Maud Contrant

Bio: Maud Contrant is an academic researcher from University of Strasbourg. The author has contributed to research in topics: microRNA & Outbreak. The author has an hindex of 4, co-authored 8 publications receiving 189 citations. Previous affiliations of Maud Contrant include Centre national de la recherche scientifique.

Kaposi's Sarcoma Herpesvirus microRNAs Target Caspase 3 and Regulate Apoptosis

Abstract: Kaposi's sarcoma herpesvirus (KSHV) encodes a cluster of twelve micro (mi)RNAs, which are abundantly expressed during both latent and lytic infection. Previous studies reported that KSHV is able to inhibit apoptosis during latent infection; we thus tested the involvement of viral miRNAs in this process. We found that both HEK293 epithelial cells and DG75 cells stably expressing KSHV miRNAs were protected from apoptosis. Potential cellular targets that were significantly down-regulated upon KSHV miRNAs expression were identified by microarray profiling. Among them, we validated by luciferase reporter assays, quantitative PCR and western blotting caspase 3 (Casp3), a critical factor for the control of apoptosis. Using site-directed mutagenesis, we found that three KSHV miRNAs, miR-K12-1, 3 and 4-3p, were responsible for the targeting of Casp3. Specific inhibition of these miRNAs in KSHV-infected cells resulted in increased expression levels of endogenous Casp3 and enhanced apoptosis. Altogether, our results suggest that KSHV miRNAs directly participate in the previously reported inhibition of apoptosis by the virus, and are thus likely to play a role in KSHV-induced oncogenesis.

Structural and Functional Motifs in Influenza Virus RNAs.

Abstract: Influenza A viruses (IAV) are responsible for recurrent influenza epidemics and occasional devastating pandemics in humans and animals. They belong to the Orthomyxoviridae family and their genome consists of eight (-) sense viral RNA (vRNA) segments of different lengths coding for at least 11 viral proteins. A heterotrimeric polymerase complex is bound to the promoter consisting of the 13 5'-terminal and 12 3'-terminal nucleotides of each vRNA, while internal parts of the vRNAs are associated with multiple copies of the viral nucleoprotein (NP), thus forming ribonucleoproteins (vRNP). Transcription and replication of vRNAs result in viral mRNAs (vmRNAs) and complementary RNAs (cRNAs), respectively. Complementary RNAs are the exact positive copies of vRNAs; they also form ribonucleoproteins (cRNPs) and are intermediate templates in the vRNA amplification process. On the contrary, vmRNAs have a 5' cap snatched from cellular mRNAs and a 3' polyA tail, both gained by the viral polymerase complex. Hence, unlike vRNAs and cRNAs, vmRNAs do not have a terminal promoter able to recruit the viral polymerase. Furthermore, synthesis of at least two viral proteins requires vmRNA splicing. Except for extensive analysis of the viral promoter structure and function and a few, mostly bioinformatics, studies addressing the vRNA and vmRNA structure, structural studies of the influenza A vRNAs, cRNAs, and vmRNAs are still in their infancy. The recent crystal structures of the influenza polymerase heterotrimeric complex drastically improved our understanding of the replication and transcription processes. The vRNA structure has been mainly studied in vitro using RNA probing, but its structure has been very recently studied within native vRNPs using crosslinking and RNA probing coupled to next generation RNA sequencing. Concerning vmRNAs, most studies focused on the segment M and NS splice sites and several structures initially predicted by bioinformatics analysis have now been validated experimentally and their role in the viral life cycle demonstrated. This review aims to compile the structural motifs found in the different RNA classes (vRNA, cRNA, and vmRNA) of influenza viruses and their function in the viral replication cycle.

Importance of the RNA secondary structure for the relative accumulation of clustered viral microRNAs

Abstract: Micro (mi)RNAs are small non-coding RNAs with key regulatory functions. Recent advances in the field allowed researchers to identify their targets. However, much less is known regarding the regulation of miRNAs themselves. The accumulation of these tiny regulators can be modulated at various levels during their biogenesis from the transcription of the primary transcript (pri-miRNA) to the stability of the mature miRNA. Here, we studied the importance of the pri-miRNA secondary structure for the regulation of mature miRNA accumulation. To this end, we used the Kaposi's sarcoma herpesvirus, which encodes a cluster of 12 pre-miRNAs. Using small RNA profiling and quantitative northern blot analysis, we measured the absolute amount of each mature miRNAs in different cellular context. We found that the difference in expression between the least and most expressed viral miRNAs could be as high as 60-fold. Using high-throughput selective 2′-hydroxyl acylation analyzed by primer extension, we then determined the secondary structure of the long primary transcript. We found that highly expressed miRNAs derived from optimally structured regions within the pri-miRNA. Finally, we confirmed the importance of the local structure by swapping stem-loops or by targeted mutagenesis of selected miRNAs, which resulted in a perturbed accumulation of the mature miRNA.

The expression of a viral microRNA is regulated by clustering to allow optimal B cell transformation

Abstract: The Epstein-Barr virus (EBV) transforms B cells by expressing latent proteins and the BHRF1 microRNA cluster. MiR-BHRF1-3, its most transforming member, belongs to the recently identified group of weakly expressed microRNAs. We show here that miR-BHRF1-3 displays an unusually low propensity to form a stem-loop structure, an effect potentiated by miR-BHRF1-3's proximity to the BHRF1 polyA site. Cloning miR-BHRF1-2 or a cellular microRNA, but not a ribozyme, 5' of miR-BHRF1-3 markedly enhanced its expression. However, a virus carrying mutated miR-BHRF1-2 seed regions expressed miR-BHRF1-3 at normal levels and was fully transforming. Therefore, miR-BHRF1-2's role during transformation is independent of its seed regions, revealing a new microRNA function. Increasing the distance between miR-BHRF1-2 and miR-BHRF1-3 in EBV enhanced miR-BHRF1-3's expression but decreased its transforming potential. Thus, the expression of some microRNAs must be restricted to a narrow range, as achieved by placing miR-BHRF1-3 under the control of miR-BHRF1-2.

Cis regulation within a cluster of viral microRNAs

Abstract: MicroRNAs (miRNAs) are small regulatory RNAs involved in virtually all biological processes. Although a large number of them are co-expressed from clusters, little is known regarding the impact of this organization on the regulation of their accumulation. In this study, we set to decipher the regulatory mechanism controlling the expression of the ten clustered pre-miRNAs from Kaposis sarcoma associated herpesvirus (KSHV). We measured in vitro the efficiency of cleavage of each individual pre-miRNA by the Microprocessor. Strikingly, pre-miR-K1 and -K3 were the most efficiently cleaved pre-miRNAs but their mature forms accumulated at low levels, indicating that they might perform an additional function. A mutational analysis showed that they are indeed important for the optimal expression of the whole set of miRNAs. We showed that this solely relies on the presence of a canonical pre-miRNA at this localization since we could functionally replace pre-miR-K1 by a heterologous pre-miRNA. Further in vitro processing analysis suggests that the two stem-loops act in cis and that the cluster is cleaved in a sequential manner. Finally, we showed that we can exploit this feature of the cluster to inhibit the expression of the whole set of miRNAs by targeting the pre-miR-K1 with LNA-based antisense oligonucleotides.

Regulation of microRNA biogenesis and its crosstalk with other cellular pathways

Abstract: MicroRNAs (miRNAs) are short non-coding RNAs that inhibit the expression of target genes by directly binding to their mRNAs. miRNAs are transcribed as precursor molecules, which are subsequently cleaved by the endoribonucleases Drosha and Dicer. Mature miRNAs are bound by a member of the Argonaute (AGO) protein family to form the RNA-induced silencing complex (RISC) in a process termed RISC loading. Advances in structural analyses of Drosha and Dicer complexes enabled elucidation of the mechanisms that drive these molecular machines. Transcription of miRNAs, their processing by Drosha and Dicer and RISC loading are key steps in miRNA biogenesis, and various additional factors facilitate, support or inhibit these processes. Recent work has revealed that regulatory factors not only coordinate individual miRNA processing steps but also connect miRNA biogenesis with other cellular processes. Protein phosphorylation, for example, links miRNA biogenesis to various signalling pathways, and such modifications are often associated with disease. Furthermore, not all miRNAs follow canonical processing routes, and many non-canonical miRNA biogenesis pathways have recently been characterized.

MicroRNAs: Target Recognition and Regulatory Functions

Abstract: MicroRNAs (miRNAs) are endogenous ∼23 nt RNAs that play important gene-regulatory roles in animals and plants by pairing to the mRNAs of protein-coding genes to direct their posttranscriptional repression. This review outlines the current understanding of miRNA target recognition in animals and discusses the widespread impact of miRNAs on both the expression and evolution of protein-coding genes.

Virus-Encoded microRNAs: An Overview and a Look to the Future

Abstract: MicroRNAs (miRNAs) are small RNAs that play important roles in the regulation of gene expression. First described as posttranscriptional gene regulators in eukaryotic hosts, virus-encoded miRNAs were later uncovered. It is now apparent that diverse virus families, most with DNA genomes, but at least some with RNA genomes, encode miRNAs. While deciphering the func- tions of viral miRNAs has lagged behind their discovery, recent functional studies are bringing into focus these roles. Some of the best characterized viral miRNA functions include subtle roles in prolonging the longevity of infected cells, evading the immune response, and regulating the switch to lytic infection. Notably, all of these functions are particularly important during persis- tent infections. Furthermore, an emerging view of viral miRNAs suggests two distinct groups exist. In the first group, viral miRNAs mimic host miRNAs and take advantage of conserved networks of host miRNA target sites. In the larger second group, viral miRNAs do not share common target sites conserved for host miRNAs, and it remains unclear what fraction of these targeted transcripts are beneficial to the virus. Recent insights from multiple virus families have revealed new ways of interacting with the host miRNA machinery including noncanonical miRNA biogenesis and new mechanisms of posttranscriptional cis gene regulation. Exciting challeng- es await the field, including determining the most relevant miRNA targets and parlaying our current understanding of viral miRNAs into new therapeutic strategies. To accomplish these goals and to better grasp miRNA function, new in vivo models that recapitulate persistent infections associated with viral pathogens are required.