Publications by authors named "Roni Winkler"

8 Publications

  • Page 1 of 1

SARS-CoV-2 uses a multipronged strategy to impede host protein synthesis.

Nature 2021 06 12;594(7862):240-245. Epub 2021 May 12.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

The coronavirus SARS-CoV-2 is the cause of the ongoing pandemic of COVID-19. Coronaviruses have developed a variety of mechanisms to repress host mRNA translation to allow the translation of viral mRNA, and concomitantly block the cellular innate immune response. Although several different proteins of SARS-CoV-2 have previously been implicated in shutting off host expression, a comprehensive picture of the effects of SARS-CoV-2 infection on cellular gene expression is lacking. Here we combine RNA sequencing, ribosome profiling and metabolic labelling of newly synthesized RNA to comprehensively define the mechanisms that are used by SARS-CoV-2 to shut off cellular protein synthesis. We show that infection leads to a global reduction in translation, but that viral transcripts are not preferentially translated. Instead, we find that infection leads to the accelerated degradation of cytosolic cellular mRNAs, which facilitates viral takeover of the mRNA pool in infected cells. We reveal that the translation of transcripts that are induced in response to infection (including innate immune genes) is impaired. We demonstrate this impairment is probably mediated by inhibition of nuclear mRNA export, which prevents newly transcribed cellular mRNA from accessing ribosomes. Overall, our results uncover a multipronged strategy that is used by SARS-CoV-2 to take over the translation machinery and to suppress host defences.
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http://dx.doi.org/10.1038/s41586-021-03610-3DOI Listing
June 2021

Comprehensive annotations of human herpesvirus 6A and 6B genomes reveal novel and conserved genomic features.

Elife 2020 01 16;9. Epub 2020 Jan 16.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

Human herpesvirus-6 (HHV-6) A and B are ubiquitous betaherpesviruses, infecting the majority of the human population. They encompass large genomes and our understanding of their protein coding potential is far from complete. Here, we employ ribosome-profiling and systematic transcript-analysis to experimentally define HHV-6 translation products. We identify hundreds of new open reading frames (ORFs), including upstream ORFs (uORFs) and internal ORFs (iORFs), generating a complete unbiased atlas of HHV-6 proteome. By integrating systematic data from the prototypic betaherpesvirus, human cytomegalovirus, we uncover numerous uORFs and iORFs conserved across betaherpesviruses and we show uORFs are enriched in late viral genes. We identified three highly abundant HHV-6 encoded long non-coding RNAs, one of which generates a non-polyadenylated stable intron appearing to be a conserved feature of betaherpesviruses. Overall, our work reveals the complexity of HHV-6 genomes and highlights novel features conserved between betaherpesviruses, providing a rich resource for future functional studies.
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http://dx.doi.org/10.7554/eLife.50960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6964970PMC
January 2020

Deciphering the "mA Code" via Antibody-Independent Quantitative Profiling.

Cell 2019 07 27;178(3):731-747.e16. Epub 2019 Jun 27.

Department of Molecular Genetics, Weizmann Institute of Science, 7610001 Rehovot, Israel. Electronic address:

N6-methyladenosine (mA) is the most abundant modification on mRNA and is implicated in critical roles in development, physiology, and disease. A major limitation has been the inability to quantify mA stoichiometry and the lack of antibody-independent methodologies for interrogating mA. Here, we develop MAZTER-seq for systematic quantitative profiling of m6A at single-nucleotide resolution at 16%-25% of expressed sites, building on differential cleavage by an RNase. MAZTER-seq permits validation and de novo discovery of mA sites, calibration of the performance of antibody-based approaches, and quantitative tracking of mA dynamics in yeast gametogenesis and mammalian differentiation. We discover that m6A stoichiometry is "hard coded" in cis via a simple and predictable code, accounting for 33%-46% of the variability in methylation levels and allowing accurate prediction of mA loss and acquisition events across evolution. MAZTER-seq allows quantitative investigation of mA regulation in subcellular fractions, diverse cell types, and disease states.
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http://dx.doi.org/10.1016/j.cell.2019.06.013DOI Listing
July 2019

Long Noncoding RNA MALAT1 Regulates Cancer Glucose Metabolism by Enhancing mTOR-Mediated Translation of TCF7L2.

Cancer Res 2019 05 26;79(10):2480-2493. Epub 2019 Mar 26.

Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel Canada (IMRIC), Hebrew University-Hadassah Medical School, Jerusalem, Israel.

Reprogrammed glucose metabolism of enhanced aerobic glycolysis (or the Warburg effect) is known as a hallmark of cancer. The roles of long noncoding RNAs (lncRNA) in regulating cancer metabolism at the level of both glycolysis and gluconeogenesis are mostly unknown. We previously showed that lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) acts as a proto-oncogene in hepatocellular carcinoma (HCC). Here, we investigated the role of MALAT1 in regulating cancer glucose metabolism. MALAT1 upregulated the expression of glycolytic genes and downregulated gluconeogenic enzymes by enhancing the translation of the metabolic transcription factor TCF7L2. MALAT1-enhanced TCF7L2 translation was mediated by upregulation of SRSF1 and activation of the mTORC1-4EBP1 axis. Pharmacological or genetic inhibition of mTOR and Raptor or expression of a hypophosphorylated mutant version of eIF4E-binding protein (4EBP1) resulted in decreased expression of TCF7L2. MALAT1 expression regulated TCF7L2 mRNA association with heavy polysomes, probably through the TCF7L2 5'-untranslated region (UTR), as determined by polysome fractionation and 5'UTR-reporter assays. Knockdown of TCF7L2 in MALAT1-overexpressing cells and HCC cell lines affected their metabolism and abolished their tumorigenic potential, suggesting that the effects of MALAT1 on glucose metabolism are essential for its oncogenic activity. Taken together, our findings suggest that MALAT1 contributes to HCC development and tumor progression by reprogramming tumor glucose metabolism. SIGNIFICANCE: These findings show that lncRNA MALAT1 contributes to HCC development by regulating cancer glucose metabolism, enhancing glycolysis, and inhibiting gluconeogenesis via elevated translation of the transcription factor TCF7L2.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-1432DOI Listing
May 2019

Publisher Correction: m6A modification controls the innate immune response to infection by targeting type I interferons.

Nat Immunol 2019 02;20(2):243

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

In the version of this article initially published, the penultimate sentence of the abstract included a typographical error ('cxgenes'). The correct word is 'genes'. The error has been corrected in the HTML and PDF version of the article.
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http://dx.doi.org/10.1038/s41590-019-0314-4DOI Listing
February 2019

mA modification controls the innate immune response to infection by targeting type I interferons.

Nat Immunol 2019 02 17;20(2):173-182. Epub 2018 Dec 17.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

N-methyladenosine (mA) is the most common mRNA modification. Recent studies have revealed that depletion of mA machinery leads to alterations in the propagation of diverse viruses. These effects were proposed to be mediated through dysregulated methylation of viral RNA. Here we show that following viral infection or stimulation of cells with an inactivated virus, deletion of the mA 'writer' METTL3 or 'reader' YTHDF2 led to an increase in the induction of interferon-stimulated genes. Consequently, propagation of different viruses was suppressed in an interferon-signaling-dependent manner. Significantly, the mRNA of IFNB, the gene encoding the main cytokine that drives the type I interferon response, was mA modified and was stabilized following repression of METTL3 or YTHDF2. Furthermore, we show that mA-mediated regulation of interferon genes was conserved in mice. Together, our findings uncover the role mA serves as a negative regulator of interferon response by dictating the fast turnover of interferon mRNAs and consequently facilitating viral propagation.
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http://dx.doi.org/10.1038/s41590-018-0275-zDOI Listing
February 2019

The m1A landscape on cytosolic and mitochondrial mRNA at single-base resolution.

Nature 2017 11 25;551(7679):251-255. Epub 2017 Oct 25.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

Modifications on mRNA offer the potential of regulating mRNA fate post-transcriptionally. Recent studies suggested the widespread presence of N-methyladenosine (mA), which disrupts Watson-Crick base pairing, at internal sites of mRNAs. These studies lacked the resolution of identifying individual modified bases, and did not identify specific sequence motifs undergoing the modification or an enzymatic machinery catalysing them, rendering it challenging to validate and functionally characterize putative sites. Here we develop an approach that allows the transcriptome-wide mapping of mA at single-nucleotide resolution. Within the cytosol, mA is present in a low number of mRNAs, typically at low stoichiometries, and almost invariably in tRNA T-loop-like structures, where it is introduced by the TRMT6/TRMT61A complex. We identify a single mA site in the mitochondrial ND5 mRNA, catalysed by TRMT10C, with methylation levels that are highly tissue specific and tightly developmentally controlled. mA leads to translational repression, probably through a mechanism involving ribosomal scanning or translation. Our findings suggest that mA on mRNA, probably because of its disruptive impact on base pairing, leads to translational repression, and is generally avoided by cells, while revealing one case in mitochondria where tight spatiotemporal control over mA levels was adopted as a potential means of post-transcriptional regulation.
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http://dx.doi.org/10.1038/nature24456DOI Listing
November 2017

Global mRNA polarization regulates translation efficiency in the intestinal epithelium.

Science 2017 09 10;357(6357):1299-1303. Epub 2017 Aug 10.

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.

Asymmetric messenger RNA (mRNA) localization facilitates efficient translation in cells such as neurons and fibroblasts. However, the extent and importance of mRNA polarization in epithelial tissues are unclear. Here, we used single-molecule transcript imaging and subcellular transcriptomics to uncover global apical-basal intracellular polarization of mRNA in the mouse intestinal epithelium. The localization of mRNAs did not generally overlap protein localization. Instead, ribosomes were more abundant on the apical sides, and apical transcripts were consequently more efficiently translated. Refeeding of fasted mice elicited a basal-to-apical shift in polarization of mRNAs encoding ribosomal proteins, which was associated with a specific boost in their translation. This led to increased protein production, required for efficient nutrient absorption. These findings reveal a posttranscriptional regulatory mechanism involving dynamic polarization of mRNA and polarized translation.
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http://dx.doi.org/10.1126/science.aan2399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955215PMC
September 2017