Développement de lignées de poissons zébrés transgéniques pour l'étude du rôle de la protéine F dans la pathogenèse de l'hépatite C

Le virus de l’hépatite C (VHC) est une des principales causes d’hépatite chronique. La protéine F du VHC est codée par un cadre de lecture alternatif du gène de la capside, Core. La protéine F a été découverte après que l’on ait associé Core à plusieurs des fonctions pathogènes du VHC. Nous proposons donc que certaines fonctions biologiques et pathogènes attribuées à la protéine Core résultent de l’activité de la protéine F. Nous avons choisi de développer trois lignées de poissons zébrés (Danio rerio) qui expriment différentes versions de la protéine F afin d’étudier les effets de la protéine F et leur incidence dans la pathogenèse du VHC. Deux versions de la séquence codant pour la protéine F (AF11 et AUG26) et une version mutante du gène core (CoremutI) ont été introduites sur les vecteurs d’un système d’expression répressible spécifique au foie. Ces vecteurs ont été co-injectés dans des embryons unicellulaires de poissons zébrés pour générer les poissons fondateurs des lignées transgéniques. 19, 21 et 36 poissons ont été choisis comme fondateurs pour les lignées AF11, AUG26 et CoremutI respectivement. De ce nombre, 9, 11 et 11 poissons ont atteint la maturité, dans l’ordre pour les mêmes lignées, et seront croisés pour donner naissance à des lignées transgéniques stables. Les résultats de ces expériences nous permettront de mieux cerner les propriétés biologiques de la protéine F et de définir son rôle dans la pathogenèse du VHC.Hepatitis C virus (HCV) is a major cause of liver steatosis, fibrosis and hepatocellular carcinoma. HCV F protein is expressed from an alternative reading frame within the Core sequence. F protein was discovered after many of the pathogenic determinants of HCV had been associated with the effects of Core. Hence, we propose that a part of the functions attributed to Core result from the activity of the F protein. We produced and selected 19, 21 and 36 transgenic zebrafish (Danio rerio) to give rise to 3 independent lines expressing different versions of the F protein. Of these founders, 9, 11 and 11 were raised to maturity and will be bred to generate stable transgenic lines. Characterizing the phenotype of these transgenic fish will help determine the precise role of the F protein in the pathogenesis of hepatitis C

Novel HIV-1 Recombinant Forms in Antenatal Cohort, Montreal, Quebec, Canada

Near full-length genomes of 4 unclassified HIV-1 variants infecting patients enrolled in an antenatal cohort in Canada were obtained by sequencing. All 4 variants showed original recombination profiles, including A1/A2/J, A1/D, and A1/G/J/CRF11_cpx structures. Identification of these variants highlights the growing prevalence of unique recombinant forms of HIV-1 in North America

Misregulation of an activity-dependent splicing network as a common mechanism underlying autism spectrum disorders

A key challenge in understanding and ultimately treating autism is to identify common molecular mechanisms underlying this genetically heterogeneous disorder. Transcriptomic profiling of autistic brains has revealed correlated misregulation of the neuronal splicing regulator nSR100/SRRM4 and its target microexon splicing program in more than one-third of analyzed individuals. To investigate whether nSR100 misregulation is causally linked to autism, we generated mutant mice with reduced levels of this protein and its target splicing program. Remarkably, these mice display multiple autistic-like features, including altered social behaviors, synaptic density, and signaling. Moreover, increased neuronal activity, which is often associated with autism, results in a rapid decrease in nSR100 and splicing of microexons that significantly overlap those misregulated in autistic brains. Collectively, our results provide evidence that misregulation of an nSR100-dependent splicing network controlled by changes in neuronal activity is causally linked to a substantial fraction of autism cases.This work was supported by CIHR grants to S.P.C. (MOP#111199 and MOP#142340), B.J.B. (MOP#14609 and FDN#148434), and M.A.W.(MOP#12346), and an ERC Starting Grant to M.I. (ERC-StG-LS2-637591). M.Q.V. was supported by CIHR and OGS scholarships. T.G.-P. was supported by EMBO and OIRM fellowship

The Tumor Suppressor <i>Adenomatous Polyposis Coli (apc)</i> Is Required for Neural Crest-Dependent Craniofacial Development in Zebrafish

Neural crest (NC) is a unique vertebrate cell type arising from the border of the neural plate and epidermis that gives rise to diverse tissues along the entire body axis. Roberto Mayor and colleagues have made major contributions to our understanding of NC induction, delamination, and migration. We report that a truncating mutation of the classical tumor suppressor Adenomatous Polyposis Coli (apc) disrupts craniofacial development in zebrafish larvae, with a marked reduction in the cranial neural crest (CNC) cells that contribute to mandibular and hyoid pharyngeal arches. While the mechanism is not yet clear, the altered expression of signaling molecules that guide CNC migration could underlie this phenotype. For example, apcmcr/mcr larvae express substantially higher levels of complement c3, which Mayor and colleagues showed impairs CNC cell migration when overexpressed. However, we also observe reduction in stroma-derived factor 1 (sdf1/cxcl12), which is required for CNC migration into the head. Consistent with our previous work showing that APC directly enhances the activity of glycogen synthase kinase 3 (GSK-3) and, independently, that GSK-3 phosphorylates multiple core mRNA splicing factors, we identify 340 mRNA splicing variations in apc mutant zebrafish, including a splice variant that deletes a conserved domain in semaphorin 3f (sema3f), an axonal guidance molecule and a known regulator of CNC migration. Here, we discuss potential roles for apc in CNC development in the context of some of the seminal findings of Mayor and colleagues

The germ cell-specific RNA binding protein RBM46 is essential for spermatogonial differentiation in mice.

Control over gene expression is exerted, in multiple stages of spermatogenesis, at the post-transcriptional level by RNA binding proteins (RBPs). We identify here an essential role in mammalian spermatogenesis and male fertility for 'RNA binding protein 46' (RBM46). A highly evolutionarily conserved gene, Rbm46 is also essential for fertility in both flies and fish. We found Rbm46 expression was restricted to the mouse germline, detectable in males in the cytoplasm of premeiotic spermatogonia and meiotic spermatocytes. To define its requirement for spermatogenesis, we generated Rbm46 knockout (KO, Rbm46-/-) mice; although male Rbm46-/- mice were viable and appeared grossly normal, they were infertile. Testes from adult Rbm46-/- mice were small, with seminiferous tubules containing only Sertoli cells and few undifferentiated spermatogonia. Using genome-wide unbiased high throughput assays RNA-seq and 'enhanced crosslinking immunoprecipitation' coupled with RNA-seq (eCLIP-seq), we discovered RBM46 could bind, via a U-rich conserved consensus sequence, to a cohort of mRNAs encoding proteins required for completion of differentiation and subsequent meiotic initiation. In summary, our studies support an essential role for RBM46 in regulating target mRNAs during spermatogonia differentiation prior to the commitment to meiosis in mice

An atlas of alternative splicing profiles and functional associations reveals new regulatory programs and genes that simultaneously express multiple major isoforms

Alternative splicing (AS) generates remarkable regulatory and proteomic complexity in metazoans. However, the functions of most AS events are not known, and programs of regulated splicing remain to be identified. To address these challenges, we describe the Vertebrate Alternative Splicing and Transcription Database (VastDB), the largest resource of genome-wide, quantitative profiles of AS events assembled to date. VastDB provides readily accessible quantitative information on the inclusion levels and functional associations of AS events detected in RNA-seq data from diverse vertebrate cell and tissue types, as well as developmental stages. The VastDB profiles reveal extensive new intergenic and intragenic regulatory relationships among different classes of AS and previously unknown and conserved landscapes of tissue-regulated exons. Contrary to recent reports concluding that nearly all human genes express a single major isoform, VastDB provides evidence that at least 48% of multiexonic protein-coding genes express multiple splice variants that are highly regulated in a cell/tissue-specific manner, and that >18% of genes simultaneously express multiple major isoforms across diverse cell and tissue types. Isoforms encoded by the latter set of genes are generally coexpressed in the same cells and are often engaged by translating ribosomes. Moreover, they are encoded by genes that are significantly enriched in functions associated with transcriptional control, implying they may have an important and wide-ranging role in controlling cellular activities. VastDB thus provides an unprecedented resource for investigations of AS function and regulation.This work has been supported by grants from the European Research Council (ERC-StG-LS2-637591 to M.I.), the Spanish Ministry of Economy and Competitiveness (Centro de Excelencia Severo Ochoa 2013–2017, SEV-2012-0208 to the CRG, SEV-2015-0505 to the CNIC, and BFU2014-55076-P to M.I.). J.T. holds a FPI Severo Ochoa fellowship and was supported by Grant SVP-2014-068315. K.C.H. was supported by an Ontario Graduate Scholarship and a CIHR Frederick Banting and Charles Best Canada Graduate Doctoral Award. Y.M. holds an EMBO LTF fellowshi

RBM46 sequence-specific binding to a specific cohort of mRNAs encoding factors required for RNA processing and meiosis.

(A) FLAG-stained western blot of lysates from Rbm46WT/WT and Rbm46FLAG/FLAG showing input and immunoprecipitated proteins, respectively. Band at the expected protein size is indicated by arrow. (B) Genomic distribution of RBM46 binding in the testis, displayed as percentages of binding to each sequence feature. (C) Representative UCSC genome browser view showing exonic binding of RBM46 across Dazl and Meioc. (D) WebLogo showing U-rich RNA binding sequence motif identified using mCross as the most enriched sequence at the RBM46 crosslink sites. (E) Gene ontology analysis performed using EnrichR showing the top biological processes of RBM46-bound mRNAs.</p

Genes involved in cell cycle regulation were deregulated in <i>Rbm46</i>^<i>-/-</i> testes at P8.

(A) Heatmap of 200 genes with >2-fold changes in Rbm46-/- relative to Rbm46+/+ controls at P8. (B) Gene ontology terms for biological processes enriched in genes that are downregulated (top) or upregulated (bottom) in Rbm46-/-. Circle size and numbers correspond to the number of genes that are differentially expressed and represented in a GO term over to the total number of genes listed in the GO term. (C) Expression level of genes involved in spermatogonia differentiation and somatic cell markers. P-values are DESeq2 adjusted p-values comparing Rbm46+/+ to Rbm46-/- testes.</p

List of genes bound by RBM46 at P21 whose mRNAs were differentially expressed in <i>Rbm46</i>^-/- testes at P8.

(XLSX)</p