Constance Ciaudo

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Ciaudo

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Constance

ORCID

0000-0002-0857-4506

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Publications 1 - 10 of 20

Translation is required for miRNA‐dependent decay of endogenous transcripts
Biasini, Adriano; Abdulkarim, Baroj; de Pretis, Stefano; et al. (2021)
The EMBO Journal
Post-transcriptional repression of gene expression by miRNAs occurs through transcript destabilization or translation inhibition. mRNA decay is known to account for most miRNA-dependent repression. However, because transcript decay occurs co-translationally, whether target translation is a requirement for miRNA-dependent transcript destabilization remains unknown. To decouple these two molecular processes, we used cytosolic long noncoding RNAs (lncRNAs) as models for endogenous transcripts that are not translated. We show that, despite interacting with the miRNA-loaded RNA-induced silencing complex, the steady-state abundance and decay rates of these transcripts are minimally affected by miRNA loss. To further validate the apparent requirement of translation for miRNA-dependent decay, we fused two lncRNA candidates to the 3'-end of a protein-coding gene reporter and found this results in their miRNA-dependent destabilization. Further analysis revealed that the few natural lncRNAs whose levels are regulated by miRNAs in mESCs tend to associate with translating ribosomes, and possibly represent misannotated micropeptides, further substantiating the necessity of target translation for miRNA-dependent transcript decay. In summary, our analyses suggest that translation is required for miRNA-dependent transcript destabilization, and demonstrate that the levels of coding and noncoding transcripts are differently affected by miRNAs.
Inhibition of FGF and TGF-β Pathways in hESCs Identify STOX2 as a Novel SMAD2/4 Cofactor
Renz, Peter F.; Spies, Daniel; Tsikrika, Panagiota; et al. (2020)
Biology
The fibroblast growth factor (FGF) and the transforming growth factor-β (TGF-β) pathways are both involved in the maintenance of human embryonic stem cells (hESCs) and regulate the onset of their differentiation. Their converging functions have suggested that these pathways might share a wide range of overlapping targets. Published studies have focused on the long-term effects (24–48 h) of FGF and TGF-β inhibition in hESCs, identifying direct and indirect target genes. In this study, we focused on the earliest transcriptome changes occurring between 3 and 9 h after FGF and TGF-β inhibition to identify direct target genes only. Our analysis clearly shows that only a handful of target transcripts are common to both pathways. This is surprising in light of the previous literature, and has implications for models of cell signaling in human pluripotent cells. In addition, we identified STOX2 as a novel primary target of the TGF-β signaling pathway. We show that STOX2 might act as a novel SMAD2/4 cofactor. Taken together, our results provide insights into the effect of cell signaling on the transcription profile of human pluripotent cells.
Fast In Vitro Procedure to Identify Extraembryonic Differentiation Defect of Mouse Embryonic Stem Cells
Ngondo, Richard P.; Cohen-Tannoudji, Michel; Ciaudo, Constance (2020)
STAR Protocols
Mouse embryonic stem cells (mESCs) are a powerful model to study early mouse development. These blastocyst-derived cells can maintain pluripotency and differentiate into the three embryonic germ layers and an extraembryonic layer, the extraembryonic endoderm (ExEn), which shares similar molecular markers to the definitive endoderm. Here, we present a fast procedure to identify a differentiation defect of mESCs toward ExEn in vitro through the molecular and cellular characterization of embryoid bodies, followed by direct differentiation of mESCs into ExEn.
Argonaute 2 Is Required for Extra-embryonic Endoderm Differentiation of Mouse Embryonic Stem Cells
Ngondo, Richard P.; Cirera-Salinas, Daniel; Yu, Jian; et al. (2018)
Stem Cell Reports
In mouse, although four Argonaute (AGO) proteins with partly overlapping functions in small-RNA pathways exist, only Ago2 deficiency causes embryonic lethality. To investigate the role of AGO2 during mouse early development, we generated Ago2-deficient mouse embryonic stem cells (mESCs) and performed a detailed characterization of their differentiation potential. Ago2 disruption caused a global reduction of microRNAs, which resulted in the misregulation of only a limited number of transcripts. We demonstrated, both in vivo and in vitro, that AGO2 is dispensable for the embryonic germ-layer formation. However, Ago2-deficient mESCs showed a specific defect during conversion into extra-embryonic endoderm cells. We proved that this defect is cell autonomous and can be rescued by both a catalytically active and an inactive Ago2, but not by Ago2 deprived of its RNA binding capacity or by Ago1 overexpression. Overall, our results suggest a role for AGO2 in stem cell differentiation.
The solution structure of Dead End bound to AU-rich RNA reveals an unusual mode of tandem RRM-RNA recognition required for mRNA regulation
Duszczyk, Malgorzata M.; Wischnewski, Harry; Kazeeva, Tamara; et al. (2022)
Nature Communications
Dead End (DND1) is an RNA-binding protein essential for germline development through its role in post-transcriptional gene regulation. The molecular mechanisms behind selection and regulation of its targets are unknown. Here, we present the solution structure of DND1's tandem RNA Recognition Motifs (RRMs) bound to AU-rich RNA. The structure reveals how an NYAYUNN element is specifically recognized, reconciling seemingly contradictory sequence motifs discovered in recent genome-wide studies. RRM1 acts as a main binding platform, including atypical extensions to the canonical RRM fold. RRM2 acts cooperatively with RRM1, capping the RNA using an unusual binding pocket, leading to an unusual mode of tandem RRM-RNA recognition. We show that the consensus motif is sufficient to mediate upregulation of a reporter gene in human cells and that this process depends not only on RNA binding by the RRMs, but also on DND1's double-stranded RNA binding domain (dsRBD), which is dispensable for binding of a subset of targets in cellulo. Our results point to a model where DND1 target selection is mediated by a non-canonical mode of AU-rich RNA recognition by the tandem RRMs and a role for the dsRBD in the recruitment of effector complexes responsible for target regulation.
Prediction of the miRNA interactome – Established methods and upcoming perspectives
Schäfer, Moritz; Ciaudo, Constance (2020)
Computational and Structural Biotechnology Journal
MicroRNAs (miRNAs) are well-studied small noncoding RNAs involved in post-transcriptional gene regulation in a wide range of organisms, including mammals. Their function is mediated by base pairing with their target RNAs. Although many features required for miRNA-mediated repression have been described, the identification of functional interactions is still challenging. In the last two decades, numerous Machine Learning (ML) models have been developed to predict their putative targets. In this review, we summarize the biological knowledge and the experimental data used to develop these ML models. Recently, Deep Neural Network-based models have also emerged in miRNA interaction modeling. We thus outline established and emerging models to give a perspective on the future developments needed to improve the identification of genes directly regulated by miRNAs.
Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair
Savic, Natasa; Ringnalda, Femke C.A.S.; Lindsay, Helen; et al. (2018)
eLife
Argonaute proteins regulate a specific network of genes through KLF4 in mouse embryonic stem cells
Müller, Madlen; Schaefer, Moritz; Fäh, Tara; et al. (2022)
Stem Cell Reports
The Argonaute proteins (AGOs) are well known for their role in post-transcriptional gene silencing in the microRNA (miRNA) pathway. Here we show that in mouse embryonic stem cells, AGO1&2 serve additional functions that go beyond the miRNA pathway. Through the combined deletion of both Agos, we identified a specific set of genes that are uniquely regulated by AGOs but not by the other miRNA biogenesis factors. Deletion of Ago2&1 caused a global reduction of the repressive histone mark H3K27me3 due to downregulation at protein levels of Polycomb repressive complex 2 components. By integrating chromatin accessibility, prediction of transcription factor binding sites, and chromatin immunoprecipitation sequencing data, we identified the pluripotency factor KLF4 as a key modulator of AGO1&2-regulated genes. Our findings revealed a novel axis of gene regulation that is mediated by noncanonical functions of AGO proteins that affect chromatin states and gene expression using mechanisms outside the miRNA pathway.
The Role of RNA Interference in Stem Cell Biology: Beyond the Mutant Phenotypes
Bodak, Maxime; Cirera-Salinas, Daniel; Luitz, Janina; et al. (2017)
Journal of Molecular Biology
Complex gene regulation systems ensure the maintenance of cellular identity during early development in mammals. Eukaryotic small RNAs have emerged as critical players in RNA interference (RNAi) by mediating gene silencing during embryonic stem cell self-renewal. Most of the proteins involved in the biogenesis of small RNAs are essential for proliferation and differentiation into the three germ layers of mouse embryonic stem cells. In the last decade, new functions for some RNAi proteins, independent of their roles in RNAi pathways, have been demonstrated in different biological systems. In parallel, new concepts in stem cell biology have emerged. Here, we review and integrate the current understanding of how RNAi proteins regulate stem cell identity with the new advances in the stem cell field and the recent non-canonical functions of the RNAi proteins. Finally, we propose a reevaluation of all RNAi mutant phenotypes, as non-canonical (small non-coding RNA independent) functions may contribute to the molecular mechanisms governing mouse embryonic stem cells commitment.
Vector Integration Sites Identification for Gene-Trap Screening in Mammalian Haploid Cells
Yu, Jian; Ciaudo, Constance (2017)
Scientific Reports
Forward genetic screens using retroviral (or transposon) gene-trap vectors in a haploid genome revolutionized the investigation of molecular networks in mammals. However, the sequencing data generated by Phenotypic interrogation followed by Tag sequencing (PhiT-seq) were not well characterized. The analysis of human and mouse haploid screens allowed us to describe PhiT-seq data and to define quality control steps. Moreover, we identified several blind spots in both haploid genomes where gene-trap vectors can hardly integrate. Integration of transcriptomic data improved the performance of candidate gene identification. Furthermore, we experimented with various statistical tests to account for biological replicates in PhiT-seq and investigated the effect of normalization methods and other parameters on the performance. Finally, we developed: VISITs, a dedicated pipeline for analyzing PhiT-seq data

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Constance Ciaudo

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