Publications by authors named "Ella Hartenian"

10 Publications

  • Page 1 of 1

The molecular virology of coronaviruses.

J Biol Chem 2020 09 13;295(37):12910-12934. Epub 2020 Jul 13.

Department of Molecular and Cell Biology, University of California, Berkeley, California, USA; Department of Plant and Microbial Biology, University of California, Berkeley, California, USA; Howard Hughes Medical Institute, University of California, Berkeley, California, USA. Electronic address:

Few human pathogens have been the focus of as much concentrated worldwide attention as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of COVID-19. Its emergence into the human population and ensuing pandemic came on the heels of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), two other highly pathogenic coronavirus spillovers, which collectively have reshaped our view of a virus family previously associated primarily with the common cold. It has placed intense pressure on the collective scientific community to develop therapeutics and vaccines, whose engineering relies on a detailed understanding of coronavirus biology. Here, we present the molecular virology of coronavirus infection, including its entry into cells, its remarkably sophisticated gene expression and replication mechanisms, its extensive remodeling of the intracellular environment, and its multifaceted immune evasion strategies. We highlight aspects of the viral life cycle that may be amenable to antiviral targeting as well as key features of its biology that await discovery.
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http://dx.doi.org/10.1074/jbc.REV120.013930DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7489918PMC
September 2020

RNA decay during gammaherpesvirus infection reduces RNA polymerase II occupancy of host promoters but spares viral promoters.

PLoS Pathog 2020 02 7;16(2):e1008269. Epub 2020 Feb 7.

Department of Molecular and Cell Biology, University of California Berkeley, CA, United States of America.

In mammalian cells, widespread acceleration of cytoplasmic mRNA degradation is linked to impaired RNA polymerase II (Pol II) transcription. This mRNA decay-induced transcriptional repression occurs during infection with gammaherpesviruses including Kaposi's sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68), which encode an mRNA endonuclease that initiates widespread RNA decay. Here, we show that MHV68-induced mRNA decay leads to a genome-wide reduction of Pol II occupancy at mammalian promoters. This reduced Pol II occupancy is accompanied by down-regulation of multiple Pol II subunits and TFIIB in the nucleus of infected cells, as revealed by mass spectrometry-based global measurements of protein abundance. Viral genes, despite the fact that they require Pol II for transcription, escape transcriptional repression. Protection is not governed by viral promoter sequences; instead, location on the viral genome is both necessary and sufficient to escape the transcriptional repression effects of mRNA decay. We propose a model in which the ability to escape from transcriptional repression is linked to the localization of viral DNA within replication compartments, providing a means for these viruses to counteract decay-induced transcript loss.
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http://dx.doi.org/10.1371/journal.ppat.1008269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7032723PMC
February 2020

Feedback to the central dogma: cytoplasmic mRNA decay and transcription are interdependent processes.

Crit Rev Biochem Mol Biol 2019 08 27;54(4):385-398. Epub 2019 Oct 27.

Department of Molecular & Cell Biology, University of California, Berkeley, CA, USA.

Transcription and RNA decay are key determinants of gene expression; these processes are typically considered as the uncoupled beginning and end of the messenger RNA (mRNA) lifecycle. Here we describe the growing number of studies demonstrating interplay between these spatially disparate processes in eukaryotes. Specifically, cells can maintain mRNA levels by buffering against changes in mRNA stability or transcription, and can also respond to virally induced accelerated decay by reducing RNA polymerase II gene expression. In addition to these global responses, there is also evidence that mRNAs containing a premature stop codon can cause transcriptional upregulation of homologous genes in a targeted fashion. In each of these systems, RNA binding proteins (RBPs), particularly those involved in mRNA degradation, are critical for cytoplasmic to nuclear communication. Although their specific mechanistic contributions are yet to be fully elucidated, differential trafficking of RBPs between subcellular compartments are likely to play a central role in regulating this gene expression feedback pathway.
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http://dx.doi.org/10.1080/10409238.2019.1679083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6871655PMC
August 2019

Changes in mRNA abundance drive shuttling of RNA binding proteins, linking cytoplasmic RNA degradation to transcription.

Elife 2018 10 3;7. Epub 2018 Oct 3.

Department of Molecular and Cell Biology, University of California, Berkeley, United States.

Alterations in global mRNA decay broadly impact multiple stages of gene expression, although signals that connect these processes are incompletely defined. Here, we used tandem mass tag labeling coupled with mass spectrometry to reveal that changing the mRNA decay landscape, as frequently occurs during viral infection, results in subcellular redistribution of RNA binding proteins (RBPs) in human cells. Accelerating Xrn1-dependent mRNA decay through expression of a gammaherpesviral endonuclease drove nuclear translocation of many RBPs, including poly(A) tail-associated proteins. Conversely, cells lacking Xrn1 exhibited changes in the localization or abundance of numerous factors linked to mRNA turnover. Using these data, we uncovered a new role for relocalized cytoplasmic poly(A) binding protein in repressing recruitment of TATA binding protein and RNA polymerase II to promoters. Collectively, our results show that changes in cytoplasmic mRNA decay can directly impact protein localization, providing a mechanism to connect seemingly distal stages of gene expression.
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http://dx.doi.org/10.7554/eLife.37663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6203436PMC
October 2018

Orthologous CRISPR-Cas9 enzymes for combinatorial genetic screens.

Nat Biotechnol 2018 02 18;36(2):179-189. Epub 2017 Dec 18.

Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.

Combinatorial genetic screening using CRISPR-Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current methods suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from Staphylococcus aureus and Streptococcus pyogenes. We used machine learning to establish S. aureus Cas9 sgRNA design rules and paired S. aureus Cas9 with S. pyogenes Cas9 to achieve dual targeting in a high fraction of cells. We also developed a lentiviral vector and cloning strategy to generate high-complexity pooled dual-knockout libraries to identify synthetic lethal and buffering gene pairs across multiple cell types, including MAPK pathway genes and apoptotic genes. Our orthologous approach also enabled a screen combining gene knockouts with transcriptional activation, which revealed genetic interactions with TP53. The "Big Papi" (paired aureus and pyogenes for interactions) approach described here will be widely applicable for the study of combinatorial phenotypes.
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http://dx.doi.org/10.1038/nbt.4048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5800952PMC
February 2018

The structural basis of flagellin detection by NAIP5: A strategy to limit pathogen immune evasion.

Science 2017 11;358(6365):888-893

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.

Robust innate immune detection of rapidly evolving pathogens is critical for host defense. Nucleotide-binding domain leucine-rich repeat (NLR) proteins function as cytosolic innate immune sensors in plants and animals. However, the structural basis for ligand-induced NLR activation has so far remained unknown. NAIP5 (NLR family, apoptosis inhibitory protein 5) binds the bacterial protein flagellin and assembles with NLRC4 to form a multiprotein complex called an inflammasome. Here we report the cryo-electron microscopy structure of the assembled ~1.4-megadalton flagellin-NAIP5-NLRC4 inflammasome, revealing how a ligand activates an NLR. Six distinct NAIP5 domains contact multiple conserved regions of flagellin, prying NAIP5 into an open and active conformation. We show that innate immune recognition of multiple ligand surfaces is a generalizable strategy that limits pathogen evolution and immune escape.
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http://dx.doi.org/10.1126/science.aao1140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5842810PMC
November 2017

Genetic and Proteomic Interrogation of Lower Confidence Candidate Genes Reveals Signaling Networks in β-Catenin-Active Cancers.

Cell Syst 2016 09;3(3):302-316.e4

Broad Institute of Harvard and MIT, 415 Main Street, Cambridge, MA 02142, USA; Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 450 Brookline Avenue, Boston, MA 02215, USA. Electronic address:

Genome-scale expression studies and comprehensive loss-of-function genetic screens have focused almost exclusively on the highest confidence candidate genes. Here, we describe a strategy for characterizing the lower confidence candidates identified by such approaches. We interrogated 177 genes that we classified as essential for the proliferation of cancer cells exhibiting constitutive β-catenin activity and integrated data for each of the candidates, derived from orthogonal short hairpin RNA (shRNA) knockdown and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated gene editing knockout screens, to yield 69 validated genes. We then characterized the relationships between sets of these genes using complementary assays: medium-throughput stable isotope labeling by amino acids in cell culture (SILAC)-based mass spectrometry, yielding 3,639 protein-protein interactions, and a CRISPR-mediated pairwise double knockout screen, yielding 375 combinations exhibiting greater- or lesser-than-additive phenotypic effects indicating genetic interactions. These studies identify previously unreported regulators of β-catenin, define functional networks required for the survival of β-catenin-active cancers, and provide an experimental strategy that may be applied to define other signaling networks.
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http://dx.doi.org/10.1016/j.cels.2016.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5455996PMC
September 2016

Genetic screens and functional genomics using CRISPR/Cas9 technology.

FEBS J 2015 Apr 16;282(8):1383-93. Epub 2015 Mar 16.

Department of Molecular and Cellular Biology, University of California Berkeley, Berkeley, CA, USA.

Functional genomics attempts to understand the genome by perturbing the flow of information from DNA to RNA to protein, in order to learn how gene dysfunction leads to disease. CRISPR/Cas9 technology is the newest tool in the geneticist's toolbox, allowing researchers to edit DNA with unprecedented ease, speed and accuracy, and representing a novel means to perform genome-wide genetic screens to discover gene function. In this review, we first summarize the discovery and characterization of CRISPR/Cas9, and then compare it to other genome engineering technologies. We discuss its initial use in screening applications, with a focus on optimizing on-target activity and minimizing off-target effects. Finally, we comment on future challenges and opportunities afforded by this technology.
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http://dx.doi.org/10.1111/febs.13248DOI Listing
April 2015

Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation.

Nat Biotechnol 2014 Dec 3;32(12):1262-7. Epub 2014 Sep 3.

Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

Components of the prokaryotic clustered, regularly interspaced, short palindromic repeats (CRISPR) loci have recently been repurposed for use in mammalian cells. The CRISPR-associated (Cas)9 can be programmed with a single guide RNA (sgRNA) to generate site-specific DNA breaks, but there are few known rules governing on-target efficacy of this system. We created a pool of sgRNAs, tiling across all possible target sites of a panel of six endogenous mouse and three endogenous human genes and quantitatively assessed their ability to produce null alleles of their target gene by antibody staining and flow cytometry. We discovered sequence features that improved activity, including a further optimization of the protospacer-adjacent motif (PAM) of Streptococcus pyogenes Cas9. The results from 1,841 sgRNAs were used to construct a predictive model of sgRNA activity to improve sgRNA design for gene editing and genetic screens. We provide an online tool for the design of highly active sgRNAs for any gene of interest.
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http://dx.doi.org/10.1038/nbt.3026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4262738PMC
December 2014

Genome-scale CRISPR-Cas9 knockout screening in human cells.

Science 2014 Jan 12;343(6166):84-87. Epub 2013 Dec 12.

Broad Institute of MIT and Harvard, 7 Cambridge Center, MA 02142, USA.

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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http://dx.doi.org/10.1126/science.1247005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4089965PMC
January 2014