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The SARS-Cov-2 RNA–Protein Interactome in Infected Human Cells

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ARTICLES https://doi.org/10.1038/s41564-020-00846-z The SARS-CoV-2 RNA–protein interactome in infected human cells Nora Schmidt 1,10, Caleb A. Lareau 2,10, Hasmik Keshishian3,10, Sabina Ganskih 1, Cornelius Schneider4,5, Thomas Hennig6, Randy Melanson3, Simone Werner1, Yuanjie Wei1, Matthias Zimmer1, Jens Ade 1, Luisa Kirschner6, Sebastian Zielinski 1, Lars Dölken1,6, Eric S. Lander3,7,8, Neva Caliskan1,9, Utz Fischer1,5, Jörg Vogel 1,4, Steven A. Carr3, Jochen Bodem 6 ✉ and Mathias Munschauer 1 ✉ Characterizing the interactions that SARS-CoV-2 viral RNAs make with host cell proteins during infection can improve our understanding of viral RNA functions and the host innate immune response. Using RNA antisense purification and mass spec- trometry, we identified up to 104 human proteins that directly and specifically bind to SARS-CoV-2 RNAs in infected human cells. We integrated the SARS-CoV-2 RNA interactome with changes in proteome abundance induced by viral infection and linked interactome proteins to cellular pathways relevant to SARS-CoV-2 infections. We demonstrated by genetic perturba- tion that cellular nucleic acid-binding protein (CNBP) and La-related protein 1 (LARP1), two of the most strongly enriched viral RNA binders, restrict SARS-CoV-2 replication in infected cells and provide a global map of their direct RNA contact sites. Pharmacological inhibition of three other RNA interactome members, PPIA, ATP1A1, and the ARP2/3 complex, reduced viral replication in two human cell lines. The identification of host dependency factors and defence strategies as presented in this work will improve the design of targeted therapeutics against SARS-CoV-2. he rapid spread of a new severe acute respiratory between viral and host proteins has revealed cellular pathways rele- syndrome-related coronavirus (SARS-CoV-2) around the vant to productive infection12. However, these studies cannot reveal globe has led to a worldwide spike in a SARS-like respira- how viral RNA is regulated during infection or how host cell RNA T 1 13 tory illness termed coronavirus disease 2019 (COVID-19) . To date, metabolism is remodelled to enable virus replication . more than one million lives have been lost due to COVID-19. A We sought to obtain an unbiased and quantitative picture of the detailed understanding of the molecular interactions and perturba- cellular proteins that directly bind to SARS-CoV-2 RNAs in infected tions occurring during SARS-CoV-2 infection is required to under- human cells. Recent RNA capture and quantitative mass spectrom- stand the biology of SARS-CoV-2 and design therapeutic strategies. etry (MS) approaches14–17 applied ultraviolet (UV) crosslinking to SARS-CoV-2 is an enveloped, positive-sense, single-stranded create covalent bonds between RNA molecules and the proteins RNA virus that, upon infection of a host cell, deploys a they directly interact with. Unlike chemical crosslinking, UV irradi- ‘translation-ready’ RNA molecule, which uses the protein synthe- ation does not stabilize protein–protein or RNA–RNA interactions, sis machinery of the host to express a set of viral proteins crucial making it a preferable choice for dissecting direct RNA–pro- for replication2. Replication of the full-length viral genome and tein interactions18,19. RNA antisense purification and quantitative transcription of subgenomic RNAs both involve the synthesis of mass spectrometry (RAP–MS) combines UV crosslinking with a negative-strand RNA intermediates3. In common with other RNA highly denaturing purification procedure and is ideally suited to viruses, SARS-CoV-2 is dependent on effectively engaging host cell capture and identify only those proteins that bind directly to factors such as regulators of RNA stability, processing, localization SARS-CoV-2 RNAs14,15. and translation to facilitate replication and production of progeny. The host cell, on the other hand, must detect the pathogen and Results activate appropriate innate immune response pathways to restrict Capturing SARS-CoV-2 RNAs in infected human cells. To purify virus infection4. SARS-CoV-2 RNAs and the complement of directly crosslinked Studies on SARS-CoV-2-infected human cells to date have cellular proteins from infected human cells, we designed a pool of focused on characterizing expression or modification changes biotinylated DNA oligonucleotides antisense to the positive-sense in the host cell transcriptome5,6 or proteome7–9. Further, interac- SARS-CoV-2 RNA and its subgenomic messenger RNAs. As a cel- tions between recombinant viral proteins and host proteins have lular system, we selected the human liver cell line Huh7, which been identified in uninfected cells10,11. Mapping of the interactions is naturally permissive to both SARS-CoV-1 and SARS-CoV-2 1Helmholtz Institute for RNA-based Infection Research, Helmholtz-Center for Infection Research, Würzburg, Germany. 2School of Medicine, Stanford University, Palo Alto, CA, USA. 3Broad Institute of MIT and Harvard, Cambridge, MA, USA. 4Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany. 5Department of Biochemistry, University of Würzburg, Würzburg, Germany. 6Institute for Virology and Immunobiology, Julius-Maximilians-University Würzburg, Würzburg, Germany. 7Department of Biology, MIT, Cambridge, MA, USA. 8Department of Systems Biology, Harvard Medical School, Boston, MA, USA. 9Faculty of Medicine, University of Würzburg, Würzburg, Germany. 10These authors contributed equally: Nora Schmidt, Caleb A. Lareau, Hasmik Keshishian. ✉e-mail: [email protected]; [email protected] Nature MICROBIOLOgy | VOL 6 | March 2021 | 339–353 | www.nature.com/naturemicrobiology 339 ARTICLES NATURE MICROBIOLOGY a UV crosslink Lysis and denaturing antisense purification Quantitative LC–MS/MS Identification Quantitation Crosslinked protein Benzonase elution, SARS-CoV-2 95% TMT labelling of peptides RNA Intensity m/z AAA SARS- CoV-2 Crosslinked RNA Antisense capture 5% probes NHS beads RNase H elution, Reads Infected covalent protein capture human cell b c Expanded RNA interactome Core RNA interactome 4 Adjusted P value Adjusted P value –2 –3 NSP6 10–1 10–2 10–3 10–4 10 10 CNBP PPIA NSP6 EIF3H ORF3a N EIF1AY S FUBP3 NSP15 LARP4 HNRNPA3 STIP1 RPS4X PUM1 RPS26 S YBX1 ATXN2L NSP9 YBX3 smORF_G043944 2 NSP15 IGF2BP1 N PABPC4 HNRNPAB SYNCRIP GALNT1 G3BP2 ATP1A1 NSP10 NSP12 YTHDF2 RPS11 NSP1 ORF3a STOM RTCB EEF2 M PABPC1 EIF4G1 NSP5 SND1 MSI2 ORF9b M SERBP1 NSP8 EIF4B HNRNPA0 UPF1 CCT3 NSP3 EIF4H RPL21 MOV10 RPS12 CSDE1 RPS5 –4 –2 NSP16 2 4 A1CF NSP10 RBMS2 PCBP2 RAB6A RPL3 RAB6D APOE PPP1CC EIF3G RPS2 EIF3C SCFD1 RPL28 NSP12 EEF1A1 CAPRIN1 RPL8 DDX3X RPL18A EIF3E PFN1 TMT ratio (SARS-CoV-2/RMRP) replicate 2 RPS14 CNBP 2 –2 NSP16 NSP1 RPP30 RPS10 ACTA1 RPP25 RPL15 LASP1 Log SARS-CoV-2 proteins LSM14A TAGLN2 POP1 HNRNPA1 HNRNPA2B1 IGF2BP2 ORF9b POP4 Human SARS-CoV-2 RNA LIN28B RPL36AL interactome (adjusted P < 0.05) RYDEN NSP3 POP7 STRAP PGK1 NSP9 PEBP1 RMRP subunits (adjusted P < 0.05) ACTR2 RAB7A ANXA1 ALMS1 USO1 ACTB –4 RPL6 EGFR CFL1 SRI DDX1 G3BP1 HDLBP EIF3L EIF5A HNRNPL Log2 TMT ratio (SARS-CoV-2/RMRP) replicate 1 PPP1CB GSPT1 RPL13 GDI2 RPS3 RBM47 GPI YWHAZ RPL7A PURB Fig. 1 | RNA–protein interactome of SARS-CoV-2 in infected human cells. a, Outline of the RAP–MS method to identify proteins bound to SARS-CoV-2 RNA and their crosslinked RNA sequences. b, Quantification of SARS-CoV-2 RNA-interacting proteins relative to RMRP-interacting proteins. The scatter plot of log2-transformed TMT ratios from two biological replicates is shown. The grey dots represent all proteins detected with two or more unique peptides. c, Proteins enriched in SARS-CoV-2 RNA purifications (Supplementary Table 1). Left: core SARS-CoV-2 RNA interactome (adjusted P < 0.05). Left and right: expanded SARS-CoV-2 RNA interactome. Significantly enriched proteins are highlighted in teal; SARS-CoV-2-encoded proteins are highlighted in magenta. Adjusted P value: two-tailed moderated t-test. replication20,21. SARS-CoV-2 preferentially infects cells in the originating from SARS-CoV-2 RNA made up 93 and 92% of all respiratory tract, but infection of multiple organs, including the mapped reads in 2 highly correlated replicate experiments (r = 0.994; liver, has been reported22. Extended Data Fig. 1b,c). To test if our pool of antisense capture probes was suitable for To identify proteins that specifically interact with SARS-CoV-2 the purification of SARS-CoV-2 RNAs from infected Huh7 cells, we RNAs as opposed to non-specific background proteins, we com- performed RAP–MS 24 h after infection when viral replication levels pared the protein content of SARS-CoV-2 RNA purifications to were high21. We implemented a covalent protein capture step after that of an unrelated control ribonucleoprotein complex of known the release of SARS-CoV-2 RNA-bound proteins, which enabled composition. As the control, we used the endogenously expressed us to identify RNA sequences crosslinked to purified proteins human ribonuclease mitochondrial RNA processing (RMRP) RNA (Fig. 1a and Methods). Protein-crosslinked RNA fragments mapped and purified both SARS-CoV-2 RNA and RMRP from infected to the entire length of the viral genome with near-complete sequence Huh7 cells. RMRP was selected for several reasons: (1) RMRP inter- coverage, indicating that interactions across all viral RNA acts with approximately ten well-known proteins that serve as an regions were captured (Extended Data Fig. 1a). Sequencing reads internal control15,23; (2) RMRP is not translated; and (3) RMRP does 340 Nature MICROBIOLOgy | VOL 6 | March 2021 | 339–353 | www.nature.com/naturemicrobiology NATURE MICROBIOLOGY ARTICLES not globally bind to mRNA. Hence, RMRP-binding proteins are dis- aimed at capturing proteins that crosslink to RNA36 (Supplementary tinct from the group of proteins expected to bind to SARS-CoV-2 Table 2). Comparing this expanded SARS-CoV-2 RNA interactome RNAs, making it an ideal control for the discovery of unknown with the poly(A)-RNA interactome in Huh7 cells37, revealed high interactors.
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  • WO 2019/079361 Al 25 April 2019 (25.04.2019) W 1P O PCT

    WO 2019/079361 Al 25 April 2019 (25.04.2019) W 1P O PCT

    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization I International Bureau (10) International Publication Number (43) International Publication Date WO 2019/079361 Al 25 April 2019 (25.04.2019) W 1P O PCT (51) International Patent Classification: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, C12Q 1/68 (2018.01) A61P 31/18 (2006.01) DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, C12Q 1/70 (2006.01) HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, (21) International Application Number: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, PCT/US2018/056167 OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (22) International Filing Date: SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, 16 October 2018 (16. 10.2018) TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (25) Filing Language: English (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (26) Publication Language: English GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, (30) Priority Data: UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, 62/573,025 16 October 2017 (16. 10.2017) US TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, ΓΕ , IS, IT, LT, LU, LV, (71) Applicant: MASSACHUSETTS INSTITUTE OF MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TECHNOLOGY [US/US]; 77 Massachusetts Avenue, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, Cambridge, Massachusetts 02139 (US).
  • RNA Dynamics in Alzheimer's Disease

    RNA Dynamics in Alzheimer's Disease

    molecules Review RNA Dynamics in Alzheimer’s Disease Agnieszka Rybak-Wolf 1,* and Mireya Plass 2,3,4,* 1 Max Delbrück Center for Molecular Medicine (MDC), Berlin Institute for Medical Systems Biology (BIMSB), 10115 Berlin, Germany 2 Gene Regulation of Cell Identity, Regenerative Medicine Program, Bellvitge Institute for Biomedical Research (IDIBELL), L’Hospitalet del Llobregat, 08908 Barcelona, Spain 3 Program for Advancing Clinical Translation of Regenerative Medicine of Catalonia, P-CMR[C], L’Hospitalet del Llobregat, 08908 Barcelona, Spain 4 Center for Networked Biomedical Research on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), 28029 Madrid, Spain * Correspondence: [email protected] (A.R.-W.); [email protected] (M.P.) Abstract: Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder that heavily burdens healthcare systems worldwide. There is a significant requirement to understand the still unknown molecular mechanisms underlying AD. Current evidence shows that two of the major features of AD are transcriptome dysregulation and altered function of RNA binding proteins (RBPs), both of which lead to changes in the expression of different RNA species, including microRNAs (miRNAs), circular RNAs (circRNAs), long non-coding RNAs (lncRNAs), and messenger RNAs (mRNAs). In this review, we will conduct a comprehensive overview of how RNA dynamics are altered in AD and how this leads to the differential expression of both short and long RNA species. We will describe how RBP expression and function are altered in AD and how this impacts the expression of different RNA species. Furthermore, we will also show how changes in the abundance Citation: Rybak-Wolf, A.; Plass, M.