Constance Ciaudo

Antiviral RNA Interference in Mammalian Cells

Viral Defenses In plants and invertebrates, RNA interference (RNAi) functions as an innate antiviral defense mechanism. Viruses that infect plants and invertebrates have evolved viral suppressors of RNAi (VSRs) that disable the RNAi pathway. Whether mammals use RNAi as a defense against viruses has been less clear (see the Perspective by Sagan and Sarnow). Li et al. (p. 231) and Maillard et al. (p. 235) studied mammalian cell lines and baby mice productively infected with RNA viruses and observed the production of virus-derived small RNAs (vsRNAs). When the putative VSR proteins of the infecting viruses were disabled, host RNAi-derived vsRNAs were much increased and the viruses were rapidly cleared and unable to mount a full-blown infection. Thus, RNAi also has an innate antiviral function in mammals. Certain mammalian cells can use RNA interference in the innate defense against invading viruses. [Also see Perspective by Sagan and Sarnow] In antiviral RNA interference (RNAi), the DICER enzyme processes virus-derived double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that guide ARGONAUTE proteins to silence complementary viral RNA. As a counterdefense, viruses deploy viral suppressors of RNAi (VSRs). Well-established in plants and invertebrates, the existence of antiviral RNAi remains unknown in mammals. Here, we show that undifferentiated mouse cells infected with encephalomyocarditis virus (EMCV) or Nodamura virus (NoV) accumulate ~22-nucleotide RNAs with all the signature features of siRNAs. These derive from viral dsRNA replication intermediates, incorporate into AGO2, are eliminated in Dicer knockout cells, and decrease in abundance upon cell differentiation. Furthermore, genetically ablating a NoV-encoded VSR that antagonizes DICER during authentic infections reduces NoV accumulation, which is rescued in RNAi-deficient mouse cells. We conclude that antiviral RNAi operates in mammalian cells.

RNAi-Dependent and Independent Control of LINE1 Accumulation and Mobility in Mouse Embryonic Stem Cells

At the request of the authors, PLOS Genetics is retracting this publication following an investigation into concerns that were raised regarding the assembly of Fig 4 and S4 Fig, and the statistical analysis used in Fig 2A. The text below has been agreed to by the authors and editors. The corresponding author, Olivier Voinnet, was originally alerted to errors that occurred during the assembly of Fig 4 (panel A) and S4 Fig (panels A and F). These errors have been corrected using the original raw data, and a correction notice was published accordingly. Further analysis of the paper revealed flaws in the interpretation of the transposition data presented in Fig 2A. In the originally submitted version, the L1 copy number was only presented for the DCR P10 and DCR P30 cells, and a T-test performed on the two datasets showed that the L1 copy number was statistically higher in DCR cells than in control cells. During the last stage of the review process, additional datasets were added and a second T-test was then used to establish the statistical analysis published in the final version of the paper. However, it was later realized that T-tests are not appropriate for comparing more than two datasets. At the recommendation of the ETH statistics helpdesk, a suitable Analysis of Variance (ANOVA) test with multiple comparisons was then conducted on the Dcr P30 and Dcr P30 datasets, providing a p-value of 0.0501, which is at the margin of the threshold of significance. The ANOVA test conducted on the Dcr P10 and Dcr P30 datasets revealed a statistically significant p-value of 0.0018. The statistical issue regarding the L1 copy number in DCR versus control ES cells is currently being addressed using a new set of cells and a direct GFP-based transposition assay. This issue will hopefully be clarified in the near future via the submission of an amended study for peer-review. Based on the present uncertainty revealed by the corrected statistical analysis of the L1 copy number—a key element of this paper—and on the previous errors in the figures, the authors have collectively decided to retract this study. Constance Ciaudo and Olivier Voinnet take full responsibility for the mistakes on this paper and wish to apologize. They also wish to state that none of the above-mentioned mistakes involved any of the co-authors from the Curie Institute, whose contributions to the paper were restricted to the bioinformatics analysis of small RNAs (NS, CJC, EB) and the generation of reagents including an ES cell line required for the study (EH, IO). All authors regret the inconvenience caused.

Dynamics in Transcriptomics: Advancements in RNA-seq Time Course and Downstream Analysis

Analysis of gene expression has contributed to a plethora of biological and medical research studies. Microarrays have been intensively used for the profiling of gene expression during diverse developmental processes, treatments and diseases. New massively parallel sequencing methods, often named as RNA-sequencing (RNA-seq) are extensively improving our understanding of gene regulation and signaling networks. Computational methods developed originally for microarrays analysis can now be optimized and applied to genome-wide studies in order to have access to a better comprehension of the whole transcriptome. This review addresses current challenges on RNA-seq analysis and specifically focuses on new bioinformatics tools developed for time series experiments. Furthermore, possible improvements in analysis, data integration as well as future applications of differential expression analysis are discussed.

Embryonic stem cell-specific microRNAs contribute to pluripotency by inhibiting regulators of multiple differentiation pathways

The findings that microRNAs (miRNAs) are essential for early development in many species and that embryonic miRNAs can reprogram somatic cells into induced pluripotent stem cells suggest that these miRNAs act directly on transcriptional and chromatin regulators of pluripotency. To elucidate the transcription regulatory networks immediately downstream of embryonic miRNAs, we extended the motif activity response analysis approach that infers the regulatory impact of both transcription factors (TFs) and miRNAs from genome-wide expression states. Applying this approach to multiple experimental data sets generated from mouse embryonic stem cells (ESCs) that did or did not express miRNAs of the ESC-specific miR-290-295 cluster, we identified multiple TFs that are direct miRNA targets, some of which are known to be active during cell differentiation. Our results provide new insights into the transcription regulatory network downstream of ESC-specific miRNAs, indicating that these miRNAs act on cell cycle and chromatin regulators at several levels and downregulate TFs that are involved in the innate immune response.

Generation of a Knockout Mouse Embryonic Stem Cell Line Using a Paired CRISPR/Cas9 Genome Engineering Tool

CRISPR/Cas9, originally discovered as a bacterial immune system, has recently been engineered into the latest tool to successfully introduce site-specific mutations in a variety of different organisms. Composed only of the Cas9 protein as well as one engineered guide RNA for its functionality, this system is much less complex in its setup and easier to handle than other guided nucleases such as Zinc-finger nucleases or TALENs.Here, we describe the simultaneous transfection of two paired CRISPR sgRNAs-Cas9 plasmids, in mouse embryonic stem cells (mESCs), resulting in the knockout of the selected target gene. Together with a four primer-evaluation system, it poses an efficient way to generate new independent knockout mouse embryonic stem cell lines.

Noncanonical function of DGCR8 controls mESC exit from pluripotency

Mouse embryonic stem cells (mESCs) deficient for DGCR8, a key component of the microprocessor complex, present strong differentiation defects. However, the exact reasons impairing their commitment remain elusive. The analysis of newly generated mutant mESCs revealed that DGCR8 is essential for the exit from the pluripotency state. To dissociate canonical versus noncanonical functions of DGCR8, we complemented the mutant mESCs with a phosphomutant DGCR8, which restored microRNA levels but did not rescue the exit from pluripotency defect. Integration of omics data and RNA immunoprecipitation experiments established DGCR8 as a direct interactor of Tcf7l1 mRNA, a core component of the pluripotency network. Finally, we found that DGCR8 facilitated the splicing of Tcf7l1, an event necessary for the differentiation of mESCs. Our data reveal a new noncanonical function of DGCR8 in the modulation of the alternative splicing of Tcf7l1 mRNA in addition to its established function in microRNA biogenesis.

Dicer, a new regulator of pluripotency exit and LINE‐1 elements in mouse embryonic stem cells

A gene regulation network orchestrates processes ensuring the maintenance of cellular identity and genome integrity. Small RNAs generated by the RNAse III DICER have emerged as central players in this network. Moreover, deletion of Dicer in mice leads to early embryonic lethality. To better understand the underlying mechanisms leading to this phenotype, we generated Dicer‐deficient mouse embryonic stem cells (mESCs). Their detailed characterization revealed an impaired differentiation potential, and incapacity to exit from the pluripotency state. We also observed a strong accumulation of LINE‐1 (L1s) transcripts, which was translated at protein level and led to an increased L1s retrotransposition. Our findings reveal Dicer as a new essential player that sustains mESCs self‐renewal and genome integrity by controlling L1s regulation.

Regulation of LINE-1 in mammals

Abstract Transposable elements (TEs) are mobile DNA elements that represent almost half of the human genome. Transposition of TEs has been implicated as a source of genome evolution and acquisition of new traits but also as an origin of diseases. The activity of these elements is therefore tightly regulated during the life cycle of each individual, and many recent discoveries involved the genetic and epigenetic mechanisms in their control. In this review, we present recent findings in this field of research, focusing on the case of one specific family of TEs: the long-interspersed nuclear elements-1 (LINE-1 or L1). LINE-1 elements are the most representative class of retrotransposons in mammalian genomes. We illustrate how these elements are conserved between mice and humans, and how they are regulated during the life cycle. Additionally, recent advances in genome-wide sequencing approaches allow us not only to better understand the regulation of LINE-1 but also highlight new issues specifically at the bioinformatics level. Therefore, we discuss the state of the art in analyzing such bioinformatics datasets to identify epigenetic regulators of repeated elements in the human genomes.

The Role of RNA Interference in Stem Cell Biology: Beyond the Mutant Phenotypes

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.

Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair

The CRISPR-Cas9 targeted nuclease technology allows the insertion of genetic modifications with single base-pair precision. The preference of mammalian cells to repair Cas9-induced DNA double-strand breaks via error-prone end-joining pathways rather than via homology-directed repair mechanisms, however, leads to relatively low rates of precise editing from donor DNA. Here we show that spatial and temporal co-localization of the donor template and Cas9 via covalent linkage increases the correction rates up to 24-fold, and demonstrate that the effect is mainly caused by an increase of donor template concentration in the nucleus. Enhanced correction rates were observed in multiple cell types and on different genomic loci, suggesting that covalently linking the donor template to the Cas9 complex provides advantages for clinical applications where high-fidelity repair is desired.