Hepatitis C virus RNA is 5'-capped with flavin adenine dinucleotide.

RNA viruses have evolved elaborate strategies to protect their genomes, including 5' capping. However, until now no RNA 5' cap has been identified for hepatitis C virus1,2 (HCV), which causes chronic infection, liver cirrhosis and cancer3. Here we demonstrate that the cellular metabolite flavin adenine dinucleotide (FAD) is used as a non-canonical initiating nucleotide by the viral RNA-dependent RNA polymerase, resulting in a 5'-FAD cap on the HCV RNA. The HCV FAD-capping frequency is around 75%, which is the highest observed for any RNA metabolite cap across all kingdoms of life4-8. FAD capping is conserved among HCV isolates for the replication-intermediate negative strand and partially for the positive strand. It is also observed in vivo on HCV RNA isolated from patient samples and from the liver and serum of a human liver chimeric mouse model. Furthermore, we show that 5'-FAD capping protects RNA from RIG-I mediated innate immune recognition but does not stabilize the HCV RNA. These results establish capping with cellular metabolites as a novel viral RNA-capping strategy, which could be used by other viruses and affect anti-viral treatment outcomes and persistence of infection.

Characterising the RNA-binding protein atlas of the mammalian brain uncovers RBM5 misregulation in mouse models of Huntington's disease.

RNA-binding proteins (RBPs) are key players regulating RNA processing and are associated with disorders ranging from cancer to neurodegeneration. Here, we present a proteomics workflow for large-scale identification of RBPs and their RNA-binding regions in the mammalian brain identifying 526 RBPs. Analysing brain tissue from males of the Huntington's disease (HD) R6/2 mouse model uncovered differential RNA-binding of the alternative splicing regulator RBM5. Combining several omics workflows, we show that RBM5 binds differentially to transcripts enriched in pathways of neurodegeneration in R6/2 brain tissue. We further find these transcripts to undergo changes in splicing and demonstrate that RBM5 directly regulates these changes in human neurons derived from embryonic stem cells. Finally, we reveal that RBM5 interacts differently with several known huntingtin interactors and components of huntingtin aggregates. Collectively, we demonstrate the applicability of our method for capturing RNA interactor dynamics in the contexts of tissue and disease.

Differential activity of nucleotide analogs against tick-borne encephalitis and yellow fever viruses in human cell lines.

With no approved antiviral therapies, the continuous emergence and re-emergence of tick-borne encephalitis virus (TBEV) and yellow fever virus (YFV) is a rising concern. We performed head-to-head comparisons of the antiviral activity of available nucleos(t)ide analogs (nucs) using relevant human cell lines. Eight existing nucs inhibited TBEV and/or YFV with differential activity between cell lines and viruses. Remdesivir, uprifosbuvir and sofosbuvir were the most potent drugs against TBEV and YFV in liver cells, but they had reduced activity in neural cells, whereas galidesivir retained uniform activity across cell lines and viruses. Ribavirin, valopicitabine, molnupiravir and GS-6620 exhibited only moderate antiviral activity. We found antiviral activity for drugs previously reported as inactive, demonstrating the importance of using human cell lines and comparative experimental assays when screening the activity of nucs. The relatively high antiviral activity of remdesivir, sofosbuvir and uprifosbuvir against TBEV and YFV merits further investigation in clinical studies.

Identification of the viral and cellular microRNA interactomes during SARS-CoV-2 infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has had a tremendous impact worldwide. Mapping virus-host interactions is critical to understand disease progression. MicroRNAs (miRNAs) are important RNA regulators, but their interaction with SARS-CoV-2 RNA was not experimentally investigated. Here, using Argonaute (AGO) cross-linking immunoprecipitation combined with RNA proximity ligation (CLEAR-CLIP), we provide unbiased mapping of SARS-CoV-2/miRNA interactions. We identified six main regions on the viral RNA bound primarily by one specific miRNA. Targeted mutagenesis and AGO1-3 knockdown demonstrated that these interactions are not critical for virus production. Moreover, we identified perturbed regulation of cellular miRNA interactions during infection, including non-compensated viral sequestration of the miR-15 family. Transcriptome analysis further showed that mRNAs targeted by this miRNA family are derepressed. This work delineates the interphase between miRNA regulation and SARS-CoV-2 infection and further contributes to deciphering the full molecular interactome of this virus.

A cis-regulatory-directed pipeline for the identification of genes involved in cardiac development and disease.

BACKGROUND: Congenital heart diseases are the major cause of death in newborns, but the genetic etiology of this developmental disorder is not fully known. The conventional approach to identify the disease-causing genes focuses on screening genes that display heart-specific expression during development. However, this approach would have discounted genes that are expressed widely in other tissues but may play critical roles in heart development. RESULTS: We report an efficient pipeline of genome-wide gene discovery based on the identification of a cardiac-specific cis-regulatory element signature that points to candidate genes involved in heart development and congenital heart disease. With this pipeline, we retrieve 76% of the known cardiac developmental genes and predict 35 novel genes that previously had no known connectivity to heart development. Functional validation of these novel cardiac genes by RNAi-mediated knockdown of the conserved orthologs in Drosophila cardiac tissue reveals that disrupting the activity of 71% of these genes leads to adult mortality. Among these genes, RpL14, RpS24, and Rpn8 are associated with heart phenotypes. CONCLUSIONS: Our pipeline has enabled the discovery of novel genes with roles in heart development. This workflow, which relies on screening for non-coding cis-regulatory signatures, is amenable for identifying developmental and disease genes for an organ without constraining to genes that are expressed exclusively in the organ of interest.

TWIST1 and chromatin regulatory proteins interact to guide neural crest cell differentiation.

Protein interaction is critical molecular regulatory activity underlining cellular functions and precise cell fate choices. Using TWIST1 BioID-proximity-labeling and network propagation analyses, we discovered and characterized a TWIST-chromatin regulatory module (TWIST1-CRM) in the neural crest cells (NCC). Combinatorial perturbation of core members of TWIST1-CRM: TWIST1, CHD7, CHD8, and WHSC1 in cell models and mouse embryos revealed that loss of the function of the regulatory module resulted in abnormal differentiation of NCCs and compromised craniofacial tissue patterning. Following NCC delamination, low level of TWIST1-CRM activity is instrumental to stabilize the early NCC signatures and migratory potential by repressing the neural stem cell programs. High level of TWIST1 module activity at later phases commits the cells to the ectomesenchyme. Our study further revealed the functional interdependency of TWIST1 and potential neurocristopathy factors in NCC development.

Shaping the head and face during development relies on a complex ballet of molecular signals that orchestrates the movement and specialization of various groups of cells. In animals with a backbone for example, neural crest cells (NCCs for short) can march long distances from the developing spine to become some of the tissues that form the skull and cartilage but also the pigment cells and nervous system. NCCs mature into specific cell types thanks to a complex array of factors which trigger a precise sequence of binary fate decisions at the right time and place. Amongst these factors, the protein TWIST1 can set up a cascade of genetic events that control how NCCs will ultimately form tissues in the head. To do so, the TWIST1 protein interacts with many other molecular actors, many of which are still unknown. To find some of these partners, Fan et al. studied TWIST1 in the NCCs of mice and cells grown in the lab. The experiments showed that TWIST1 interacted with CHD7, CHD8 and WHSC1, three proteins that help to switch genes on and off, and which contribute to NCCs moving across the head during development. Further work by Fan et al. then revealed that together, these molecular actors are critical for NCCs to form cells that will form facial bones and cartilage, as opposed to becoming neurons. This result helps to show that there is a trade-off between NCCs forming the face or being part of the nervous system. One in three babies born with a birth defect shows anomalies of the head and face: understanding the exact mechanisms by which NCCs contribute to these structures may help to better predict risks for parents, or to develop new approaches for treatment.

Evolutionary selection of pestivirus variants with altered or no microRNA dependency.

Host microRNA (miRNA) dependency is a hallmark of the human pathogen hepatitis C virus (HCV) and was also described for the related pestiviruses, which are important livestock pathogens. The liver-specific miR-122 binds within the HCV 5' untranslated region (UTR), whereas the broadly expressed let-7 and miR-17 families bind two sites (S1 and S2, respectively) in the pestiviral 3' UTR. Here, we dissected the mechanism of miRNA dependency of the pestivirus bovine viral diarrhea virus (BVDV). Argonaute 2 (AGO2) and miR-17 binding were essential for viral replication, whereas let-7 binding was mainly required for full translational efficiency. Furthermore, using seed site randomized genomes and evolutionary selection experiments, we found that tropism could be redirected to different miRNAs. AGO cross-linking and immunoprecipitation (CLIP) experiments and miRNA antagonism demonstrated that these alternative variants bound and depended on the corresponding miRNAs. Interestingly, we also identified miRNA-independent variants that were obtained through acquisition of compensatory mutations near the genomic 3' terminus. Rescue experiments demonstrated that miRNA binding and 3' mutagenesis contribute to replication through mutually exclusive mechanisms. Altogether, our findings suggest that pestiviruses, although capable of miRNA-independent replication, took advantage of miRNAs as essential host factors, suggesting a favorable path during evolutionary adaptation.

TWIST1 Homodimers and Heterodimers Orchestrate Lineage-Specific Differentiation.

The extensive array of basic helix-loop-helix (bHLH) transcription factors and their combinations as dimers underpin the diversity of molecular function required for cell type specification during embryogenesis. The bHLH factor TWIST1 plays pleiotropic roles during development. However, which combinations of TWIST1 dimers are involved and what impact each dimer imposes on the gene regulation network controlled by TWIST1 remain elusive. In this work, proteomic profiling of human TWIST1-expressing cell lines and transcriptome analysis of mouse cranial mesenchyme have revealed that TWIST1 homodimers and heterodimers with TCF3, TCF4, and TCF12 E-proteins are the predominant dimer combinations. Disease-causing mutations in TWIST1 can impact dimer formation or shift the balance of different types of TWIST1 dimers in the cell, which may underpin the defective differentiation of the craniofacial mesenchyme. Functional analyses of the loss and gain of TWIST1-E-protein dimer activity have revealed previously unappreciated roles in guiding lineage differentiation of embryonic stem cells: TWIST1-E-protein heterodimers activate the differentiation of mesoderm and neural crest cells, which is accompanied by the epithelial-to-mesenchymal transition. At the same time, TWIST1 homodimers maintain the stem cells in a progenitor state and block entry to the endoderm lineage.

Ularcirc: visualization and enhanced analysis of circular RNAs via back and canonical forward splicing.

Circular RNAs (circRNA) are a unique class of transcripts that can only be identified from sequence alignments spanning discordant junctions, commonly referred to as backsplice junctions (BSJ). Canonical splicing is also linked with circRNA biogenesis either from the parental transcript or internal to the circRNA, and is not fully utilized in circRNA software. Here we present Ularcirc, a software tool that integrates the visualization of both BSJ and forward splicing junctions and provides downstream analysis of selected circRNA candidates. Ularcirc utilizes the output of CIRI, circExplorer, or raw chimeric output of the STAR aligner and assembles BSJ count table to allow multi-sample analysis. We used Ularcirc to identify and characterize circRNA from public and in-house generated data sets and demonstrate how it can be used to (i) discover novel splicing patterns of parental transcripts, (ii) detect internal splicing patterns of circRNA, and (iii) reveal the complexity of BSJ formation. Furthermore, we identify circRNA that have potential open reading frames longer than their linear sequence. Finally, we detected and validated the presence of a novel class of circRNA generated from ApoA4 transcripts whose BSJ derive from multiple non-canonical splicing sites within coding exons. Ularcirc is accessed via https://github.com/VCCRI/Ularcirc.

Dynamics of Wnt activity on the acquisition of ectoderm potency in epiblast stem cells.

During embryogenesis, the stringent regulation of Wnt activity is crucial for the morphogenesis of the head and brain. The loss of function of the Wnt inhibitor Dkk1 results in elevated Wnt activity, loss of ectoderm lineage attributes from the anterior epiblast, and the posteriorisation of anterior germ layer tissue towards the mesendoderm. The modulation of Wnt signalling may therefore be crucial for the allocation of epiblast cells to ectoderm progenitors during gastrulation. To test this hypothesis, we examined the lineage characteristics of epiblast stem cells (EpiSCs) that were derived and maintained under different signalling conditions. We showed that suppression of Wnt activity enhanced the ectoderm propensity of the EpiSCs. Neuroectoderm differentiation of these EpiSCs was further empowered by the robust re-activation of Wnt activity. Therefore, during gastrulation, the tuning of the signalling activities that mediate mesendoderm differentiation is instrumental for the acquisition of ectoderm potency in the epiblast.

Functional Interplay between RNA Viruses and Non-Coding RNA in Mammals.

Exploring virus⁻host interactions is key to understand mechanisms regulating the viral replicative cycle and any pathological outcomes associated with infection. Whereas interactions at the protein level are well explored, RNA interactions are less so. Novel sequencing methodologies have helped uncover the importance of RNA⁻protein and RNA⁻RNA interactions during infection. In addition to messenger RNAs (mRNAs), mammalian cells express a great number of regulatory non-coding RNAs, some of which are crucial for regulation of the immune system whereas others are utilized by viruses. It is thus becoming increasingly clear that RNA interactions play important roles for both sides in the arms race between virus and host. With the emerging field of RNA therapeutics, such interactions are promising antiviral targets. In this review, we discuss direct and indirect RNA interactions occurring between RNA viruses or retroviruses and host non-coding transcripts upon infection. In addition, we review RNA virus derived non-coding RNAs affecting immunological and metabolic pathways of the host cell typically to provide an advantage to the virus. The relatively few known examples of virus⁻host RNA interactions suggest that many more await discovery.

A gene regulatory network anchored by LIM homeobox 1 for embryonic head development.

Development of the embryonic head is driven by the activity of gene regulatory networks of transcription factors. LHX1 is a homeobox transcription factor that plays an essential role in the formation of the embryonic head. The loss of LHX1 function results in anterior truncation of the embryo caused by the disruption of morphogenetic movement of tissue precursors and the dysregulation of WNT signaling activity. Profiling the gene expression pattern in the Lhx1 mutant embryo revealed that tissues in anterior germ layers acquire posterior tissue characteristics, suggesting LHX1 activity is required for the allocation and patterning of head precursor tissues. Here, we used LHX1 as an entry point to delineate its transcriptional targets and interactors and construct a LHX1-anchored gene regulatory network. Using a gain-of-function approach, we identified genes that immediately respond to Lhx1 activation. Meta-analysis of the datasets of LHX1-responsive genes and genes expressed in the anterior tissues of mouse embryos at head-fold stage, in conjunction with published Xenopus embryonic LHX1 (Xlim1) ChIP-seq data, has pinpointed the putative transcriptional targets of LHX1 and an array of genetic determinants functioning together in the formation of the mouse embryonic head.

Formation of the Embryonic Head in the Mouse: Attributes of a Gene Regulatory Network.

The embryonic head is the first major body part to be constructed during embryogenesis. The allocation and the assembly of the progenitor tissues, which start at gastrulation, are accompanied by the spatiotemporal activity of transcription factors and signaling pathways that drives lineage specification, germ layer formation, and cell/tissue movement. The morphogenesis, regionalization, and patterning of the brain and craniofacial structures rely on the function of LIM-domain, homeodomain, and basic helix-loop-helix transcription factors. These factors constitute the central nodes of a gene regulatory network (GRN) which encompasses and intersects with signaling pathways involved with head formation. It is predicted that the functional output of this "head GRN" impacts on cellular function and cell-cell interactions that are essential for lineage differentiation and tissue modeling, which are key processes underpinning the formation of the head.

Conditional restoration and inactivation of Rbm47 reveal its tissue-context requirement for viability and growth.

Rbm47 encodes a RNA binding protein that is necessary for Cytidine to Uridine RNA editing. Rbm47(gt/gt) mutant mice that harbor inactivated Rbm47 display poor viability. Here it was determined that the loss of Rbm47(gt/gt) offspring is due to embryonic lethality at mid-gestation. It was further showed that growth of the surviving Rbm47(gt/gt) mutants is impaired. Rbm47 is expressed in both the visceral endoderm and the definitive endoderm. Using the utility of the switchable FlEx gene-trap cassette and the activity of Cre and FLP recombinases to generate mice that conditionally inactivate and restore Rbm47 function in tissue-specific manner, it was demonstrated that Rbm47 function is required in the embryo proper, and not the visceral endoderm, for viability and growth. genesis 54:115-122, 2016. © 2016 Wiley Periodicals, Inc.

Generation of genome-edited mouse epiblast stem cells via a detour through ES cell-chimeras.

Conventionally, mouse epiblast stem cells (EpiSCs) are derived directly from the epiblast or ectoderm germ layer of the post-implantation embryo. Self-renewing and multipotent EpiSC-like stem cells can also be derived by the conversion of embryonic stem cells (ESCs) via the provision of culture conditions that enable the maintenance of the EpiSCs. Here, we outline an experimental procedure for deriving EpiSCs from post-implantation chimeric embryos that are generated using genome-edited ESCs. This strategy enables the production of EpiSCs where (i) no genetically modified animals or ESCs are available, (ii) the impact of the genetic modification on post-implantation development, which may influence the property of the EpiSCs, is requisite knowledge for using the EpiSC for a specific investigation, and (iii) multiple editing of the genome is desirable to modify the biological attributes of the EpiSCs for studying, for example, the gene network activity on the trajectory of lineage differentiation and tissue morphogenesis.

Context-specific function of the LIM homeobox 1 transcription factor in head formation of the mouse embryo.

Lhx1 encodes a LIM homeobox transcription factor that is expressed in the primitive streak, mesoderm and anterior mesendoderm of the mouse embryo. Using a conditional Lhx1 flox mutation and three different Cre deleters, we demonstrated that LHX1 is required in the anterior mesendoderm, but not in the mesoderm, for formation of the head. LHX1 enables the morphogenetic movement of cells that accompanies the formation of the anterior mesendoderm, in part through regulation of Pcdh7 expression. LHX1 also regulates, in the anterior mesendoderm, the transcription of genes encoding negative regulators of WNT signalling, such as Dkk1, Hesx1, Cer1 and Gsc. Embryos carrying mutations in Pcdh7, generated using CRISPR-Cas9 technology, and embryos without Lhx1 function specifically in the anterior mesendoderm displayed head defects that partially phenocopied the truncation defects of Lhx1-null mutants. Therefore, disruption of Lhx1-dependent movement of the anterior mesendoderm cells and failure to modulate WNT signalling both resulted in the truncation of head structures. Compound mutants of Lhx1, Dkk1 and Ctnnb1 show an enhanced head truncation phenotype, pointing to a functional link between LHX1 transcriptional activity and the regulation of WNT signalling. Collectively, these results provide comprehensive insight into the context-specific function of LHX1 in head formation: LHX1 enables the formation of the anterior mesendoderm that is instrumental for mediating the inductive interaction with the anterior neuroectoderm and LHX1 also regulates the expression of factors in the signalling cascade that modulate the level of WNT activity.

Head formation: OTX2 regulates Dkk1 and Lhx1 activity in the anterior mesendoderm.

The Otx2 gene encodes a paired-type homeobox transcription factor that is essential for the induction and the patterning of the anterior structures in the mouse embryo. Otx2 knockout embryos fail to form a head. Whereas previous studies have shown that Otx2 is required in the anterior visceral endoderm and the anterior neuroectoderm for head formation, its role in the anterior mesendoderm (AME) has not been assessed specifically. Here, we show that tissue-specific ablation of Otx2 in the AME phenocopies the truncation of the embryonic head of the Otx2 null mutant. Expression of Dkk1 and Lhx1, two genes that are also essential for head formation, is disrupted in the AME of the conditional Otx2-deficient embryos. Consistent with the fact that Dkk1 is a direct target of OTX2, we showed that OTX2 can interact with the H1 regulatory region of Dkk1 to activate its expression. Cross-species comparative analysis, RT-qPCR, ChIP-qPCR and luciferase assays have revealed two conserved regions in the Lhx1 locus to which OTX2 can bind to activate Lhx1 expression. Abnormal development of the embryonic head in Otx2;Lhx1 and Otx2;Dkk1 compound mutant embryos highlights the functional intersection of Otx2, Dkk1 and Lhx1 in the AME for head formation.

C to U RNA editing mediated by APOBEC1 requires RNA-binding protein RBM47.

Cytidine (C) to Uridine (U) RNA editing is a post-transcriptional modification that is accomplished by the deaminase APOBEC1 and its partnership with the RNA-binding protein A1CF. We identify and characterise here a novel RNA-binding protein, RBM47, that interacts with APOBEC1 and A1CF and is expressed in tissues where C to U RNA editing occurs. RBM47 can substitute for A1CF and is necessary and sufficient for APOBEC1-mediated editing in vitro. Editing is further impaired in Rbm47-deficient mutant mice. These findings suggest that RBM47 and APOBEC1 constitute the basic machinery for C to U RNA editing.

Purkinje cells and Bergmann glia are primary targets of the TRα1 thyroid hormone receptor during mouse cerebellum postnatal development.

Thyroid hormone is necessary for normal development of the central nervous system, as shown by the severe mental retardation syndrome affecting hypothyroid patients with low levels of active thyroid hormone. The postnatal defects observed in hypothyroid mouse cerebellum are recapitulated in mice heterozygous for a dominant-negative mutation of Thra, the gene encoding the ubiquitous TRα1 receptor. Using CRE/loxP-mediated conditional expression approach, we found that this mutation primarily alters the differentiation of Purkinje cells and Bergmann glia, two cerebellum-specific cell types. These primary defects indirectly affect cerebellum development in a global manner. Notably, the inward migration and terminal differentiation of granule cell precursors is impaired. Therefore, despite the broad distribution of its receptors, thyroid hormone targets few cell types that exert a predominant role in the network of cellular interactions that govern normal cerebellum maturation.