, mammalian reovirus can be recognized and attacked by the host-cell interferon response network. Similarly, many viruses have developed resistance mechanisms to counteract the host-cell response at different points of this response. Reflecting the complexity of the interferon signaling pathways as well as the resulting antiviral response, viruses can&mdash;and often have&mdash;evolved many determinants to interfere with this innate immune response and allow viral replication. In the last few years, it has been evidenced that mammalian reovirus encodes many different determinants that are involved in regulating the induction of the interferon response or in interfering with the action of interferon-stimulated gene products. In this brief review, we present our current understanding of the different reovirus proteins known to be involved, introduce their postulated modes of action, and raise current questions that may lead to further investigations

Blood Pressure Measurement in Morbid Obesity: Comparison between Forearm and Intra-Arterial Assessment of Blood pressure

Background: Obesity is associated with high blood pressure. Measurement of blood pressure (BP) in morbid obesity with a larger cuff bladder may be erroneous due to the anatomy of their upper limb and/or a nonsuitable cuff bladder. The objective of the study was to compare a non-invasive technique of BP measurement on the forearm with an appropriate standard blood cuff bladder to an invasive method (intra-arterial measurement in the radial artery) in two groups of subjects; non obese (NO) and morbidly obese (MO). Methods: Twenty-seven NO subjects with a mean body mass index (BMI) of 25,6 &#177; 2,7 kg/m2 were evaluated using two different techniques for blood pressure measurement. Measures were performed, in a supine position, at the forearm level with a standard cuff bladder (Welch Allyn, NY, USA) and on the opposite forearm using an intra-arterial access. The NO group was compared to 20 MO subjects with a mean BMI of 5,1 &#177; 8,7 kg/m2. The MO group was evaluated during a bariatric surgery procedure, with regular intervals (10 to 15 min) using the same two methods described above. Results: A difference was observed between the two techniques of blood pressure measurements (P < 0.05 and P < 0.01 intra-arterial vs. cuff bladder for the NO and the MO groups respectively) (see Table). Correlations between the two techniques were observed in the NO group (P < 0.001) for the systolic BP (r=0,823) and the diastolic BP (r=0,769). Similar correlations were also observed in the MO group for the systolic BP (r=0,792, P < 0,001) and the diastolic BP (r=0,830, P < 0,001; Table). *** Table in Full Text PDF. *** Conclusion: Although different, BP measurements at the forearm level in MO subjects seems to be clinically valid. This technique may be used in this specific population as an alternative for the measurement of BP