The chicken miR-150 targets the avian orthologue of the functional zebrafish MYB 3'UTR target site

International audienceBackground: The c-myb proto-oncogene is the founding member of a family of transcription factors involved principally in haematopoiesis, in diverse organisms, from zebrafish to mammals. Its deregulation has been implicated in human leukaemogenesis and other cancers. The expression of c-myb is tightly regulated by post-transcriptional mechanisms involving microRNAs. MicroRNAs are small, highly conserved non-coding RNAs that inhibit translation and decrease mRNA stability by binding to regulatory motifs mostly located in the 3'UTR of target mRNAs conserved throughout evolution. MYB is an evolutionarily conserved miR-150 target experimentally validated in mice, humans and zebrafish. However, the functional miR-150 sites of humans and mice are orthologous, whereas that of zebrafish is different. Results: We identified the avian mature miRNA-150-5P, Gallus gallus gga-miR-150 from chicken leukocyte small-RNA libraries and showed that, as expected, the gga-miR-150 sequence was highly conserved, including the seed region sequence present in the other miR-150 sequences listed in miRBase. Reporter assays showed that gga-miR-150 acted on the avian MYB 3'UTR and identified the avian MYB target site involved in gga-miR-150 binding. A comparative in silico analysis of the miR-150 target sites of MYB 3'UTRs from different species led to the identification of a single set of putative target sites in amphibians and zebrafish, whereas two sets of putative target sites were identified in chicken and mammals. However, only the target site present in the chicken MYB 3'UTR that was identical to that in zebrafish was functional, despite the additional presence of mammalian target sites in chicken. This specific miR-150 site usage was not cell-type specific and persisted when the chicken c-myb 3'UTR was used in the cell system to identify mammalian target sites, showing that this miR-150 target site usage was intrinsic to the chicken c-myb 3'UTR. Conclusion: Our study of the avian MYB/gga-miR-150 interaction shows a conservation of miR-150 target site functionality between chicken and zebrafish that does not extend to mammals

Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein

Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma virus-specific antibodies in rabbits after immunization. Inoculations by the intradermal route protected animals against virulent RHDV and myxoma virus challenges

GaHV-2 ICP22 protein is expressed from a bicistronic transcript regulated by three GaHV-2 microRNAs

International audienceHerpesviruses have a lifecycle consisting of successive lytic, latent and reactivation phases. Only three infected cell proteins (ICPs) have been described for the oncogenic Marek's disease virus (or Gallid herpes virus 2, GaHV-2): ICP4, ICP22 and ICP27. We focus here on ICP22, confirming its cytoplasmic location and showing that ICP22 is expressed during productive phases of the lifecycle, via a bicistronic transcript encompassing the US10 gene. We also identified the unique promoter controlling ICP22 expression, and its core promoter, containing functional responsive elements including E-box, ETS-1 and GATA elements involved in ICP22 transactivation. ICP22 gene expression was weakly regulated by DNA methylation and activated by ICP4 or ICP27 proteins. We also investigated the function of GaHV-2 ICP22. We found that this protein repressed transcription from its own promoter and from those of IE ICP4 and ICP27, and the late gK promoter. Finally, we investigated posttranscriptional ICP22 regulation by GaHV-2 microRNAs. We found that mdv1-miR-M5-3p and -M1-5p downregulated ICP22 mRNA expression during latency, whereas, unexpectedly, mdv1-miR-M4-5p upregulated the expression of the protein ICP22, indicating a tight regulation of ICP22 expression by microRNAs

Alternative splicing and nonsense-mediated decay regulate telomerase reverse transcriptase (TERT) expression during virus-induced lymphomagenesis in vivo

<p>Abstract</p> <p>Background</p> <p>Telomerase activation, a critical step in cell immortalization and oncogenesis, is partly regulated by alternative splicing. In this study, we aimed to use the Marek's disease virus (MDV) T-cell lymphoma model to evaluate TERT regulation by splicing during lymphomagenesis <it>in vivo</it>, from the start point to tumor establishment.</p> <p>Results</p> <p>We first screened cDNA libraries from the chicken MDV lymphoma-derived MSB-1 T- cell line, which we compared with B (DT40) and hepatocyte (LMH) cell lines. The chTERT splicing pattern was cell line-specific, despite similar high levels of telomerase activity. We identified 27 alternative transcripts of chicken TERT (chTERT). Five were in-frame alternative transcripts without <it>in vitro </it>telomerase activity in the presence of viral or chicken telomerase RNA (vTR or chTR), unlike the full-length transcript. Nineteen of the 22 transcripts with a premature termination codon (PTC) harbored a PTC more than 50 nucleotides upstream from the 3' splice junction, and were therefore predicted targets for nonsense-mediated decay (NMD). The major PTC-containing alternatively spliced form identified in MSB1 (ie10) was targeted to the NMD pathway, as demonstrated by UPF1 silencing. We then studied three splicing events separately, and the balance between in-frame alternative splice variants (d5f and d10f) plus the NMD target i10ec and constitutively spliced chTERT transcripts during lymphomagenesis induced by MDV indicated that basal telomerase activity in normal T cells was associated with a high proportion of in-frame non functional isoforms and a low proportion of constitutively spliced chTERT. Telomerase upregulation depended on an increase in active constitutively spliced chTERT levels and coincided with a switch in alternative splicing from an in-frame variant to NMD-targeted variants.</p> <p>Conclusions</p> <p>TERT regulation by splicing plays a key role in telomerase upregulation during lymphomagenesis, through the sophisticated control of constitutive and alternative splicing. Using the MDV T-cell lymphoma model, we identified a chTERT splice variant as a new NMD target.</p

Etude d'un coronavirus, le virus de la gastro-enterite transmissible du porc : identification des genes structuraux et non-structuraux et localisation d'un site antigenique majeur sur la sequence de la glycoproteine de spicule E2

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

p53 transactive le promoteur du gène LAT encodant le cluster 2 de miARN du virus oncogène aviaire GaHV2.

National audienc

Etude comparative de l'interaction de la sous-unité vTR de MDV et de la sous-unité cTR du poulet avec la télomérase

La maladie de Marek est un lymphome T du poulet causé par l'herpesvirus de la maladie de Marek. Ma thèse a consisté à caractériser la première sous-unité ARN télomérase virale (vTR), que nous avons mise en évidence dans le génome de la souche MDV-RB1B. La télomérase est une ribonucléoprotéine qui assure l'élongation des télomères et qui est détectée dans plus de 85% des cancers humains. Nous avons réalisé une étude comparative de vTR avec la composante aviaire cTR, homologue à 88%. L'étude de l'expression de vTR a fait ressortir que son promoteur pourrait être une combinaison d'un promoteur similaire à celui de cTR et d'un promoteur potentiellement inductible pendant la tumorigenèse. Par ailleurs, nous avons démontré la fonctionnalité du gène vTR qui semble plus efficace que cTR. Par mutagenèse dirigée, nous avons confirmé que le domaine CR1 de vTR correspondait bien au domaine matriciel, que l'intégrité du pseudonoeud était nécessaire pour l'activité télomérase et enfin que la boîte H assurait la localisation nucléolaire de vTR. Au vue de l'implication de la télomérase dans de nombreux cancers, vTR pourrait être un facteur déterminant dans la tumorigenèse viro-induite.The Marek's disease is a T lymphoma induced by a herpesvirus, the Marek's disease virus. The aim of my phD consisted of the characterization of the first viral RNA telomerase component identified in the very virulent MDV-RB1B strain. The telomerase is a ribonucleoprotein that is involved in telomere lengthening and that is detected at least in 85% of human cancers. In order to characterize vTR, I made a comparative study of this gene with its avian ortholog cTR, which is 88%.homologous to vTR. We realized an expression study of vTR, which led us to hypothesize that the promoter region of vTR could be a combination of a promoter similar to cTR and a promoter, which could be induced during tumorigenesis. Otherwise, we demonstrated the fonctionnality of vTR, which thus seems to be more efficient than cTR. We also confirmed by a mutagenesis study that the CR1 domain of vTR is the template sequence, that the integrity of the pseudonoeud domain is essential for the telomerase activity and that the H box of vTR permits the nucleolar localisation of vTR in cells. According to the telomerase involvement in cancers, vTR could be considered as a determinant factor in the tumorigenesis induced by MDV.TOURS-BU Sciences Pharmacie (372612104) / SudocSudocFranceF

Etude de la régulation transcriptionnelle de la sous-unité ARN virale de la télomérase, vTR, codée par le virus de la maladie de Marek et des sous-unités cellulaires aviaires, chTR chTERT, de la télomérase

Marek s disease virus (MDV) induces a highly malignant T-lymphoma in chickens. The viral genome encodes a viral telomerase RNA subunit (vTR). The telomerase complex consists of a protein subunit (TERT) and a RNA subunit (TR). The active complex compensates for the progressive telomere shortening that occurs during mitosis. An up-regulation of telomerase activity is associated with an increase in vTR gene expression in chickens infected with MDV. The thesis work focused on transcriptional regulation of vTR, chTR and chTERT. We demonstrated that vTR promoter is up to 2-fold more efficient than the chTR promoter in avian cells. Furthermore, transactivation assays and ChIP assays demonstrated the involvement of the c-Myc oncoprotein in the transcriptional regulation of vTR through the E-box 3 element. Otherwise, the study of chTERT promoter activity showed that a c-Myb element is involved in the repression of chTERT expression in avian cells.Le virus de la maladie de Marek (MDV), responsable du développement de tumeurs lymphoïdes de type T chez le poulet, code le gène de la sous-unité ARN de la télomérase (TR). Responsable de l élongation des télomères, la télomérase est composée d une partie protéique (TERT) et d une partie ARN (TR). Une augmentation de l activité télomérase est associée à une forte expression de vTR dans les leucocytes sanguins de poulets infectés par le MDV. Le travail de thèse a consisté en l étude de la régulation transcriptionnelle des gènes vTR, chTR et chTERT. Ainsi, l efficacité transcriptionnelle du promoteur de vTR est au moins 2 fois plus importante que chTR. Des essais de ChIP et de surexpression ont montré que la protéine c-Myc était responsable de la transactivation du promoteur de vTR via sa boîte E3. Par ailleurs, l étude de l efficacité du promoteur de chTERT a montré que sa répression, dans les lignées cellulaires testées, impliquait un site liant le facteur c-Myb.TOURS-BU Sciences Pharmacie (372612104) / SudocSudocFranceF

Etude des mécanismes de maturation de la polyprotéine du virus de la maladie hémorragique du lapin (RHDV)

TOURS-BU Sciences Pharmacie (372612104) / SudocSudocFranceF

Alternative splicing of a viral mirtron differentially affects the expression of other microRNAs from its cluster and of the host transcript

Interplay between alternative splicing and the Microprocessor may have differential effects on the expression of intronic miRNAs organized into clusters. We used a viral model the LAT long non-coding RNA (LAT lncRNA) of Marek's disease oncogenic herpesvirus (MDV-1), which has the mdv1-miR-M8-M6-M7-M10 cluster embedded in its first intron to assess the impact of splicing modifications on the biogenesis of each of the miRNAs from the cluster. Drosha silencing and alternative splicing of an extended exon 2 of the LAT lncRNA from a newly identified 3 splice site (SS) at the end of the second miRNA of the cluster showed that mdv1-miR-M6 was a 5-tailed mirtron. We have thus identified the first 5-tailed mirtron within a cluster of miRNAs for which alternative splicing is directly associated with differential expression of the other miRNAs of the cluster, with an increase in intronic mdv1-miR-M8 expression and a decrease in expression of the exonic mdv1-miR-M7, and indirectly associated with regulation of the host transcript. According to the alternative 3SS used for the host intron splicing, the mdv1-miR-M6 is processed as a mirtron by the spliceosome, dispatching the other miRNAs of the cluster into intron and exon, or as a canonical miRNA by the Microprocessor complex. The viral mdv1-miR-M6 mirtron is the first mirtron described that can also follow the canonical pathway