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

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.

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The Antisense Transcriptomes of Human Cells

Science ◽

10.1126/science.1163853 ◽

2008 ◽

Vol 322 (5909) ◽

pp. 1855-1857 ◽

Cited By ~ 345

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.

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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

10.1101/2020.05.20.105528 ◽

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.

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Faculty Opinions recommendation of A genome-wide RNAi screen for modifiers of the circadian clock in human cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.2062956.1646054 ◽

2010 ◽

Author(s):

Jean Clairambault ◽

Pasquale Innominato

Keyword(s):

Circadian Clock ◽

Rnai Screen ◽

Human Cells ◽

Genome Wide ◽

A Genome

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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 ◽

10.1128/msphere.00914-20 ◽

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

Journal of Virology ◽

10.1128/jvi.02145-19 ◽

2020 ◽

Vol 94 (9) ◽

Cited By ~ 2

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.

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

10.1101/758201 ◽

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.

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A High-Resolution Genome-Wide CRISPR/Cas9 Viability Screen Reveals Structural Features and Contextual Diversity of the Human Cell-Essential Proteome

Molecular and Cellular Biology ◽

10.1128/mcb.00302-17 ◽

2017 ◽

Vol 38 (1) ◽

Cited By ~ 20

Author(s):

Thierry Bertomeu ◽

Jasmin Coulombe-Huntington ◽

Andrew Chatr-aryamontri ◽

Karine G. Bourdages ◽

Etienne Coyaud ◽

...

Keyword(s):

Stress Responses ◽

B Cell Lymphoma ◽

Human Cells ◽

Essential Genes ◽

Analysis Tool ◽

Contextual Diversity ◽

Genome Wide ◽

A Genome ◽

Alternatively Spliced ◽

Alternatively Spliced Exons

ABSTRACT To interrogate genes essential for cell growth, proliferation and survival in human cells, we carried out a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 screen in a B-cell lymphoma line using a custom extended-knockout (EKO) library of 278,754 single-guide RNAs (sgRNAs) that targeted 19,084 RefSeq genes, 20,852 alternatively spliced exons, and 3,872 hypothetical genes. A new statistical analysis tool called robust analytics and normalization for knockout screens (RANKS) identified 2,280 essential genes, 234 of which were unique. Individual essential genes were validated experimentally and linked to ribosome biogenesis and stress responses. Essential genes exhibited a bimodal distribution across 10 different cell lines, consistent with a continuous variation in essentiality as a function of cell type. Genes essential in more lines had more severe fitness defects and encoded the evolutionarily conserved structural cores of protein complexes, whereas genes essential in fewer lines formed context-specific modules and encoded subunits at the periphery of essential complexes. The essentiality of individual protein residues across the proteome correlated with evolutionary conservation, structural burial, modular domains, and protein interaction interfaces. Many alternatively spliced exons in essential genes were dispensable and were enriched for disordered regions. Fitness defects were observed for 44 newly evolved hypothetical reading frames. These results illuminate the contextual nature and evolution of essential gene functions in human cells.

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Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells

Science ◽

10.1126/science.1247005 ◽

2013 ◽

Vol 343 (6166) ◽

pp. 84-87 ◽

Cited By ~ 2545

Author(s):

Ophir Shalem ◽

Neville E. Sanjana ◽

Ella Hartenian ◽

Xi Shi ◽

David A. Scott ◽

...

Keyword(s):

Pluripotent Stem Cells ◽

Human Cells ◽

High Rate ◽

Guide Rnas ◽

Genome Wide ◽

A Genome ◽

Melanoma Model ◽

Wide Scale ◽

High Level ◽

Genome Scale

The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.

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