Publications by authors named "Markus Landthaler"

78 Publications

Transcriptomic profiling of SARS-CoV-2 infected human cell lines identifies HSP90 as target for COVID-19 therapy.

iScience 2021 Mar 6;24(3):102151. Epub 2021 Feb 6.

Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association, Hannoversche Str 28, 10115 Berlin, Germany.

Detailed knowledge of the molecular biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is crucial for understanding of viral replication, host responses, and disease progression. Here, we report gene expression profiles of three SARS-CoV- and SARS-CoV-2-infected human cell lines. SARS-CoV-2 elicited an approximately two-fold higher stimulation of the innate immune response compared to SARS-CoV in the human epithelial cell line Calu-3, including induction of miRNA-155. Single-cell RNA sequencing of infected cells showed that genes induced by virus infections were broadly upregulated, whereas interferon beta/lambda genes, a pro-inflammatory cytokines such as IL-6, were expressed only in small subsets of infected cells. Temporal analysis suggested that transcriptional activities of interferon regulatory factors precede those of nuclear factor κB. Lastly, we identified heat shock protein 90 (HSP90) as a protein relevant for the infection. Inhibition of the HSP90 activity resulted in a reduction of viral replication and pro-inflammatory cytokine expression in primary human airway epithelial cells.
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http://dx.doi.org/10.1016/j.isci.2021.102151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7866843PMC
March 2021

CRNKL1 Is a Highly Selective Regulator of Intron-Retaining HIV-1 and Cellular mRNAs.

mBio 2021 01 19;12(1). Epub 2021 Jan 19.

Institute of Clinical and Molecular Virology, University Hospital Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany

The HIV-1 Rev protein is a nuclear export factor for unspliced and incompletely spliced HIV-1 RNAs. Without Rev, these intron-retaining RNAs are trapped in the nucleus. A genome-wide screen identified nine proteins of the spliceosome, which all enhanced expression from the HIV-1 unspliced RNA after CRISPR/Cas knockdown. Depletion of DHX38, WDR70, and four proteins of the Prp19-associated complex (ISY1, BUD31, XAB2, and CRNKL1) resulted in a more than 20-fold enhancement of unspliced HIV-1 RNA levels in the cytoplasm. Targeting of CRNKL1, DHX38, and BUD31 affected nuclear export efficiencies of the HIV-1 unspliced RNA to a much larger extent than splicing. Transcriptomic analyses further revealed that CRNKL1 also suppresses cytoplasmic levels of a subset of cellular mRNAs, including some with selectively retained introns. Thus, CRNKL1-dependent nuclear retention is a novel cellular mechanism for the regulation of cytoplasmic levels of intron-retaining HIV-1 mRNAs, which HIV-1 may have harnessed to direct its complex splicing pattern. To regulate its complex splicing pattern, HIV-1 uses the adaptor protein Rev to shuttle unspliced or partially spliced mRNA from the nucleus to the cytoplasm. In the absence of Rev, these RNAs are retained in the nucleus, but it is unclear why. Here we identify cellular proteins whose depletion enhances cytoplasmic levels of the HIV-1 unspliced RNA. Depletion of one of them, CRNKL1, also increases cytoplasmic levels of a subset of intron-retaining cellular mRNA, suggesting that CRNKL1-dependent nuclear retention may be a basic cellular mechanism exploited by HIV-1.
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http://dx.doi.org/10.1128/mBio.02525-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845644PMC
January 2021

Protein Synthesis in the Developing Neocortex at Near-Atomic Resolution Reveals Ebp1-Mediated Neuronal Proteostasis at the 60S Tunnel Exit.

Mol Cell 2021 01 22;81(2):304-322.e16. Epub 2020 Dec 22.

Institute of Medical Physics and Biophysics, Charité-Universitätsmedizin Berlin, 10117 Berlin, Germany. Electronic address:

Protein synthesis must be finely tuned in the developing nervous system as the final essential step of gene expression. This study investigates the architecture of ribosomes from the neocortex during neurogenesis, revealing Ebp1 as a high-occupancy 60S peptide tunnel exit (TE) factor during protein synthesis at near-atomic resolution by cryoelectron microscopy (cryo-EM). Ribosome profiling demonstrated Ebp1-60S binding is highest during start codon initiation and N-terminal peptide elongation, regulating ribosome occupancy of these codons. Membrane-targeting domains emerging from the 60S tunnel, which recruit SRP/Sec61 to the shared binding site, displace Ebp1. Ebp1 is particularly abundant in the early-born neural stem cell (NSC) lineage and regulates neuronal morphology. Ebp1 especially impacts the synthesis of membrane-targeted cell adhesion molecules (CAMs), measured by pulsed stable isotope labeling by amino acids in cell culture (pSILAC)/bioorthogonal noncanonical amino acid tagging (BONCAT) mass spectrometry (MS). Therefore, Ebp1 is a central component of protein synthesis, and the ribosome TE is a focal point of gene expression control in the molecular specification of neuronal morphology during development.
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http://dx.doi.org/10.1016/j.molcel.2020.11.037DOI Listing
January 2021

4EHP and GIGYF1/2 Mediate Translation-Coupled Messenger RNA Decay.

Cell Rep 2020 10;33(2):108262

Department of Biochemistry, Max Planck Institute for Developmental Biology, Max-Planck-Ring 5, D-72076 Tübingen, Germany. Electronic address:

Current models of mRNA turnover indicate that cytoplasmic degradation is coupled with translation. However, our understanding of the molecular events that coordinate ribosome transit with the mRNA decay machinery is still limited. Here, we show that 4EHP-GIGYF1/2 complexes trigger co-translational mRNA decay. Human cells lacking these proteins accumulate mRNAs with prominent ribosome pausing. They include, among others, transcripts encoding secretory and membrane-bound proteins or tubulin subunits. In addition, 4EHP-GIGYF1/2 complexes fail to reduce mRNA levels in the absence of ribosome stalling or upon disruption of their interaction with the cap structure, DDX6, and ZNF598. We further find that co-translational binding of GIGYF1/2 to the mRNA marks transcripts with perturbed elongation to decay. Our studies reveal how a repressor complex linked to neurological disorders minimizes the protein output of a subset of mRNAs.
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http://dx.doi.org/10.1016/j.celrep.2020.108262DOI Listing
October 2020

Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment.

Cell 2020 09 5;182(6):1419-1440.e23. Epub 2020 Aug 5.

Department of Infectious Diseases and Respiratory Medicine, Charité, Universitätsmedizin Berlin, Berlin, Germany; German Center for Lung Research (DZL).

Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRCD11c inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DR monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.
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http://dx.doi.org/10.1016/j.cell.2020.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405822PMC
September 2020

Loss of macpΨ Ribosomal RNA Modification Is a Major Feature of Cancer.

Cell Rep 2020 05;31(5):107611

Department of Medical Genetics, University of British Columbia, Vancouver, V6H 3N1 BC, Canada; Terry Fox Laboratory, BC Cancer, Vancouver, V5Z 1L3 BC, Canada.

The ribosome is an RNA-protein complex that is essential for translation in all domains of life. The structural and catalytic core of the ribosome is its ribosomal RNA (rRNA). While mutations in ribosomal protein (RP) genes are known drivers of oncogenesis, oncogenic rRNA variants have remained elusive. We identify a cancer-specific single-nucleotide variation in 18S rRNA at nucleotide 1248.U in up to 45.9% of patients with colorectal carcinoma (CRC) and present across >22 cancer types. This is the site of a unique hyper-modified base, 1-methyl-3-α-amino-α-carboxyl-propyl pseudouridine (macpΨ), a >1-billion-years-conserved RNA modification at the peptidyl decoding site of the ribosome. A subset of CRC tumors we call hypo-macpΨ shows sub-stoichiometric macpΨ modification, unlike normal control tissues. An macpΨ knockout model and hypo-macpΨ patient tumors share a translational signature characterized by highly abundant ribosomal proteins. Thus, macpΨ-deficient rRNA forms an uncharacterized class of "onco-ribosome" which may serve as a chemotherapeutic target for treating cancer patients.
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http://dx.doi.org/10.1016/j.celrep.2020.107611DOI Listing
May 2020

Mechanism of Virus Attenuation by Codon Pair Deoptimization.

Cell Rep 2020 04;31(4):107586

Institut für Virologie, Freie Universität Berlin, Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany. Electronic address:

Codon pair deoptimization is an efficient virus attenuation strategy, but the mechanism that leads to attenuation is unknown. The strategy involves synthetic recoding of viral genomes that alters the positions of synonymous codons, thereby increasing the number of suboptimal codon pairs and CpG dinucleotides in recoded genomes. Here we identify the molecular mechanism of codon pair deoptimization-based attenuation by studying recoded influenza A viruses. We show that suboptimal codon pairs cause attenuation, whereas the increase of CpG dinucleotides has no effect. Furthermore, we show that suboptimal codon pairs reduce both mRNA stability and translation efficiency of codon pair-deoptimized genes. Consequently, reduced protein production directly causes virus attenuation. Our study provides evidence that suboptimal codon pairs are major determinants of mRNA stability. Additionally, it demonstrates that codon pair bias can be used to increase mRNA stability and protein production of synthetic genes in many areas of biotechnology.
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http://dx.doi.org/10.1016/j.celrep.2020.107586DOI Listing
April 2020

Integrative functional genomics decodes herpes simplex virus 1.

Nat Commun 2020 04 27;11(1):2038. Epub 2020 Apr 27.

Institute for Virology and Immunobiology, Julius-Maximilians-University Würzburg, Versbacher Straße 7, 97078, Würzburg, Germany.

The predicted 80 open reading frames (ORFs) of herpes simplex virus 1 (HSV-1) have been intensively studied for decades. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identify a total of 201 transcripts and 284 ORFs including all known and 46 novel large ORFs. This includes a so far unknown ORF in the locus deleted in the FDA-approved oncolytic virus Imlygic. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of ORFs as well as N-terminal extensions (NTEs) and truncations. We show that NTEs with non-canonical start codons govern the subcellular protein localization and packaging of key viral regulators and structural proteins. We extend the current nomenclature to include all viral gene products and provide a genome browser that visualizes all the obtained data from whole genome to single-nucleotide resolution.
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http://dx.doi.org/10.1038/s41467-020-15992-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184758PMC
April 2020

The human ZC3H3 and RBM26/27 proteins are critical for PAXT-mediated nuclear RNA decay.

Nucleic Acids Res 2020 03;48(5):2518-2530

Department of Molecular Biology and Genetics, Aarhus University, C.F. Møllers Allé 3, 8000 Aarhus C, Denmark.

Recruitment of the human ribonucleolytic RNA exosome to nuclear polyadenylated (pA+) RNA is facilitated by the Poly(A) Tail eXosome Targeting (PAXT) connection. Besides its core dimer, formed by the exosome co-factor MTR4 and the ZFC3H1 protein, the PAXT connection remains poorly defined. By characterizing nuclear pA+-RNA bound proteomes as well as MTR4-ZFC3H1 containing complexes in conditions favoring PAXT assembly, we here uncover three additional proteins required for PAXT function: ZC3H3, RBM26 and RBM27 along with the known PAXT-associated protein, PABPN1. The zinc-finger protein ZC3H3 interacts directly with MTR4-ZFC3H1 and loss of any of the newly identified PAXT components results in the accumulation of PAXT substrates. Collectively, our results establish new factors involved in the turnover of nuclear pA+ RNA and suggest that these are limiting for PAXT activity.
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http://dx.doi.org/10.1093/nar/gkz1238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049725PMC
March 2020

Context-specific regulation of cell survival by a miRNA-controlled BIM rheostat.

Genes Dev 2019 12 7;33(23-24):1673-1687. Epub 2019 Nov 7.

Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin-Buch 13125, Germany.

Knockout of the ubiquitously expressed miRNA-17∼92 cluster in mice produces a lethal developmental lung defect, skeletal abnormalities, and blocked B lymphopoiesis. A shared target of miR-17∼92 miRNAs is the pro-apoptotic protein BIM, central to life-death decisions in mammalian cells. To clarify the contribution of miR-17∼92:Bim interactions to the complex miR-17∼92 knockout phenotype, we used a system of conditional mutagenesis of the nine 3' UTR miR-17∼92 seed matches. Blocking miR-17∼92:Bim interactions early in development phenocopied the lethal lung phenotype of miR-17∼92 ablation and generated a skeletal kinky tail. In the hematopoietic system, instead of causing the predicted B cell developmental block, it produced a selective inability of B cells to resist cellular stress; and prevented B and T cell hyperplasia caused by haploinsufficiency. Thus, the interaction of miR-17∼92 with a single target is essential for life, and BIM regulation by miRNAs serves as a rheostat controlling cell survival in specific physiological contexts.
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http://dx.doi.org/10.1101/gad.330134.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6942046PMC
December 2019

Single-cell RNA-sequencing of herpes simplex virus 1-infected cells connects NRF2 activation to an antiviral program.

Nat Commun 2019 10 25;10(1):4878. Epub 2019 Oct 25.

Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association, Robert-Rössle-Strasse 10, 13125, Berlin, Germany.

Herpesvirus infection initiates a range of perturbations in the host cell, which remain poorly understood at the level of individual cells. Here, we quantify the transcriptome of single human primary fibroblasts during the first hours of lytic infection with HSV-1. By applying a generalizable analysis scheme, we define a precise temporal order of early viral gene expression and propose a set-wise emergence of viral genes. We identify host cell genes and pathways relevant for infection by combining three different computational approaches: gene and pathway overdispersion analysis, prediction of cell-state transition probabilities, as well as future cell states. One transcriptional program, which correlates with increased resistance to infection, implicates the transcription factor NRF2. Consequently, Bardoxolone methyl and Sulforaphane, two known NRF2 agonists, impair virus production, suggesting that NRF2 activation restricts viral infection. Our study provides insights into early stages of HSV-1 infection and serves as a general blueprint for the investigation of heterogeneous cell states in virus infection.
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http://dx.doi.org/10.1038/s41467-019-12894-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814756PMC
October 2019

Codon bias confers stability to human mRNAs.

EMBO Rep 2019 11 3;20(11):e48220. Epub 2019 Sep 3.

Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Codon bias has been implicated as one of the major factors contributing to mRNA stability in several model organisms. However, the molecular mechanisms of codon bias on mRNA stability remain unclear in humans. Here, we show that human cells possess a mechanism to modulate RNA stability through a unique codon bias. Bioinformatics analysis showed that codons could be clustered into two distinct groups-codons with G or C at the third base position (GC3) and codons with either A or T at the third base position (AT3): the former stabilizing while the latter destabilizing mRNA. Quantification of codon bias showed that increased GC3-content entails proportionately higher GC-content. Through bioinformatics, ribosome profiling, and in vitro analysis, we show that decoupling the effects of codon bias reveals two modes of mRNA regulation, one GC3- and one GC-content dependent. Employing an immunoprecipitation-based strategy, we identify ILF2 and ILF3 as RNA-binding proteins that differentially regulate global mRNA abundances based on codon bias. Our results demonstrate that codon bias is a two-pronged system that governs mRNA abundance.
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http://dx.doi.org/10.15252/embr.201948220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6831995PMC
November 2019

Mutant FUS and ELAVL4 (HuD) Aberrant Crosstalk in Amyotrophic Lateral Sclerosis.

Cell Rep 2019 06;27(13):3818-3831.e5

Center for Life Nano Science, Istituto Italiano di Tecnologia, Viale Regina Elena 291, 00161 Rome, Italy; Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy. Electronic address:

Amyotrophic lateral sclerosis (ALS) has been genetically linked to mutations in RNA-binding proteins (RBPs), including FUS. Here, we report the RNA interactome of wild-type and mutant FUS in human motor neurons (MNs). This analysis identified a number of RNA targets. Whereas the wild-type protein preferentially binds introns, the ALS mutation causes a shift toward 3' UTRs. Neural ELAV-like RBPs are among mutant FUS targets. As a result, ELAVL4 protein levels are increased in mutant MNs. ELAVL4 and mutant FUS interact and co-localize in cytoplasmic speckles with altered biomechanical properties. Upon oxidative stress, ELAVL4 and mutant FUS are engaged in stress granules. In the spinal cord of FUS ALS patients, ELAVL4 represents a neural-specific component of FUS-positive cytoplasmic aggregates, whereas in sporadic patients it co-localizes with phosphorylated TDP-43-positive inclusions. We propose that pathological mutations in FUS trigger an aberrant crosstalk with ELAVL4 with implications for ALS.
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http://dx.doi.org/10.1016/j.celrep.2019.05.085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6613039PMC
June 2019

The Translational Landscape of the Human Heart.

Cell 2019 06 30;178(1):242-260.e29. Epub 2019 May 30.

Cardiovascular and Metabolic Sciences, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 13125 Berlin, Germany; DZHK (German Centre for Cardiovascular Research), Partner Site Berlin, 13347 Berlin, Germany; Berlin Institute of Health (BIH), 10178 Berlin, Germany; Charité-Universitätsmedizin, 10117 Berlin, Germany. Electronic address:

Gene expression in human tissue has primarily been studied on the transcriptional level, largely neglecting translational regulation. Here, we analyze the translatomes of 80 human hearts to identify new translation events and quantify the effect of translational regulation. We show extensive translational control of cardiac gene expression, which is orchestrated in a process-specific manner. Translation downstream of predicted disease-causing protein-truncating variants appears to be frequent, suggesting inefficient translation termination. We identify hundreds of previously undetected microproteins, expressed from lncRNAs and circRNAs, for which we validate the protein products in vivo. The translation of microproteins is not restricted to the heart and prominent in the translatomes of human kidney and liver. We associate these microproteins with diverse cellular processes and compartments and find that many locate to the mitochondria. Importantly, dozens of microproteins are translated from lncRNAs with well-characterized noncoding functions, indicating previously unrecognized biology.
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http://dx.doi.org/10.1016/j.cell.2019.05.010DOI Listing
June 2019

New insights into the cellular temporal response to proteostatic stress.

Elife 2018 10 12;7. Epub 2018 Oct 12.

Center for Genomics and Systems Biology, Department of Biology, New York University, New York, United States.

Maintaining a healthy proteome involves all layers of gene expression regulation. By quantifying temporal changes of the transcriptome, translatome, proteome, and RNA-protein interactome in cervical cancer cells, we systematically characterize the molecular landscape in response to proteostatic challenges. We identify shared and specific responses to misfolded proteins and to oxidative stress, two conditions that are tightly linked. We reveal new aspects of the unfolded protein response, including many genes that escape global translation shutdown. A subset of these genes supports rerouting of energy production in the mitochondria. We also find that many genes change at multiple levels, in either the same or opposing directions, and at different time points. We highlight a variety of putative regulatory pathways, including the stress-dependent alternative splicing of aminoacyl-tRNA synthetases, and protein-RNA binding within the 3' untranslated region of molecular chaperones. These results illustrate the potential of this information-rich resource.
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http://dx.doi.org/10.7554/eLife.39054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6185107PMC
October 2018

Phosphorylation of the Ribosomal Protein RPL12/uL11 Affects Translation during Mitosis.

Mol Cell 2018 10 13;72(1):84-98.e9. Epub 2018 Sep 13.

Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, 13092 Berlin, Germany; Charité-Universitätsmedizin Berlin, 10117 Berlin, Germany. Electronic address:

Emerging evidence indicates that heterogeneity in ribosome composition can give rise to specialized functions. Until now, research mainly focused on differences in core ribosomal proteins and associated factors. The effect of posttranslational modifications has not been studied systematically. Analyzing ribosome heterogeneity is challenging because individual proteins can be part of different subcomplexes (40S, 60S, 80S, and polysomes). Here we develop polysome proteome profiling to obtain unbiased proteomic maps across ribosomal subcomplexes. Our method combines extensive fractionation by sucrose gradient centrifugation with quantitative mass spectrometry. The high resolution of the profiles allows us to assign proteins to specific subcomplexes. Phosphoproteomics on the fractions reveals that phosphorylation of serine 38 in RPL12/uL11, a known mitotic CDK1 substrate, is strongly depleted in polysomes. Follow-up experiments confirm that RPL12/uL11 phosphorylation regulates the translation of specific subsets of mRNAs during mitosis. Together, our results show that posttranslational modification of ribosomal proteins can regulate translation.
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http://dx.doi.org/10.1016/j.molcel.2018.08.019DOI Listing
October 2018

Systematic Detection of Poly(A) RNA-Interacting Proteins and Their Differential Binding.

Methods Mol Biol 2018 ;1649:405-417

RNA Biology and Posttranscriptional Regulation, Max Delbrück Center for Molecular Medicine Berlin, Berlin Institute for Molecular Systems Biology, Robert-Rössle-Str. 10, 13125, Berlin, Germany.

RNA-binding proteins are dynamic posttranscriptional regulators of gene expression. Identification of mRNA-binding proteins in a given experimental setting is thus of great importance. We describe a procedure to enrich for direct poly(A) RNA protein binders by 4-thiouridine-enhanced UV cross-linking and oligo(dT) purification. Subsequent nuclease-mediated release of RNA-binding proteins (RBPs) from mRNA allows for detection of eluted proteins by mass spectrometry. In addition, we provide a comparative approach to detect differences in RBP binding activity upon a biological stimulus.
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http://dx.doi.org/10.1007/978-1-4939-7213-5_27DOI Listing
June 2018

Widespread activation of antisense transcription of the host genome during herpes simplex virus 1 infection.

Genome Biol 2017 10 31;18(1):209. Epub 2017 Oct 31.

Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association, Robert-Rössle-Strasse 10, 13125, Berlin, Germany.

Background: Herpesviruses can infect a wide range of animal species. Herpes simplex virus 1 (HSV-1) is one of the eight herpesviruses that can infect humans and is prevalent worldwide. Herpesviruses have evolved multiple ways to adapt the infected cells to their needs, but knowledge about these transcriptional and post-transcriptional modifications is sparse.

Results: Here, we show that HSV-1 induces the expression of about 1000 antisense transcripts from the human host cell genome. A subset of these is also activated by the closely related varicella zoster virus. Antisense transcripts originate either at gene promoters or within the gene body, and they show different susceptibility to the inhibition of early and immediate early viral gene expression. Overexpression of the major viral transcription factor ICP4 is sufficient to turn on a subset of antisense transcripts. Histone marks around transcription start sites of HSV-1-induced and constitutively transcribed antisense transcripts are highly similar, indicating that the genetic loci are already poised to transcribe these novel RNAs. Furthermore, an antisense transcript overlapping with the BBC3 gene (also known as PUMA) transcriptionally silences this potent inducer of apoptosis in cis.

Conclusions: We show for the first time that a virus induces widespread antisense transcription of the host cell genome. We provide evidence that HSV-1 uses this to downregulate a strong inducer of apoptosis. Our findings open new perspectives on global and specific alterations of host cell transcription by viruses.
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http://dx.doi.org/10.1186/s13059-017-1329-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5663069PMC
October 2017

Expanding the map of protein-RNA interaction sites via cell fusion followed by PAR-CLIP.

RNA Biol 2018 03 24;15(3):359-368. Epub 2018 Jan 24.

a Neuromuscular and Cardiovascular Cell Biology, Max-Delbrück-Center for Molecular Medicine , Berlin , Germany.

PAR-CLIP (photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation) facilitates the identification and mapping of protein/RNA interactions. So far, it has been limited to select cell-lines as it requires efficient 4SU uptake. To increase transcriptome complexity and thus identify additional RNA-protein interaction sites we fused HEK 293 T-Rex cells (HEK293-Y) that express the RNA binding protein YBX1 with PC12 cells expressing eGFP (PC12-eGFP). The resulting hybrids enable PAR-CLIP on a neuronally expanded transcriptome (Fusion-CLIP) and serve as a proof of principle. The fusion cells express both parental marker genes YBX1 and eGFP and the expanded transcriptome contains human and rat transcripts. PAR-CLIP of fused cells versus the parental HEK293-Y identified 768 novel RNA targets of YBX1. We were able to trace the origin of the majority of the short PAR-CLIP reads as they differentially mapped to the human and rat genome. Furthermore, Fusion-CLIP expanded the CAUC RNA binding motif of YBX1 to UCUUUNNCAUC. The fusion of HEK293-Y and PC12-eGFP cells resulted in cells with a diverse genome expressing human and rat transcripts that enabled the identification of novel YBX1 substrates. The technique allows the expansion of the HEK 293 transcriptome and makes PAR-CLIP available to fusion cells of diverse origin.
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http://dx.doi.org/10.1080/15476286.2017.1384120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5927727PMC
March 2018

Rattus norvegicus BN/SHR liver and heart left ventricle ribosomal RNA depleted directional RNA sequencing.

BMC Res Notes 2017 Aug 11;10(1):395. Epub 2017 Aug 11.

Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine, 13125, Berlin, Germany.

Objective: The spontaneously hypertensive rat strain is a frequently used disease model. In a previous study, we measured translational efficiency from this strain and BN-Lx animals. Here, we describe long RNA sequencing reads from ribosomal RNA depleted samples from the same animals. This data can be used to investigate splicing-related events.

Results: RNA was extracted from rat liver and heart left ventricle from BN-Lx and SHR/Ola rats in biological replicates. Ribosomal RNA was removed and the samples subjected to directional high-throughput RNA-sequencing. Read and alignment statistics indicate high quality of the data. The raw sequencing reads are freely available on the NCBI short read archive and can be used for further research on tissue and strain differences, or analysed together with other published high-throughput data from the same animals.
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http://dx.doi.org/10.1186/s13104-017-2716-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5553801PMC
August 2017

mRNA interactome capture in mammalian cells.

Methods 2017 08 11;126:38-43. Epub 2017 Jul 11.

RNA Biology and Posttranscriptional Regulation, Max Delbrück Center for Molecular Medicine Berlin, Berlin Institute for Molecular Systems Biology, 13125 Berlin, Germany; IRI Life Sciences, Institut für Biologie, Humboldt-Universität zu Berlin, 10115 Berlin, Germany. Electronic address:

Throughout their entire life cycle, mRNAs are associated with RNA-binding proteins (RBPs), forming ribonucleoprotein (RNP) complexes with highly dynamic compositions. Their interplay is one key to control gene regulatory mechanisms from mRNA synthesis to decay. To assay the global scope of RNA-protein interactions, we and others have published a method combining crosslinking with highly stringent oligo(dT) affinity purification to enrich proteins associated with polyadenylated RNA (poly(A)+ RNA). Identification of the poly(A)+ RNA-bound proteome (also: mRNA interactome capture) has by now been applied to a diversity of cell lines and model organisms, uncovering comprehensive repertoires of RBPs and hundreds of novel RBP candidates. In addition to determining the RBP catalog in a given biological system, mRNA interactome capture allows the examination of changes in protein-mRNA interactions in response to internal and external stimuli, altered cellular programs and disease.
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http://dx.doi.org/10.1016/j.ymeth.2017.07.006DOI Listing
August 2017

DDX54 regulates transcriptome dynamics during DNA damage response.

Genome Res 2017 08 8;27(8):1344-1359. Epub 2017 Jun 8.

Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin Institute for Medical Systems Biology, 13125 Berlin, Germany.

The cellular response to genotoxic stress is mediated by a well-characterized network of DNA surveillance pathways. The contribution of post-transcriptional gene regulatory networks to the DNA damage response (DDR) has not been extensively studied. Here, we systematically identified RNA-binding proteins differentially interacting with polyadenylated transcripts upon exposure of human breast carcinoma cells to ionizing radiation (IR). Interestingly, more than 260 proteins, including many nucleolar proteins, showed increased binding to poly(A) RNA in IR-exposed cells. The functional analysis of DDX54, a candidate genotoxic stress responsive RNA helicase, revealed that this protein is an immediate-to-early DDR regulator required for the splicing efficacy of its target IR-induced pre-mRNAs. Upon IR exposure, DDX54 acts by increased interaction with a well-defined class of pre-mRNAs that harbor introns with weak acceptor splice sites, as well as by protein-protein contacts within components of U2 snRNP and spliceosomal B complex, resulting in lower intron retention and higher processing rates of its target transcripts. Because DDX54 promotes survival after exposure to IR, its expression and/or mutation rate may impact DDR-related pathologies. Our work indicates the relevance of many uncharacterized RBPs potentially involved in the DDR.
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http://dx.doi.org/10.1101/gr.218438.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5538551PMC
August 2017

An immediate-late gene expression module decodes ERK signal duration.

Mol Syst Biol 2017 05 3;13(5):928. Epub 2017 May 3.

IRI Life Sciences & Institute for Theoretical Biology, Humboldt Universität Berlin, Berlin, Germany

The RAF-MEK-ERK signalling pathway controls fundamental, often opposing cellular processes such as proliferation and apoptosis. Signal duration has been identified to play a decisive role in these cell fate decisions. However, it remains unclear how the different early and late responding gene expression modules can discriminate short and long signals. We obtained both protein phosphorylation and gene expression time course data from HEK293 cells carrying an inducible construct of the proto-oncogene RAF By mathematical modelling, we identified a new gene expression module of immediate-late genes (ILGs) distinct in gene expression dynamics and function. We find that mRNA longevity enables these ILGs to respond late and thus translate ERK signal duration into response amplitude. Despite their late response, their GC-rich promoter structure suggested and metabolic labelling with 4SU confirmed that transcription of ILGs is induced immediately. A comparative analysis shows that the principle of duration decoding is conserved in PC12 cells and MCF7 cells, two paradigm cell systems for ERK signal duration. Altogether, our findings suggest that ILGs function as a gene expression module to decode ERK signal duration.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5448165PMC
http://dx.doi.org/10.15252/msb.20177554DOI Listing
May 2017

Transcriptome-wide Identification of RNA-binding Protein Binding Sites Using Photoactivatable-Ribonucleoside-Enhanced Crosslinking Immunoprecipitation (PAR-CLIP).

Curr Protoc Mol Biol 2017 04 3;118:27.6.1-27.6.19. Epub 2017 Apr 3.

Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany.

RNA-binding proteins (RBPs) mediate important co- and post-transcriptional gene regulation by binding pre-mRNA in a sequence- and/or structure-specific manner. For a comprehensive understanding of RBP function, transcriptome-wide mapping of the RNA-binding sites is essential, and CLIP-seq methods have been developed to elucidate protein/RNA interactions at high resolution. CLIP-seq combines protein/RNA UV-crosslinking with immunoprecipitation (CLIP) followed by high-throughput sequencing of crosslinked RNA fragments. To overcome the limitations of low RNA-protein crosslinking efficiency in standard CLIP-seq, photoactivatable-ribonucleoside-enhanced CLIP (PAR-CLIP) has been developed. Here, living cells or whole organisms are fed photo-activatable nucleoside analogs that are incorporated into nascent RNA transcripts before UV treatment. This allows greater crosslinking efficiency at comparable radiation doses for enhanced RNA recovery and separation of crosslinked target RNA fragments from background RNA degradation products. Moreover, it facilitates the generation of specific UV-induced mutations that mark the crosslinking nucleotide and allow transcriptome-wide identification of RBP binding sites at single-nucleotide resolution. © by 2017 John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpmb.35DOI Listing
April 2017

Translation of CircRNAs.

Mol Cell 2017 Apr 23;66(1):9-21.e7. Epub 2017 Mar 23.

Biological Chemistry Department, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. Electronic address:

Circular RNAs (circRNAs) are abundant and evolutionarily conserved RNAs of largely unknown function. Here, we show that a subset of circRNAs is translated in vivo. By performing ribosome footprinting from fly heads, we demonstrate that a group of circRNAs is associated with translating ribosomes. Many of these ribo-circRNAs use the start codon of the hosting mRNA, are bound by membrane-associated ribosomes, and have evolutionarily conserved termination codons. In addition, we found that a circRNA generated from the muscleblind locus encodes a protein, which we detected in fly head extracts by mass spectrometry. Next, by performing in vivo and in vitro translation assays, we show that UTRs of ribo-circRNAs (cUTRs) allow cap-independent translation. Moreover, we found that starvation and FOXO likely regulate the translation of a circMbl isoform. Altogether, our study provides strong evidence for translation of circRNAs, revealing the existence of an unexplored layer of gene activity.
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http://dx.doi.org/10.1016/j.molcel.2017.02.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5387669PMC
April 2017

JACUSA: site-specific identification of RNA editing events from replicate sequencing data.

BMC Bioinformatics 2017 Jan 3;18(1). Epub 2017 Jan 3.

Section of Bioinformatics and Systems Cardiology, Klaus Tschira Institute for Integrative Computational Cardiology at the Department of Internal Medicine III, University Hospital Heidelberg, Im Neuenheimer Feld 669, Heidelberg, 69120, Germany.

Background: RNA editing is a co-transcriptional modification that increases the molecular diversity, alters secondary structure and protein coding sequences by changing the sequence of transcripts. The most common RNA editing modification is the single base substitution (A→I) that is catalyzed by the members of the Adenosine deaminases that act on RNA (ADAR) family. Typically, editing sites are identified as RNA-DNA-differences (RDDs) in a comparison of genome and transcriptome data from next-generation sequencing experiments. However, a method for robust detection of site-specific editing events from replicate RNA-seq data has not been published so far. Even more surprising, condition-specific editing events, which would show up as differences in RNA-RNA comparisons (RRDs) and depend on particular cellular states, are rarely discussed in the literature.

Results: We present JACUSA, a versatile one-stop solution to detect single nucleotide variant positions from comparing RNA-DNA and/or RNA-RNA sequencing samples. The performance of JACUSA has been carefully evaluated and compared to other variant callers in an in silico benchmark. JACUSA outperforms other algorithms in terms of the F measure, which combines precision and recall, in all benchmark scenarios. This performance margin is highest for the RNA-RNA comparison scenario. We further validated JACUSA's performance by testing its ability to detect A→I events using sequencing data from a human cell culture experiment and publicly available RNA-seq data from Drosophila melanogaster heads. To this end, we performed whole genome and RNA sequencing of HEK-293 cells on samples with lowered activity of candidate RNA editing enzymes. JACUSA has a higher recall and comparable precision for detecting true editing sites in RDD comparisons of HEK-293 data. Intriguingly, JACUSA captures most A→I events from RRD comparisons of RNA sequencing data derived from Drosophila and HEK-293 data sets.

Conclusion: Our software JACUSA detects single nucleotide variants by comparing data from next-generation sequencing experiments (RNA-DNA or RNA-RNA). In practice, JACUSA shows higher recall and comparable precision in detecting A→I sites from RNA-DNA comparisons, while showing higher precision and recall in RNA-RNA comparisons.
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http://dx.doi.org/10.1186/s12859-016-1432-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5210316PMC
January 2017

Eyes on Translation.

Mol Cell 2016 09;63(6):918-25

Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany. Electronic address:

Translation is a fundamental biological process by which ribosomes decode genetic information into proteins. The regulation of this process plays a key role in tuning protein levels, allowing cells to respond rapidly to changes in the environment and to synthesize proteins with precise timing and at specific subcellular locations. Despite detailed biochemical and structural insight into the mechanism of protein synthesis, translational dynamics and localization in a cellular context are less well understood. Here, we summarize recent efforts to quantify and visualize translation, focusing on four publications (Morisaki et al., 2016; Wang et al., 2016; Wu et al., 2016; Yan et al., 2016) describing novel approaches to imaging in real time the synthesis of nascent peptides from individual mRNAs in living cells.
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http://dx.doi.org/10.1016/j.molcel.2016.08.031DOI Listing
September 2016

The Lupus Autoantigen La Prevents Mis-channeling of tRNA Fragments into the Human MicroRNA Pathway.

Mol Cell 2016 07 23;63(1):110-24. Epub 2016 Jun 23.

Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Regensburg 93053, Germany. Electronic address:

The Lupus autoantigen La is an RNA-binding protein that stabilizes RNA polymerase III (Pol III) transcripts and supports RNA folding and has in addition been implicated in the mammalian microRNA (miRNA) pathway. Here, we have analyzed effects of La depletion on Argonaute (Ago)-bound small RNAs in human cells. We find that in the absence of La, distinct tRNA fragments are loaded into Ago proteins. Thus, La functions as gatekeeper ensuring correct tRNA maturation and protecting the miRNA pathway from potentially functional tRNA fragments. However, one specific isoleucin pre-tRNA produces both a functional tRNA and a miRNA even when La is present. We demonstrate that the fully complementary 5' leader and 3' trailer of the pre-tRNA-Ile form a double-stranded RNA molecule that has low affinity to La. Instead, Exportin-5 (Xpo5) recognizes it as miRNA precursor and transports it into the cytoplasm for Dicer processing and Ago loading.
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http://dx.doi.org/10.1016/j.molcel.2016.05.026DOI Listing
July 2016

The mRNA-bound proteome of the early fly embryo.

Genome Res 2016 07 28;26(7):1000-9. Epub 2016 Apr 28.

Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, 13125 Berlin, Germany;

Early embryogenesis is characterized by the maternal to zygotic transition (MZT), in which maternally deposited messenger RNAs are degraded while zygotic transcription begins. Before the MZT, post-transcriptional gene regulation by RNA-binding proteins (RBPs) is the dominant force in embryo patterning. We used two mRNA interactome capture methods to identify RBPs bound to polyadenylated transcripts within the first 2 h of Drosophila melanogaster embryogenesis. We identified a high-confidence set of 476 putative RBPs and confirmed RNA-binding activities for most of 24 tested candidates. Most proteins in the interactome are known RBPs or harbor canonical RBP features, but 99 exhibited previously uncharacterized RNA-binding activity. mRNA-bound RBPs and TFs exhibit distinct expression dynamics, in which the newly identified RBPs dominate the first 2 h of embryonic development. Integrating our resource with in situ hybridization data from existing databases showed that mRNAs encoding RBPs are enriched in posterior regions of the early embryo, suggesting their general importance in posterior patterning and germ cell maturation.
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http://dx.doi.org/10.1101/gr.200386.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4937569PMC
July 2016