scholarly journals High-Throughput Fluorescence-Based Screen Identifies the Neuronal MicroRNA miR-124 as a Positive Regulator of Alphavirus Infection

2020 ◽  
Vol 94 (9) ◽  
Author(s):  
Paula López ◽  
Erika Girardi ◽  
Bryan C. Mounce ◽  
Amélie Weiss ◽  
Béatrice Chane-Woon-Ming ◽  
...  
Keyword(s):  
Binding Site ◽  
Human Cells ◽  
Viral Rna ◽  
Positive Regulation ◽  
Cellular Mirnas ◽  
Positive Regulator ◽  
Genome Wide ◽  
A Genome ◽  
Arbovirus Infection ◽  
Alphavirus Infection

ABSTRACT MicroRNAs (miRNAs) are small regulatory RNAs which act by modulating the expression of target genes. In addition to their role in maintaining essential physiological functions in the cell, miRNAs can also regulate viral infections. They can do so directly by targeting RNAs of viral origin or indirectly by targeting host mRNAs, and this can result in a positive or negative outcome for the virus. Here, we performed a fluorescence-based miRNA genome-wide screen in order to identify cellular miRNAs involved in the regulation of arbovirus infection in human cells. We identified 16 miRNAs showing a positive effect on Sindbis virus (SINV) expressing green fluorescent protein (GFP), among which were a number of neuron-specific ones such as miR-124. We confirmed that overexpression of miR-124 increases both SINV structural protein translation and viral production and that this effect is mediated by its seed sequence. We further demonstrated that the SINV genome possesses a binding site for miR-124. Both inhibition of miR-124 and silent mutations to disrupt this binding site in the viral RNA abolished positive regulation. We also proved that miR-124 inhibition reduces SINV infection in human differentiated neuronal cells. Finally, we showed that the proviral effect of miR-124 is conserved in other alphaviruses, as its inhibition reduces chikungunya virus (CHIKV) production in human cells. Altogether, our work expands the panel of positive regulation of the viral cycle by direct binding of host miRNAs to the viral RNA and provides new insights into the role of cellular miRNAs as regulators of alphavirus infection. IMPORTANCE Arthropod-borne (arbo) viruses are part of a class of pathogens that are transmitted to their final hosts by insects. Because of climate change, the habitat of some of these insects, such as mosquitoes, is shifting, thereby facilitating the emergence of viral epidemics. Among the pathologies associated with arbovirus infection, neurological diseases such as meningitis and encephalitis represent a significant health burden. Using a genome-wide miRNA screen, we identified neuronal miR-124 as a positive regulator of the Sindbis and chikungunya alphaviruses. We also showed that this effect was in part direct, thereby opening novel avenues to treat alphavirus infections.

scholarly journals High-throughput fluorescence-based screen identifies the neuronal microRNA miR-124 as a positive regulator of alphavirus infection

2019 ◽  
Author(s):  
Paula López ◽  
Erika Girardi ◽  
Bryan C. Mounce ◽  
Amélie Weiss ◽  
Béatrice Chane-Woon-Ming ◽  
...  
Keyword(s):  
Binding Site ◽  
Human Cells ◽  
Viral Rna ◽  
Viral Production ◽  
Positive Regulation ◽  
Cellular Mirnas ◽  
Positive Regulator ◽  
Genome Wide ◽  
A Genome ◽  
Alphavirus Infection

ABSTRACTMicro (mi)RNAs are small regulatory RNAs, which act as guide for the RISC complex to modulate the expression of target genes. In addition to their role in maintaining essential physiological functions in the cell, miRNAs can also regulate viral infections. They can do so directly by targeting RNAs of viral origin or indirectly by targeting RNAs from the host and this can result in a positive or negative outcome for the virus. Here, we performed a miRNA genome-wide screen in order to identify cellular miRNAs involved in the regulation of arbovirus infection in human cells. We identified sixteen miRNAs showing a positive effect on the virus, among which a number of neuron-specific ones such as miR-124. We confirmed that overexpression of miR-124 increases Sindbis virus (SINV) viral production and that this effect is mediated by its seed sequence. We further demonstrated that the SINV genome possesses a binding site for miR-124-3p. Both inhibition of miR-124-3p or silent mutations to disrupt this binding site in the viral RNA abolished the positive regulation. We also proved that miR-124 inhibition reduces SINV infection in human differentiated neuronal cells. Finally, we showed that the proviral effect of miR-124 is conserved for other medically relevant alphaviruses. Indeed, inhibition of miR-124 expression reduces chikungunya virus (CHIKV) viral production in human cells. Altogether, our work expands the panel of positive regulation of the viral cycle by direct binding of host miRNAs to the viral RNA and provides new insights into the role of cellular miRNAs as regulators of alphavirus infection.SIGNIFICANCE STATEMENTArthropod-borne (arbo) viruses are part of a class of pathogens that are transmitted to their final hosts by insects. Because of climate change, the habitat of some of these insects, such as mosquitoes, is shifting, thereby facilitating the emergence of viral epidemics. Among the pathologies associated with arboviruses infection, neurological diseases like meningitis or encephalitis represent a significant health burden. Using a genome-wide miRNA screen, we identified the neuronal miR-124 as a positive regulator of the Sindbis and chikungunya alphaviruses. We also showed that this effect was in part direct, thereby opening novel avenues to treat alphaviruses infection.

scholarly journals Timing Polymerase Pausing with TV-PRO-seq

2018 ◽  
Author(s):  
Jie Zhang ◽  
Massimo Cavallaro ◽  
Daniel Hebenstreit
Keyword(s):  
High Resolution ◽  
Human Cells ◽  
Rrna Genes ◽  
Dna Motifs ◽  
Single Base ◽  
Chromatin States ◽  
Genome Wide ◽  
A Genome ◽  
Single Base Resolution ◽  
Polymerase Pausing

Transcription of many genes in metazoans is subject to polymerase pausing, which corresponds to the transient arrest of transcriptionally engaged polymerase. It occurs mainly at promoter proximal regions and is not well understood. In particular, a genome-wide measurement of pausing times at high resolution has been lacking.We present here an extension of PRO-seq, time variant PRO-seq (TV-PRO-seq), that allowed us to estimate genome-wide pausing times at single base resolution. Its application to human cells reveals that promoter proximal pausing is surprisingly short compared to other regions and displays an intricate pattern. We also find precisely conserved pausing profiles at tRNA and rRNA genes and identified DNA motifs associated with pausing time. Finally, we show how chromatin states reflect differences in pausing times.

scholarly journals A genome‐wide screen identifies IRF2 as a key regulator of caspase‐4 in human cells

EMBO Reports ◽  
2019 ◽  
Vol 20 (9) ◽  
Author(s):  
Sacha Benaoudia ◽  
Amandine Martin ◽  
Marta Puig Gamez ◽  
Gabrielle Gay ◽  
Brice Lagrange ◽  
...  
Keyword(s):  
Human Cells ◽  
Genome Wide ◽  
A Genome

scholarly journals The Antisense Transcriptomes of Human Cells

Science ◽  
2008 ◽  
Vol 322 (5909) ◽  
pp. 1855-1857 ◽  
Author(s):  
Yiping He ◽  
Bert Vogelstein ◽  
Victor E. Velculescu ◽  
Nickolas Papadopoulos ◽  
Kenneth W. Kinzler
Keyword(s):  
Mammalian Cells ◽  
Cell Types ◽  
Human Cells ◽  
Antisense Transcripts ◽  
Genome Wide ◽  
Human Genes ◽  
A Genome ◽  
Dna Strand ◽  
The Relationship ◽  
Rna Transcript

Transcription in mammalian cells can be assessed at a genome-wide level, but it has been difficult to reliably determine whether individual transcripts are derived from the plus or minus strands of chromosomes. This distinction can be critical for understanding the relationship between known transcripts (sense) and the complementary antisense transcripts that may regulate them. Here, we describe a technique that can be used to (i) identify the DNA strand of origin for any particular RNA transcript, and (ii) quantify the number of sense and antisense transcripts from expressed genes at a global level. We examined five different human cell types and in each case found evidence for antisense transcripts in 2900 to 6400 human genes. The distribution of antisense transcripts was distinct from that of sense transcripts, was nonrandom across the genome, and differed among cell types. Antisense transcripts thus appear to be a pervasive feature of human cells, which suggests that they are a fundamental component of gene regulation.

scholarly journals Systematic discovery and functional interrogation of SARS-CoV-2 viral RNA-host protein interactions during infection

2020 ◽  
Author(s):  
Ryan A. Flynn ◽  
Julia A. Belk ◽  
Yanyan Qi ◽  
Yuki Yasumoto ◽  
Cameron O. Schmitz ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Viral Rna ◽  
Host Protein ◽  
List Type ◽  
Genome Wide ◽  
A Genome

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a pandemic with growing global mortality. There is an urgent need to understand the molecular pathways required for host infection and anti-viral immunity. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with viral ChIRP-MS data from three other positive-sense RNA viruses defined pan-viral and SARS-CoV-2-specific host interactions. Functional interrogation of these factors with a genome-wide CRISPR screen revealed that the vast majority of viral RNA-binding proteins protect the host from virus-induced cell death, and we identified known and novel anti-viral proteins that regulate SARS-CoV-2 pathogenicity. Finally, our RNA-centric approach demonstrated a physical connection between SARS-CoV-2 RNA and host mitochondria, which we validated with functional and electron microscopy data, providing new insights into a more general virus-specific protein logic for mitochondrial interactions. Altogether, these data provide a comprehensive catalogue of SARS-CoV-2 RNA-host protein interactions, which may inform future studies to understand the mechanisms of viral pathogenesis, as well as nominate host pathways that could be targeted for therapeutic benefit.Highlights· ChIRP-MS of SARS-CoV-2 RNA identifies a comprehensive viral RNA-host protein interaction network during infection across two species· Comparison to RNA-protein interaction networks with Zika virus, dengue virus, and rhinovirus identify SARS-CoV-2-specific and pan-viral RNA protein complexes and highlights distinct intracellular trafficking pathways· Intersection of ChIRP-MS and genome-wide CRISPR screens identify novel SARS-CoV-2-binding proteins with pro- and anti-viral function· Viral RNA-RNA and RNA-protein interactions reveal specific SARS-CoV-2-mediated mitochondrial dysfunction during infection

scholarly journals Genome-Wide Identification of NBS-Encoding Resistance Genes in Sunflower (Helianthus annuus L.)

Genes ◽  
2018 ◽  
Vol 9 (8) ◽  
pp. 384 ◽  
Author(s):  
Surendra Neupane ◽  
Ethan J. Andersen ◽  
Achal Neupane ◽  
Madhav P. Nepal
Keyword(s):  
Binding Site ◽  
Functional Divergence ◽  
Crop Improvement ◽  
Functional Characterization ◽  
Nucleotide Binding ◽  
Nucleotide Binding Site ◽  
Leucine Rich Repeat ◽  
Genome Wide ◽  
A Genome ◽  
Encoding Genes

Nucleotide Binding Site—Leucine-Rich Repeat (NBS-LRR) genes encode disease resistance proteins involved in plants’ defense against their pathogens. Although sunflower is affected by many diseases, only a few molecular details have been uncovered regarding pathogenesis and resistance mechanisms. Recent availability of sunflower whole genome sequences in publicly accessible databases allowed us to accomplish a genome-wide identification of Toll-interleukin-1 receptor-like Nucleotide-binding site Leucine-rich repeat (TNL), Coiled Coil (CC)-NBS-LRR (CNL), Resistance to powdery mildew 8 (RPW8)-NBS-LRR (RNL) and NBS-LRR (NL) protein encoding genes. Hidden Markov Model (HMM) profiling of 52,243 putative protein sequences from sunflower resulted in 352 NBS-encoding genes, among which 100 genes belong to CNL group including 64 genes with RX_CC like domain, 77 to TNL, 13 to RNL, and 162 belong to NL group. We also identified signal peptides and nuclear localization signals present in the identified genes and their homologs. We found that NBS genes were located on all chromosomes and formed 75 gene clusters, one-third of which were located on chromosome 13. Phylogenetic analyses between sunflower and Arabidopsis NBS genes revealed a clade-specific nesting pattern in CNLs, with RNLs nested in the CNL-A clade, and species-specific nesting pattern for TNLs. Surprisingly, we found a moderate bootstrap support (BS = 50%) for CNL-A clade being nested within TNL clade making both the CNL and TNL clades paraphyletic. Arabidopsis and sunflower showed 87 syntenic blocks with 1049 high synteny hits between chromosome 5 of Arabidopsis and chromosome 6 of sunflower. Expression data revealed functional divergence of the NBS genes with basal level tissue-specific expression. This study represents the first genome-wide identification of NBS genes in sunflower paving avenues for functional characterization and potential crop improvement.

scholarly journals Genome-wide CRISPR-Cas9 screen reveals the importance of the heparan sulfate pathway and the conserved oligomeric golgi complex for synthetic dsRNA uptake and Sindbis virus infection

2020 ◽  
Author(s):  
Olivier Petitjean ◽  
Erika Girardi ◽  
Richard Patryk Ngondo ◽  
Vladimir Lupashin ◽  
Sébastien Pfeffer
Keyword(s):  
Cell Death ◽  
Heparan Sulfate ◽  
Cell Survival ◽  
Sindbis Virus ◽  
Human Cells ◽  
Innate Immune ◽  
Genome Wide ◽  
A Genome ◽  
Conserved Oligomeric Golgi Complex ◽  
Conserved Oligomeric Golgi

AbstractDouble 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.

scholarly journals A Host Susceptibility Gene, DR1, Facilitates Influenza A Virus Replication by Suppressing Host Innate Immunity and Enhancing Viral RNA Replication

2015 ◽  
Vol 89 (7) ◽  
pp. 3671-3682 ◽  
Author(s):  
Shih-Feng Hsu ◽  
Wen-Chi Su ◽  
King-Song Jeng ◽  
Michael M. C. Lai
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Susceptibility Gene ◽  
Influenza Virus Infection ◽  
Rna Replication ◽  
Viral Rna ◽  
Host Susceptibility ◽  
Viral Rdrp ◽  
Genome Wide ◽  
A Genome

ABSTRACTInfluenza A virus (IAV) depends on cellular factors to complete its replication cycle; thus, investigation of the factors utilized by IAV may facilitate antiviral drug development. To this end, a cellular transcriptional repressor, DR1, was identified from a genome-wide RNA interference (RNAi) screen. Knockdown (KD) of DR1 resulted in reductions of viral RNA and protein production, demonstrating that DR1 acts as a positive host factor in IAV replication. Genome-wide transcriptomic analysis showed that there was a strong induction of interferon-stimulated gene (ISG) expression after prolonged DR1 KD. We found that beta interferon (IFN-β) was induced by DR1 KD, thereby activating the JAK-STAT pathway to turn on ISG expression, which led to a strong inhibition of IAV replication. This result suggests that DR1 in normal cells suppresses IFN induction, probably to prevent undesired cytokine production, but that this suppression may create a milieu that favors IAV replication once cells are infected. Furthermore, biochemical assays of viral RNA replication showed that DR1 KD suppressed viral RNA replication. We also showed that DR1 associated with all three subunits of the viral RNA-dependent RNA polymerase (RdRp) complex, indicating that DR1 may interact with individual components of the viral RdRp complex to enhance viral RNA replication. Thus, DR1 may be considered a novel host susceptibility gene for IAV replication via a dual mechanism, not only suppressing the host defense to indirectly favor IAV replication but also directly facilitating viral RNA replication.IMPORTANCEInvestigations of virus-host interactions involved in influenza A virus (IAV) replication are important for understanding viral pathogenesis and host defenses, which may manipulate influenza virus infection or prevent the emergence of drug resistance caused by a high error rate during viral RNA replication. For this purpose, a cellular transcriptional repressor, DR1, was identified from a genome-wide RNAi screen as a positive regulator in IAV replication. In the current studies, we showed that DR1 suppressed the gene expression of a large set of host innate immunity genes, which indirectly facilitated IAV replication in the event of IAV infection. Besides this scenario, DR1 also directly enhanced the viral RdRp activity, likely through associating with individual components of the viral RdRp complex. Thus, DR1 represents a novel host susceptibility gene for IAV replication via multiple functions, not only suppressing the host defense but also enhancing viral RNA replication. DR1 may be a potential target for drug development against influenza virus infection.

scholarly journals Genome-Wide CRISPR-Cas9 Screen Reveals the Importance of the Heparan Sulfate Pathway and the Conserved Oligomeric Golgi Complex for Synthetic Double-Stranded RNA Uptake and Sindbis Virus Infection

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Olivier Petitjean ◽  
Erika Girardi ◽  
Richard Patryk Ngondo ◽  
Vladimir Lupashin ◽  
Sébastien Pfeffer
Keyword(s):  
Cell Death ◽  
Heparan Sulfate ◽  
Cell Survival ◽  
Sindbis Virus ◽  
Human Cells ◽  
Double Stranded Rna ◽  
Genome Wide ◽  
A Genome ◽  
Conserved Oligomeric Golgi Complex ◽  
Conserved Oligomeric Golgi

ABSTRACT 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 knockout led to a decrease of extracellular HS that specifically affected dsRNA transfection efficiency and reduced viral production, which explains the increased cell survival of these mutants. IMPORTANCE When 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 RNA (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.

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