Biology subjects

Ngondo, R. P.

Publications and source records attributed to Ngondo, R. P..

4 recordsLinked to original sources

bioRxiv2021

Integrative analysis allows a global and precise identification of functional miRNA target genes in mESCs

MicroRNA (miRNA) loaded Argonaute (AGO) complexes regulate gene expression via direct base pairing with their mRNA targets. Current prediction approaches identified that between 20 to 60% of mammalian transcriptomes are regulated by miRNAs, but it remains largely unknown which fraction of these interactions are functional in a specific cellular context. Here, we integrated transcriptome data from a set of miRNA-depleted mouse embryonic stem cell (mESC) lines with published miRNA interaction predictions and AGO-binding profiles. This integrative approach, combined with molecular validation data, identified that only 6% of expressed genes are functionally and directly regulated by miRNAs in mESCs. In addition, analyses of the stem cell-specific miR-290-295 cluster target genes identified TFAP4 as an important transcription factor for early development. The extensive datasets developed in this study will support the development of improved predictive models for miRNA-mRNA functional interactions.

systems biology↗

bioRxiv2021

AGO1 regulates major satellite transcripts and H3K9me3 distribution at pericentromeric regions in mESCs

Argonaute proteins (AGOs), that play an essential role in cytosolic post-transcriptional gene silencing, have been also reported to function in nuclear processes like transcriptional activation or repression, alternative splicing and, chromatin organization. As most of these studies have been conducted in human cancer cell lines, the relevance of AGOs nuclear functions in the context of mouse early embryonic development remains uninvestigated. Here, we examined a possible role of the AGO1 protein on the distribution of constitutive heterochromatin in mouse Embryonic Stem Cells (mESCs). We observed a specific redistribution of the repressive histone mark H3K9me3 and the heterochromatin protein HP1, away from pericentromeric regions upon Ago1 depletion. Furthermore, we demonstrated that major satellite transcripts are strongly upregulated in Ago1_KO mESCs and that their levels are partially restored upon AGO1 rescue. We also observed a similar redistribution of H3K9me3 and HP1 in Drosha_KO mESCs, suggesting a role for microRNAs (miRNAs) in the regulation of heterochromatin distribution in mESCs. Finally, we showed that specific miRNAs with complementarity to major satellites can partially regulate the expression of these transcripts. Summary blurbDepletion of AGO1 in mESCs leads to a redistribution of H3K9me3 and HP1 away from pericentromeric regions and is accompanied by an upregulation of major satellites transcripts.

cell biology↗

bioRxiv2021

CLUH interactome reveals an association to SPAG5 and a proximity to the translation of mitochondrial protein

Mitochondria require thousands of proteins to fulfil their essential function in energy production and other fundamental biological processes. These proteins are mostly encoded by the nuclear genome, translated in the cytoplasm before being imported into the organelle. RNA binding proteins (RBPs) are central players in the regulation of this process by affecting mRNA translation, stability or localization. CLUH is an RBP recognizing specifically mRNAs coding for mitochondrial proteins, but its precise molecular function and interacting partners remain undiscovered in mammals. Here we reveal for the first time CLUH interactome in mammalian cells. Using both co-IP and BioID proximity-labeling approaches, we identify novel molecular partners interacting stably or transiently with CLUH in HCT116 cells and mouse embryonic stem cells. We reveal a stable RNA-independent interaction of CLUH with itself and with SPAG5 in cytosolic granular structures. More importantly, we uncover an unexpected proximity of CLUH to mitochondrial proteins and their cognate mRNAs in the cytosol. Additionally, our data highlight the importance of CLUH TPR domain for its interactions with both proteins and mRNAs. Overall, through the analysis of CLUH interactome, our study sheds a new light on CLUH molecular function by highlighting its association to the translation and subcellular localization of some mRNAs coding for mitochondrial proteins.

molecular biology↗

bioRxiv2020

Genome-wide CRISPR-Cas9 screen reveals common factors in dsRNA and Sindbis virus-induced cell death

Double stranded RNA (dsRNA) is the hallmark of many viral infections. dsRNA is produced either by RNA viruses during replication or by DNA viruses upon convergent transcription. Synthetic dsRNA is also able to mimic viral-induced activation of innate immune response and cell death. In this study, we employed a genome-wide CRISPR-Cas9 loss of function screen based on cell survival in order to identify genes implicated in the host response to dsRNA. By challenging HCT116 human cells with either synthetic dsRNA or Sindbis virus (SINV), we identified the heparan sulfate (HS) pathway as a crucial factor for dsRNA entry and we validated SINV dependency on HS. Interestingly, we uncovered a novel role for COG4, a component of the Conserved Oligomeric Golgi (COG) complex, as a factor involved in cell survival to both dsRNA and SINV in human cells. We showed that COG4 knock-out led to a decrease of extracellular HS, specifically affected dsRNA transfection efficiency and reduced viral production, explaining the increased cell survival of these mutants. ImportanceWhen facing a viral infection, the organism has to put in place a number of defense mechanisms in order to clear the pathogen from the cell. At the early phase of this preparation for fighting against the invader, the innate immune response is triggered by the sensing of danger signals. Among those molecular cues, double-stranded (dsRNA) is a very potent inducer of different reactions at the cellular level that can ultimately lead to cell death. Using a genome-wide screening approach, we set to identify genes involved in dsRNA entry, sensing and apoptosis induction in human cells. This allowed us to determine that the heparan sulfate pathway and the Conserved Oligomeric Golgi complex are key determinants allowing entry of both dsRNA and viral nucleic acid leading to cell death.

microbiology↗