Changes of Protein Expression after CRISPR/Cas9 Knockout of miRNA-142 in Cell Lines Derived from Diffuse Large B-Cell Lymphoma

Background: As microRNA-142 (miR-142) is the only human microRNA gene where mutations have consistently been found in about 20% of all cases of diffuse large B-cell lymphoma (DLBCL), we wanted to determine the impact of miR-142 inactivation on protein expression of DLBCL cell lines. Methods: miR-142 was deleted by CRISPR/Cas9 knockout in cell lines from DLBCL. Results: By proteome analyses, miR-142 knockout resulted in a consistent up-regulation of 52 but also down-regulation of 41 proteins in GC-DLBCL lines BJAB and SUDHL4. Various mitochondrial ribosomal proteins were up-regulated in line with their pro-tumorigenic properties, while proteins necessary for MHC-I presentation were down-regulated in accordance with the finding that miR-142 knockout mice have a defective immune response. CFL2, CLIC4, STAU1, and TWF1 are known targets of miR-142, and we could additionally confirm AKT1S1, CCNB1, LIMA1, and TFRC as new targets of miR-142-3p or -5p. Conclusions: Seed-sequence mutants of miR-142 confirmed potential targets and novel targets of miRNAs can be identified in miRNA knockout cell lines. Due to the complex contribution of miRNAs within cellular regulatory networks, in particular when miRNAs highly present in RISC complexes are replaced by other miRNAs, primary effects on gene expression may be covered by secondary layers of regulation

Étude des mécanismes de régulation de biogenèse des microARN exprimés par l’Hrepèsvirus associé au sarcome de Kaposi

MicroRNAs (miRNAs) are important regulators of gene expression expressed by almost all eukaryotes and also some of their associated viruses. In this project, we have focused on the biogenesis of a miRNA cluster expressed by the human oncovirus, Kaposi’s sarcoma associated herpesvirus (KSHV), with the aim to decipher the post-transcriptional molecular mechanisms controlling their processing. We have shown that some of the viral precursor miRNAs (pre-miRNAs) can act as cis regulatory elements and are required for the optimal processing of the remaining pre-miRNAs within the cluster. An approach to inhibit the global expression of the cluster was developed based on these findings. In addition, we were interested in potential implication of protein co-factors HNRL1 and SUGP1 that could regulate the miRNA expression in trans.Les microARN (miARN) sont d’importants régulateurs de l’expression génique exprimés par presque tous les eucaryotes, ainsi que certains de leurs virus associés. Dans le cadre de ce projet, nous avons étudié la biogenèse d’un cluster de miARN exprimé par un oncovirus humain, l’Herpèsvirus associé au sarcome de Kaposi (KSHV). L’objectif était de caractériser les mécanismes de régulation post-transcriptionnelle qui régissent la maturation ce de cluster. Nous avons démontré que certains des miARN précurseurs (pré-miARN) servent d’éléments régulateurs en cis et sont nécessaires pour l’expression de la totalité du cluster. En se basant sur ces résultats, nous avons développé une méthode d’inhibition globale de l’expression du cluster. Finalement, nous avons également étudié l’implication de potentiels co-facteurs HNRL1 et SUGP1 qui pourraient réguler la biogenèse des miARN en trans

Étude des mécanismes de régulation de biogenèse des microARN exprimés par l’Hrepèsvirus associé au sarcome de Kaposi

Les microARN (miARN) sont d’importants régulateurs de l’expression génique exprimés par presque tous les eucaryotes, ainsi que certains de leurs virus associés. Dans le cadre de ce projet, nous avons étudié la biogenèse d’un cluster de miARN exprimé par un oncovirus humain, l’Herpèsvirus associé au sarcome de Kaposi (KSHV). L’objectif était de caractériser les mécanismes de régulation post-transcriptionnelle qui régissent la maturation ce de cluster. Nous avons démontré que certains des miARN précurseurs (pré-miARN) servent d’éléments régulateurs en cis et sont nécessaires pour l’expression de la totalité du cluster. En se basant sur ces résultats, nous avons développé une méthode d’inhibition globale de l’expression du cluster. Finalement, nous avons également étudié l’implication de potentiels co-facteurs HNRL1 et SUGP1 qui pourraient réguler la biogenèse des miARN en trans.MicroRNAs (miRNAs) are important regulators of gene expression expressed by almost all eukaryotes and also some of their associated viruses. In this project, we have focused on the biogenesis of a miRNA cluster expressed by the human oncovirus, Kaposi’s sarcoma associated herpesvirus (KSHV), with the aim to decipher the post-transcriptional molecular mechanisms controlling their processing. We have shown that some of the viral precursor miRNAs (pre-miRNAs) can act as cis regulatory elements and are required for the optimal processing of the remaining pre-miRNAs within the cluster. An approach to inhibit the global expression of the cluster was developed based on these findings. In addition, we were interested in potential implication of protein co-factors HNRL1 and SUGP1 that could regulate the miRNA expression in trans

Cis regulation within a cluster of viral microRNAs

Correction: Volume49, Issue18 Page10804-10805 Article Number711910 DOI10.1093/nar/gkab806MicroRNAs (miRNAs) are small regulatory RNAs involved in virtually all biological processes. Although many 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 a regulatory mechanism controlling the expression of the ten clustered pre-miRNAs from Kaposi's sarcoma associated herpesvirus (KSHV). We measured in vitro the efficiency of cleavage of each individual pre-miRNA by the Microprocessor and found that pre-miR-K1 and -K3 were the most efficiently cleaved pre-miRNAs. A mutational analysis showed that, in addition to producing mature miRNAs, they are also important for the optimal expression of the whole set of miRNAs. We showed that this feature depends on the presence of a canonical pre-miRNA at this location 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 exploited this characteristic of the cluster to inhibit the expression of the whole set of miRNAs by targeting the premiR-K1 with LNA-based antisense oligonucleotides in cells either expressing a synthetic construct or latently infected with KSHV.Peer reviewe