Adenovirus-mediated ubiquitination alters protein–RNA binding and aids viral RNA processing

Abstract

Viruses promote infection by hijacking the ubiquitin machinery of the host to counteract or redirect cellular processes. Adenovirus encodes two early proteins, E1B55K and E4orf6, that together co-opt a cellular ubiquitin ligase complex to overcome host defences and promote virus production. Adenovirus mutants lacking E1B55K or E4orf6 display defects in viral RNA processing and protein production, but previously identified substrates of the redirected ligase do not explain these phenotypes. Here, we used a quantitative proteomics approach to identify substrates of E1B55K/E4orf6-mediated ubiquitination that facilitate RNA processing. While all currently known cellular substrates of E1B55K and E4orf6 are degraded by the proteasome, we uncovered RNA-binding proteins as high-confidence substrates that are not decreased in overall abundance. We focused on two RNA-binding proteins, RALY and hnRNP-C, which we confirm are ubiquitinated without degradation. Knockdown of RALY and hnRNP-C increased levels of viral RNA splicing, protein abundance and progeny production during infection with E1B55K-deleted virus. Furthermore, infection with E1B55K-deleted virus resulted in an increased interaction of hnRNP-C with viral RNA and attenuation of viral RNA processing. These data suggest that viral-mediated ubiquitination of RALY and hnRNP-C relieves a restriction on viral RNA processing and reveal an unexpected role for non-degradative ubiquitination in the manipulation of cellular processes during virus infection.

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Fig. 1: E1B55K deletion or inhibition of cullin-mediated ubiquitination decreases Ad late RNA splicing and overall RNA processing.
Fig. 2: Unbiased proteomics reveals RBPs among putative non-degraded substrates of the Ad E1B55K–E4orf6 complex.
Fig. 3: RALY and hnRNP-C are non-degraded substrates of Ad E1B55K/E4orf6-mediated ubiquitination.
Fig. 4: Knockdown of RALY and hnRNP-C rescues the RNA processing defect caused by the absence of a functional E1B55K–E4orf6 complex.
Fig. 5: Interactions of hnRNP-C with viral late RNA increases in the absence of a functional E1B55K–E4orf6 complex.

Data availability

All MS data for this study have been deposited in the CHORUS database under project ID 1648, experiment IDs 3476 (K-ɛ-GG), 3477 (K-ɛ-GG-matched whole-cell proteome) and 3478 (RBR-ID of hnRNP-C). The eCLIP-seq data have been deposited in the NCBI’s Gene Expression Omnibus (GEO) and are accessible through GEO series accession number GSE145411. The eCLIP-Seq data have been deposited in the GEO97 and are accessible through GEO series accession number GSE145411. Additional supporting data are available from the corresponding author upon request. Source data are provided with this paper.

Code availability

The proteomics data were analysed using standard methods. The implementation of the analysis was performed using R software. The scripts are available from the corresponding author upon request or can be accessed via GitHub at https://github.com/JosephDybas/AdenovirusProteomics.

References

  1. 1.

    Isaacson, M. K. & Ploegh, H. L. Ubiquitination, ubiquitin-like modifiers, and deubiquitination in viral infection. Cell Host Microbe 5, 559–570 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Luo, H. Interplay between the virus and the ubiquitin–proteasome system: molecular mechanism of viral pathogenesis. Curr. Opin. Virol. 17, 1–10 (2016).

    CAS  PubMed  Google Scholar 

  3. 3.

    Dybas, J. M., Herrmann, C. & Weitzman, M. D. Ubiquitination at the interface of tumor viruses and DNA damage responses. Curr. Opin. Virol. 32, 40–47 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Komander, D. & Rape, M. The ubiquitin code. Annu. Rev. Biochem. 81, 203–229 (2012).

    CAS  PubMed  Google Scholar 

  5. 5.

    Oh, E., Akopian, D. & Rape, M. Principles of ubiquitin-dependent signaling. Annu. Rev. Cell Dev. Biol. 34, 137–162 (2018).

    CAS  PubMed  Google Scholar 

  6. 6.

    Kulathu, Y. & Komander, D. Atypical ubiquitylation—the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages. Nat. Rev. Mol. Cell Biol. 13, 508–523 (2012).

    CAS  PubMed  Google Scholar 

  7. 7.

    Petroski, M. D. & Deshaies, R. J. Function and regulation of cullin-RING ubiquitin ligases. Nat. Rev. Mol. Cell Biol. 6, 9–20 (2005).

    CAS  PubMed  Google Scholar 

  8. 8.

    Querido, E. et al. Degradation of p53 by adenovirus E4orf6 and E1B55K proteins occurs via a novel mechanism involving a cullin-containing complex. Genes Dev. 15, 3104–3117 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Harada, J. N., Shevchenko, A., Shevchenko, A., Pallas, D. C. & Berk, A. J. Analysis of the adenovirus E1B-55K-anchored proteome reveals its link to ubiquitination machinery. J. Virol. 76, 9194–9206 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Babiss, L. E. & Ginsberg, H. S. Adenovirus type 5 early region 1b gene product is required for efficient shutoff of host protein synthesis. J. Virol. 50, 202–212 (1984).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Babiss, L. E., Ginsberg, H. S. & Darnell, J. E. Jr. Adenovirus E1B proteins are required for accumulation of late viral mRNA and for effects on cellular mRNA translation and transport. Mol. Cell. Biol. 5, 2552–2558 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Halbert, D. N., Cutt, J. R. & Shenk, T. Adenovirus early region 4 encodes functions required for efficient DNA replication, late gene expression, and host cell shutoff. J. Virol. 56, 250–257 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Pilder, S., Moore, M., Logan, J. & Shenk, T. The adenovirus E1B-55K transforming polypeptide modulates transport or cytoplasmic stabilization of viral and host cell mRNAs. Mol. Cell. Biol. 6, 470–476 (1986).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Bridge, E. & Ketner, G. Redundant control of adenovirus late gene expression by early region 4. J. Virol. 63, 631–638 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Bridge, E. & Ketner, G. Interaction of adenoviral E4 and E1b products in late gene expression. Virology 174, 345–353 (1990).

    CAS  PubMed  Google Scholar 

  16. 16.

    Sandler, A. B. & Ketner, G. Adenovirus early region 4 is essential for normal stability of late nuclear RNAs. J. Virol. 63, 624–630 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Woo, J. L. & Berk, A. J. Adenovirus ubiquitin-protein ligase stimulates viral late mRNA nuclear export. J. Virol. 81, 575–587 (2007).

    CAS  PubMed  Google Scholar 

  18. 18.

    Blanchette, P. et al. Control of mRNA export by adenovirus E4orf6 and E1B55K proteins during productive infection requires E4orf6 ubiquitin ligase activity. J. Virol. 82, 2642–2651 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Querido, E. et al. Regulation of p53 levels by the E1B 55-kilodalton protein and E4orf6 in adenovirus-infected cells. J. Virol. 71, 3788–3798 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Stracker, T. H., Carson, C. T. & Weitzman, M. D. Adenovirus oncoproteins inactivate the Mre11–Rad50–NBS1 DNA repair complex. Nature 418, 348–352 (2002).

    CAS  PubMed  Google Scholar 

  21. 21.

    Baker, A., Rohleder, K. J., Hanakahi, L. A. & Ketner, G. Adenovirus E4 34k and E1b 55k oncoproteins target host DNA ligase IV for proteasomal degradation. J. Virol. 81, 7034–7040 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Dallaire, F., Blanchette, P., Groitl, P., Dobner, T. & Branton, P. E. Identification of integrin alpha3 as a new substrate of the adenovirus E4orf6/E1B 55-kilodalton E3 ubiquitin ligase complex. J. Virol. 83, 5329–5338 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Orazio, N. I., Naeger, C. M., Karlseder, J. & Weitzman, M. D. The adenovirus E1b55K/E4orf6 complex induces degradation of the Bloom helicase during infection. J. Virol. 85, 1887–1892 (2011).

    CAS  PubMed  Google Scholar 

  24. 24.

    Cathomen, T. & Weitzman, M. D. A functional complex of adenovirus proteins E1B-55kDa and E4orf6 is necessary to modulate the expression level of p53 but not its transcriptional activity. J. Virol. 74, 11407–11412 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Carson, C. T. et al. The Mre11 complex is required for ATM activation and the G2/M checkpoint. EMBO J. 22, 6610–6620 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Schwartz, R. A. et al. Distinct requirements of adenovirus E1b55K protein for degradation of cellular substrates. J. Virol. 82, 9043–9055 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Xu, G., Paige, J. S. & Jaffrey, S. R. Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling. Nat. Biotechnol. 28, 868–873 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Udeshi, N. D., Mertins, P., Svinkina, T. & Carr, S. A. Large-scale identification of ubiquitination sites by mass spectrometry. Nat. Protoc. 8, 1950–1960 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Soucy, T. A. et al. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature 458, 732–736 (2009).

    CAS  PubMed  Google Scholar 

  30. 30.

    Hori, T. et al. Covalent modification of all members of human cullin family proteins by NEDD8. Oncogene 18, 6829–6834 (1999).

    CAS  PubMed  Google Scholar 

  31. 31.

    Schwechheimer, C. NEDD8—its role in the regulation of cullin-RING ligases. Curr. Opin. Plant Biol. 45, 112–119 (2018).

    CAS  PubMed  Google Scholar 

  32. 32.

    Zhou, P. & Howley, P. M. Ubiquitination and degradation of the substrate recognition subunits of SCF ubiquitin-protein ligases. Mol. Cell 2, 571–580 (1998).

    CAS  PubMed  Google Scholar 

  33. 33.

    Tudek, A., Schmid, M. & Jensen, T. H. Escaping nuclear decay: the significance of mRNA export for gene expression. Curr. Genet. 65, 473–476 (2019).

    CAS  PubMed  Google Scholar 

  34. 34.

    Stewart, M. Polyadenylation and nuclear export of mRNAs. J. Biol. Chem. 294, 2977–2987 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Marcellus, R. C., Teodoro, J. G., Charbonneau, R., Shore, G. C. & Branton, P. E. Expression of p53 in Saos-2 osteosarcoma cells induces apoptosis which can be inhibited by Bcl-2 or the adenovirus E1B-55 kDa protein. Cell Growth Differ. 7, 1643–1650 (1996).

    CAS  PubMed  Google Scholar 

  36. 36.

    Fabregat, A. et al. The reactome pathway knowledgebase. Nucleic Acids Res. 46, D649–D655 (2018).

    CAS  PubMed  Google Scholar 

  37. 37.

    Konig, J. et al. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat. Struct. Mol. Biol. 17, 909–915 (2010).

    PubMed  PubMed Central  Google Scholar 

  38. 38.

    McCloskey, A., Taniguchi, I., Shinmyozu, K. & Ohno, M. hnRNP C tetramer measures RNA length to classify RNA polymerase II transcripts for export. Science 335, 1643–1646 (2012).

    CAS  PubMed  Google Scholar 

  39. 39.

    Zarnack, K. et al. Direct competition between hnRNP C and U2AF65 protects the transcriptome from the exonization of Alu elements. Cell 152, 453–466 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Cornella, N. et al. The hnRNP RALY regulates transcription and cell proliferation by modulating the expression of specific factors including the proliferation marker E2F1. J. Biol. Chem. 292, 19674–19692 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Rossi, A. et al. Identification and dynamic changes of RNAs isolated from RALY-containing ribonucleoprotein complexes. Nucleic Acids Res. 45, 6775–6792 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Bondy-Chorney, E. et al. RNA binding protein RALY promotes protein arginine methyltransferase 1 alternatively spliced isoform v2 relative expression and metastatic potential in breast cancer cells. Int. J. Biochem. Cell Biol. 91, 124–135 (2017).

    CAS  PubMed  Google Scholar 

  43. 43.

    van Eekelen, C. et al. Sequence dependent interaction of hnRNP proteins with late adenoviral transcripts. Nucleic Acids Res. 10, 7115–7131 (1982).

    PubMed  PubMed Central  Google Scholar 

  44. 44.

    Tenzer, S. et al. Proteome-wide characterization of the RNA-binding protein RALY-interactome using the in vivo-biotinylation-pulldown-quant (iBioPQ) approach. J. Proteome Res. 12, 2869–2884 (2013).

    CAS  PubMed  Google Scholar 

  45. 45.

    Graham, F. L., Smiley, J., Russell, W. C. & Nairn, R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. 36, 59–74 (1977).

    CAS  PubMed  Google Scholar 

  46. 46.

    Mevissen, T. E. T. & Komander, D. Mechanisms of deubiquitinase specificity and regulation. Annu. Rev. Biochem. 86, 159–192 (2017).

    CAS  PubMed  Google Scholar 

  47. 47.

    Chahal, J. S. & Flint, S. J. Timely synthesis of the adenovirus type 5 E1B 55-kilodalton protein is required for efficient genome replication in normal human cells. J. Virol. 86, 3064–3072 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Mashtalir, N. et al. Autodeubiquitination protects the tumor suppressor BAP1 from cytoplasmic sequestration mediated by the atypical ubiquitin ligase UBE2O. Mol. Cell 54, 392–406 (2014).

    CAS  PubMed  Google Scholar 

  49. 49.

    Bridge, E. & Pettersson, U. Nuclear organization of adenovirus RNA biogenesis. Exp. Cell Res. 229, 233–239 (1996).

    CAS  PubMed  Google Scholar 

  50. 50.

    Pombo, A., Ferreira, J., Bridge, E. & Carmo-Fonseca, M. Adenovirus replication and transcription sites are spatially separated in the nucleus of infected cells. EMBO J. 13, 5075–5085 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Huang, M. et al. The C-protein tetramer binds 230 to 240 nucleotides of pre-mRNA and nucleates the assembly of 40S heterogeneous nuclear ribonucleoprotein particles. Mol. Cell. Biol. 14, 518–533 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    He, C. et al. High-resolution mapping of RNA-binding regions in the nuclear proteome of embryonic stem cells. Mol. Cell 64, 416–430 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Zhang, Q. et al. RNA exploits an exposed regulatory site to inhibit the enzymatic activity of PRC2. Nat. Struct. Mol. Biol. 26, 237–247 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Ong, S. E. et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteom. 1, 376–386 (2002).

    CAS  Google Scholar 

  55. 55.

    Gorlach, M., Burd, C. G. & Dreyfuss, G. The determinants of RNA-binding specificity of the heterogeneous nuclear ribonucleoprotein C proteins. J. Biol. Chem. 269, 23074–23078 (1994).

    CAS  PubMed  Google Scholar 

  56. 56.

    Van Nostrand, E. L. et al. Principles of RNA processing from analysis of enhanced CLIP maps for 150 RNA binding proteins. Genome Biol. 21, 90 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Van Nostrand, E. L. et al. Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat. Methods 13, 508–514 (2016).

    PubMed  PubMed Central  Google Scholar 

  58. 58.

    Dallaire, F., Blanchette, P. & Branton, P. E. A proteomic approach to identify candidate substrates of human adenovirus E4orf6–E1B55K and other viral cullin-based E3 ubiquitin ligases. J. Virol. 83, 12172–12184 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Evans, V. C. et al. De novo derivation of proteomes from transcriptomes for transcript and protein identification. Nat. Methods 9, 1207–1211 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Fu, Y. R. et al. Comparison of protein expression during wild-type, and E1B-55k-deletion, adenovirus infection using quantitative time-course proteomics. J. Gen. Virol. 98, 1377–1388 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61.

    Hung, G. & Flint, S. J. Normal human cell proteins that interact with the adenovirus type 5 E1B 55kDa protein. Virology 504, 12–24 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Chanarat, S. & Mishra, S. K. Emerging roles of ubiquitin-like proteins in pre-mRNA splicing. Trends Biochem. Sci. 43, 896–907 (2018).

    CAS  PubMed  Google Scholar 

  63. 63.

    Vassileva, M. T. & Matunis, M. J. SUMO modification of heterogeneous nuclear ribonucleoproteins. Mol. Cell. Biol. 24, 3623–3632 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. 64.

    Chan, C. H. et al. The Skp2-SCF E3 ligase regulates Akt ubiquitination, glycolysis, herceptin sensitivity, and tumorigenesis. Cell 149, 1098–1111 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. 65.

    Lee, S. W. et al. Skp2-dependent ubiquitination and activation of LKB1 is essential for cancer cell survival under energy stress. Mol. Cell 57, 1022–1033 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. 66.

    Zhang, Q. et al. FBXW7 facilitates nonhomologous end-joining via K63-linked polyubiquitylation of XRCC4. Mol. Cell 61, 419–433 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. 67.

    Bischoff, J. R. et al. An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. Science 274, 373–376 (1996).

    CAS  PubMed  Google Scholar 

  68. 68.

    Goodrum, F. D. & Ornelles, D. A. p53 status does not determine outcome of E1B 55-kilodalton mutant adenovirus lytic infection. J. Virol. 72, 9479–9490 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. 69.

    O’Shea, C. C. et al. Late viral RNA export, rather than p53 inactivation, determines ONYX-015 tumor selectivity. Cancer Cell 6, 611–623 (2004).

    PubMed  Google Scholar 

  70. 70.

    Khandelia, P., Yap, K. & Makeyev, E. V. Streamlined platform for short hairpin RNA interference and transgenesis in cultured mammalian cells. Proc. Natl Acad. Sci. USA 108, 12799–12804 (2011).

    CAS  PubMed  Google Scholar 

  71. 71.

    Grifman, M. et al. Overexpression of cyclin A inhibits augmentation of recombinant adeno-associated virus transduction by the adenovirus E4orf6 protein. J. Virol. 73, 10010–10019 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. 72.

    Sobhian, B. et al. RAP80 targets BRCA1 to specific ubiquitin structures at DNA damage sites. Science 316, 1198–1202 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. 73.

    Kozarsky, K. F., Jooss, K., Donahee, M., Strauss, J. F. III. & Wilson, J. M. Effective treatment of familial hypercholesterolaemia in the mouse model using adenovirus-mediated transfer of the VLDL receptor gene. Nat. Genet. 13, 54–62 (1996).

    CAS  PubMed  Google Scholar 

  74. 74.

    Komatsu, T., Dacheux, D., Kreppel, F., Nagata, K. & Wodrich, H. A method for visualization of incoming adenovirus chromatin complexes in fixed and living cells. PLoS ONE 10, e0137102 (2015).

    PubMed  PubMed Central  Google Scholar 

  75. 75.

    Reich, N. C., Sarnow, P., Duprey, E. & Levine, A. J. Monoclonal antibodies which recognize native and denatured forms of the adenovirus DNA-binding protein. Virology 128, 480–484 (1983).

    CAS  PubMed  Google Scholar 

  76. 76.

    Sarnow, P., Sullivan, C. A. & Levine, A. J. A monoclonal antibody detecting the adenovirus type 5-E1b-58Kd tumor antigen: characterization of the E1b-58Kd tumor antigen in adenovirus-infected and -transformed cells. Virology 120, 510–517 (1982).

    CAS  PubMed  Google Scholar 

  77. 77.

    Marton, M. J., Baim, S. B., Ornelles, D. A. & Shenk, T. The adenovirus E4 17-kilodalton protein complexes with the cellular transcription factor E2F, altering its DNA-binding properties and stimulating E1A-independent accumulation of E2 mRNA. J. Virol. 64, 2345–2359 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  78. 78.

    Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A. & Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877–879 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. 79.

    Shaffer, S. M. et al. Rare cell variability and drug-induced reprogramming as a mode of cancer drug resistance. Nature 546, 431–435 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. 80.

    Poling, B. C., Price, A. M., Luftig, M. A. & Cullen, B. R. The Epstein–Barr virus miR-BHRF1 microRNAs regulate viral gene expression in cis. Virology 512, 113–123 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. 81.

    Russo, J., Heck, A. M., Wilusz, J. & Wilusz, C. J. Metabolic labeling and recovery of nascent RNA to accurately quantify mRNA stability. Methods 120, 39–48 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  82. 82.

    Price, A. M., Messinger, J. E. & Luftig, M. A. c-Myc represses transcription of Epstein–Barr virus latent membrane protein 1 early after primary B cell infection. J. Virol. 92, e01178-17 (2018).

    PubMed  PubMed Central  Google Scholar 

  83. 83.

    Dolken, L. et al. High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay. RNA 14, 1959–1972 (2008).

    PubMed  PubMed Central  Google Scholar 

  84. 84.

    Van Nostrand, E. L. et al. Robust, cost-effective profiling of RNA binding protein targets with single-end enhanced crosslinking and immunoprecipitation (seCLIP). Methods Mol. Biol. 1648, 177–200 (2017).

    PubMed  PubMed Central  Google Scholar 

  85. 85.

    Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17, 10–12 (2011).

    Google Scholar 

  86. 86.

    Smith, T., Heger, A. & Sudbery, I. UMI-tools: modeling sequencing errors in unique molecular identifiers to improve quantification accuracy. Genome Res. 27, 491–499 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  87. 87.

    Wu, T. D. & Nacu, S. Fast and SNP-tolerant detection of complex variants and splicing in short reads. Bioinformatics 26, 873–881 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  88. 88.

    Lovci, M. T. et al. Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges. Nat. Struct. Mol. Biol. 20, 1434–1442 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  89. 89.

    Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  90. 90.

    Dallaire, F. et al. The human adenovirus type 5 E4orf6/E1B55K E3 ubiquitin ligase complex can mimic E1A effects on E2F. mSphere 1, e00015-15 (2016).

    PubMed  Google Scholar 

  91. 91.

    Schwanhausser, B. et al. Global quantification of mammalian gene expression control. Nature 473, 337–342 (2011).

    PubMed  Google Scholar 

  92. 92.

    Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498–2504 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  93. 93.

    Jedrychowski, M. P. et al. Evaluation of HCD- and CID-type fragmentation within their respective detection platforms for murine phosphoproteomics. Mol. Cell. Proteom. 10, M111.009910 (2011).

    Google Scholar 

  94. 94.

    The UniProt Consortium.UniProt: a worldwide hub of protein knowledge. Nucleic Acids Res. 47, D506–D515 (2018).

    PubMed Central  Google Scholar 

  95. 95.

    The, M., MacCoss, M. J., Noble, W. S. & Kall, L. Fast and accurate protein false discovery rates on large-scale proteomics data sets with percolator 3.0. J. Am. Soc. Mass. Spectrom. 27, 1719–1727 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  96. 96.

    Wei, T. & Simko, V. corrplot: visualization of a correlation matrix. R package version 0.84 https://github.com/taiyun/corrplot (2017).

  97. 97.

    Edgar, R., Domrachev, M. & Lash, A. E. Gene expression omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 30, 207–210 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank members of the Weitzman Lab for insightful discussions and input. We thank P. Choi for help with eCLIP-seq, and G. Yeo and E. Van Nostrand for their input regarding analysis of corresponding data. We are grateful to A. Berk, P. Branton, R. Greenberg, G. Ketner, A. Levine, K. Lynch, D. Ornelles, J. Wilson and H. Wodrich for gifts of reagents. We thank S. Schaffer for technical advice, and D. Avgousti, L. Busino, P. Choi, K. Lynch and J. Weitzman for careful reading of the manuscript. We thank the UPenn Cell and Developmental Biology Microscopy Core for imaging assistance. This research was supported by NIAID grant nos. R01-AI145266 (to M.D.W.), R01-AI121321 (to M.D.W.) and R01-AI118891 (to B.A.G.), and NCI grant no. R01-CA97093 (to M.D.W.). Additional support came from the NCI T32 Training Grant in Tumor Virology T32-CA115299 (to A.M.P.) and Individual National Research Service Awards F32-AI147587 (to J.M.D.) and F32-AI138432 (to A.M.P.) from the National Institutes of Health.

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M.D.W., C.H., J.M.D. and J.C.L. conceived the project. C.H. performed the experiments and received assistance from J.C.L., A.M.P. and E.T.K. C.H. prepared the figures with input from the other authors. J.M.D. performed the bioinformatics and proteomics data analyses. J.C.L. and R.L. performed the MS experiments. A.M.P. performed RNA FISH. A.M.P., C.E.P. and C.H. performed RT–qPCR, CLIP–qPCR and eCLIP-seq sample preparation. K.E.H. performed the bioinformatics analysis of the eCLIP-seq data. M.C. performed microscopy. C.H., J.C.L. and E.T.K. performed the IP assays. C.H., J.M.D., J.C.L. and M.D.W. wrote the manuscript with input from the other authors. M.D.W. and B.A.G. supervised the research.

Corresponding author

Correspondence to Matthew D. Weitzman.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 E1B55K deletion or inhibition of Cullin-mediated ubiquitination does not decrease late RNA transcription, decay, or early RNA processing.

a, Quantification of results shown in Fig. 1b. b, Analysis of nascent transcription and mRNA half-life by labeling RNA with 4-thiouridine (4sU) for 30 min at 23.5 hpi in HeLa cells infected with WT or ΔE1B Ad5 at m.o.i. = 10. Nascent 4sU-labeled RNA was purified for RT-qPCR to determine relative transcription rates of two early (E1A and E4) and one late (MLP) viral transcripts. mRNA half-life was approximated using the ratio of nascent and total input RNA levels normalized to GAPDH. c, Analysis of decay of viral early (E1A) and late (MLP) RNA species by Actinomycin D pulse at 24 hpi in HeLa cells infected with WT or ΔE1B Ad5 at m.o.i. 10 by normalization to input levels. d, Schematic illustrating primer design to differentiate spliced and unspliced viral transcripts. e-g, HeLa cells infected with WT or ΔE1B Ad5 (MOI = 10) in the presence of DMSO or NEDDi (neddylation inhibitor MLN4924) added at 8 hours post-infection (hpi). Cells were harvested for RNA analysis at 24 hpi. e, Bar graph representing spliced RNA levels of viral early transcripts E1A by RT-qPCR, f, Bar graph representing splicing efficiency as the ratio of spliced to unspliced transcripts of E1A relative to the WT DMSO control by RT-qPCR. g, Representative RNA FISH visualizing the localization of fiber (green) and GAPDH (magenta) transcripts in relation to nuclear DNA stained with DAPI (blue). Nuclei are labeled with the classification of each cell according to the pattern of fiber used for Fig. 1d. Scale bar=10 μm. All data are representative of two (g) or three (others) biologically independent experiments. All graphs show mean+SD. Statistical significance was calculated using a paired (a) or unpaired (others), two-tailed Student’s t-test, * p < 0.05, ** p < 0.01. Source data

Extended Data Fig. 2 Quantification for number of peptides and proteins identified in di-glycine remnant profiling and whole cell proteome data sets.

a-c, HeLa cells transduced with rAd E1B55K/E4orf6 (m.o.i. = 10). a, Immunoblot of time course of E1B55K/E4of6 expression showing degradation kinetics of known substrates. hpt=hours post-transduction. Representative of 3 biologically independent experiments. b-c, Peptide and protein counts for three biologically independent replicates (dark, medium, and light blue bars). b, Total peptides and corresponding proteins identified following K-ɛ-GG antibody enrichment combined with mass spectrometry analysis at 0, 6, 8, and 10 hours post E1B55K/E4orf6 expression. c, Total proteins identified by whole cell proteomics analysis at time 0 and 10 h of E1B55K/E4orf6 expression. Source data

Extended Data Fig. 3 Di-glycine remnant profiling and whole cell proteome data for RNA-binding proteins enriched within predicted E1B55K/E4orf6 substrates.

a-b, Gene ontology analysis identified RNA-binding proteins enriched in proteins that exhibited an increase in normalized protein-based ubiquitin abundance of log2 fold change >1 following 10 h expression of E1B55K/E4orf6. a, Enriched RNA-binding proteins ubiquitination log2 fold changes (left) and whole cell protein abundance log2 fold changes (right) comparing 0 and 10 h of E1B55K/E4orf6 expression. b, Heat map showing relative ubiquitination of indicated lysine residues at 0, 6, 8, and 10 h of E1B55K/E4orf6 expression within enriched RNA-binding proteins. Colors correspond to the average z-score of the ubiquitination. All data are representative of three biologically independent experiments.

Extended Data Fig. 4 Network analysis of predicted E1B55K/E4orf6 substrates identifies a ‘RALY/hnRNP-C module’ enriched for RNA-binding proteins.

a, The Reactome-FI application in Cytoscape was utilized to generate a protein-protein interaction network for proteins increased in ubiquitination following E1B55K/E4orf6 transduction. Nodes represent proteins and edges represent Reactome-based protein-protein interactions. Node size corresponds to relative protein-based ubiquitination log2 fold change and node color corresponds to whole cell proteome log2 fold change following 10 h expression of E1B55K/E4orf6. Reactome-FI interaction module analysis was performed to identify clusters of highly interacting proteins. b, Gene ontology analysis for molecular function, as generated by ReactomeFI (FDR is Benjamini-Hochberg method), identified enrichment of RNA-binding and Poly(A) RNA-binding proteins within the RALY/hnRNP-C network module. c, RALY/hnRNP-C network module protein ubiquitin log2 fold changes (left) and whole cell protein abundance log2 fold changes (right) comparing 0 and 10 h of E1B55K/E4orf6 expression. d, Heat map showing relative ubiquitin abundance of indicated lysine residues at 0, 6, 8, and 10 h of E1B55K/E4orf6 expression from proteins within the RALY/hnRNP-C network module. Colors correspond to the average z-score of ubiquitination. All data are representative of three biologically independent experiments.

Extended Data Fig. 5

MS2 evidence for ubiquitination site localization in RALY (a) and hnRNP-C (bf) peptides. Spectra were obtained from LC-MS/MS analyses using collision-induced dissociation (CID) at 35%, and identified in MaxQuant 1.6.0.16. All modified residues can be confidently identified by confirming ions, except for hnRNP-C K198 (d), which lacks ions to distinguish between K197 and K198. Best evidence spectra were selected for annotation of b-ion (blue) and y-ion (red) series and their masses for singly- and doubly-charged fragments. All data are representative of three biologically independent experiments.

Extended Data Fig. 6 RALY and hnRNP-C are not decreased upon transduction in multiple cell lines.

a, Quantification of results shown in Fig. 3c. b, HEK293 cells transfected with the indicated constructs for 24 h, followed by denaturing IP with HA antibody, and immunoblot analysis of hnRNP-C2-Flag. c, Quantification of results shown in Fig. 3d. d, Immunoblot analysis of protein levels in HeLa cells over a time course of transduction with recombinant Ad vectors expressing only E1B55K and E4orf6 (m.o.i. = 10). e, Immunoblot analysis of protein levels in HeLa, U2OS, A549 and HEK293 cells. HeLa, U2OS and A459 cells were transduced with recombinant Ad vectors expressing only E1B55K and E4orf6 for 24 h. HEK293 cells, which contain an endogenous copy of E1B55K, were mock transfected or transfected with plasmids expressing E4orf6 or GFP. f, Bar graphs of RALY and hnRNP-C RNA levels over a time course of infection with Ad5 WT (m.o.i. = 10) relative to mock as determined by RT-qPCR. g, Quantification of results shown in Fig. 3i. All data are representative of three (four for RALY in g) biologically independent experiments. All graphs show mean+SD. Statistical significance was calculated using a paired, two-tailed Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.005. Source data

Extended Data Fig. 7 RALY and hnRNP-C single knockdowns rescue late protein, RNA and splice efficiency during infection with Ad ΔE1B.

a-d, HeLa cells transfected with control (siCTRL) or RALY and hnRNP-C (siRALY/hnRNP-C) siRNA 24 h prior to infection with Ad5 WT or ΔE1B (m.o.i. = 10), harvested at respective time points. a, Quantification of results shown in Fig. 4a. b, Extended immunoblot analysis of viral and cellular protein levels. c, Bar graph representing spliced RNA levels of viral early transcript E1A measured by RT-qPCR. d, Bar graph representing splicing efficiency (ratio of spliced to unspliced transcripts) of E1A measured by RT-qPCR. e-g, HeLa cells transfected with control siRNA (siCTRL), siRNA for RALY (siRALY), siRNA for hnRNP-C (sihnRNP-C) or siRNA for both RALY and hnRNP-C (siRALY/hnRNP-C) 24 h prior to infection with Ad5 WT or ΔE1B (m.o.i. = 10) and harvested at 24 hpi. e, Immunoblot analysis of viral and cellular protein levels. f, Bar graph representing spliced RNA levels of E1A, MLP and fiber measured by RT-qPCR. g, Bar graph representing splicing efficiency of E1A, MLP and fiber measured by RT-qPCR. h-j. HeLa cells transfected with control (siCTRL) or RALY and hnRNP-C (siRALY/hnRNP-C) siRNA 24 h prior to infection with Ad5 WT (m.o.i. = 10), treated with either DMSO or NEDDi at 8 hpi and processed at 24 hpi. h, Bar graph representing spliced RNA levels of E1A measured by RT-qPCR. i, Bar graph representing splicing efficiency as defined as the ratio of spliced to unspliced transcripts of E1A measured by RT-qPCR. j, Quantification of results shown in Fig. 4i. All data are representative of three biologically independent experiments. All graphs show mean+SD. Statistical significance was calculated using paired (a and j) or unpaired (others), two-tailed Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.005. Source data

Extended Data Fig. 8 No dramatic difference in protein localization and protein-complex formation of RALY and hnRNP-C between Ad WT and ΔE1B infection.

a, Representative images of immunofluorescence comparing the localization of RALY and hnRNP-C (both green) in mock, Ad WT and ΔE1B infection of HeLa cells (m.o.i. = 10, 24 hpi). Viral replication centers are stained by DBP or USP7 (both magenta) and nuclear DNA by DAPI (grey). Scale bar=10 μm. b, Immunoblot analysis of RALY and hnRNP-C protein complexes formed upon mock, Ad WT and ΔE1B infection of HeLa cells (m.o.i. = 10) and treatment with indicated concentrations of disuccinimidyl suberate (DSS) for 30 min at 24 hpi. c, Schematic for targeted hnRNP-C RNA-binding region identification (RBR-ID) and interactome. d, Data for RALY from hnRNP-C RBR-ID experiment comparing mock (grey), Ad5 WT (black), and ΔE1B (red) at 24 hpi (m.o.i. = 10). Shown are smoothed residue-level RBR-ID score plotted along the primary sequence (left) and smoothed residue-level fold change between crosslinked and control conditions with a significance threshold of p < 0.05 (right). RALY domain structure with ubiquitination site is shown above graphs. RNA-binding regions are highlighted in blue. e, Correlation matrix for hnRNP-C interactome between replicates of mock, Ad5 WT, and Ad5 ΔE1B. Color gradient is based on the Pearson correlation coefficient. f, Comparison of z-scores for top 20 proteins identified in hnRNP-C interactome during WT Ad5 infection (m.o.i. = 10, 24 hpi). Mock=grey, Ad5 WT = black, Ad5 ΔE1B = red. All data are representative of three biologically independent experiments. Source data

Extended Data Fig. 9 In the absence of E1B55K, hnRNP-C and RALY interact more with viral late RNA.

a, Control immunoblot for hnRNP-C CLIP-qPCR in Fig. 5b. Higher molecular weight complexes represent hnRNP-C crosslinked to RNA. * indicates antibody heavy chain. b, Schematic of Ad5 genome and viral transcription units. Amplicons for early (E1A, DBP, E4) and late (MLP, Penton, Hexon, 100 K, Fiber) transcription units indicated. c, HeLa cells infected with WT Ad5 or ΔE1B (m.o.i. = 10), UV-crosslinked and harvested at 24 hpi, subjected to hnRNP-C CLIP with 10% input as compared to Fig. 5b and RT-qPCR of indicated transcripts. d, HeLa cells infected with WT Ad5 or ΔE1B (m.o.i. = 10), without UV-crosslinking, and harvested at 24 hpi, subjected to hnRNP-C CLIP and RT-qPCR of indicated transcripts. e, HeLa cells infected with WT Ad5 or ΔE1B (m.o.i. = 10), UV-crosslinked and harvested at 24 hpi, subjected to IgG CLIP and RT-qPCR of indicated transcripts. f, HeLa cells induced for RALY-Flag expression with doxycycline for 3 days total, infected with WT Ad5 or ΔE1B (m.o.i. = 10), UV-crosslinked and harvested at 24 hpi, subjected to Flag CLIP and RT-qPCR of indicated transcripts. For all CLIP-qPCR experiments: GAPDH = negative control, hnRNP-C = positive control. g, Immunoblot for hnRNP-C CLIP-qPCR in Fig. 5c. Higher molecular weight complexes represent hnRNP-C crosslinked to RNA. * indicates antibody heavy chain. h, Number of mapped hnRNP-C eCLIP-Seq reads for the indicated conditions. i, Percentage of mapped reads that uniquely mapped to Ad5 genome for different hnRNP-C eCLIP-Seq conditions. j, PCA plot for hnRNP-C peaks mapped to host transcripts comparing mock (grey), Ad5 WT (black), and Ad5 ΔE1B (red). k, Top 6 hnRNP-C binding motifs in WT and ΔE1B infection, WT infection only, and ΔE1B infection only. n = 1043, 162 peaks, and 267 peaks, respectively. Enriched motifs were identified with HOMER Software Package using hypergeometric enrichment calculations and adjustments for multiple comparisons. All data are representative of two (h-k) or three (a-g) independent experiments. Graphs in c-f show mean+SD. Statistical significance was calculated using unpaired, two-tailed Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.005. Source data

Extended Data Fig. 10 Non-degradative ubiquitination of RNA-binding proteins promotes efficient adenoviral RNA processing.

During wild-type (WT) Ad5 infection the E1B55K/E4orf6 complex induces ubiquitination of RNA-binding proteins RALY and hnRNP-C to facilitate efficient viral late RNA processing. Ubiquitination regulates interaction of these host proteins with viral RNA to facilitate viral infection. In the absence of the E1B55K/E4orf6 complex ubiquitin ligase activity, the RBPs bind more to viral late mRNAs and limit RNA processing and protein production. RNP = ribonucleoprotein.

Supplementary information

Supplementary Information

Supplementary Sections 1 and 2: literature review of identified RBPs and primer sequences.

Reporting Summary

Supplementary Tables

Supplementary Table 1: Complete di-glycine remnant and WCP data. Normalized and log2-transformed proteomics data for K-ɛ-GG peptide-based quantification (tab 1), transformation to K-ɛ-GG protein-based quantification (tab 2) and whole-cell proteome quantification. Data were generated from three biologically independent experiments. Fold changes were calculated in log2 and P values were calculated using unpaired, two-sided Student’s t-tests. Supplementary Table 2: Di-glycine remnant and WCP data for predicted E1B55K/E4orf6 substrates. Normalized and log2-transformed proteomics data for K-ɛ-GG protein-based (tab 1) and peptide-based (tab 2) quantifications for predicted E1B55K/E4orf6 substrates. Data were generated from three biologically independent experiments. Significance tests for whole-cell proteome abundance changes were calculated using unpaired, two-sided Student’s t-tests. K-ɛ-GG abundance changes were normalized to whole-cell proteome abundance changes and significance was calculated using one-sided Student’s t-tests with a null hypothesis of 0 fold change. Supplementary Table 3: Reactome network protein–protein interactome analysis results. Reactome FI network analysis results of the proteins that exhibited increased protein-based ubiquitination (n = 120). The Reactome FI clustering algorithm was used to divide the interactome into modules (tab 1). Reactome FI was used to perform GO and pathway enrichment analysis. The GO ‘Molecular Functions’ is included for modules (tab 2) and the entire network (tab 5). The GO ‘Biological Processes’ is included for modules (tab 3) and the entire network (tab 6). The Reactome Pathway analysis is included for modules (tab 4) and the entire network (tab 7). Data xwere generated from three biologically independent experiments. Supplementary Table 4: SILAC ratios for chymotryptic and tryptic peptides from targeted hnRNP-C RBR-ID analysis. Heavy/light ratios indicate the proportion of peptides crosslinked to RNA after UV light treatment. Data represent three biological replicates and two technical replicates (n = 6) per digestion condition. Chymotryptic (tab 1) and tryptic (tab 2) data were processed independently in Proteome Discoverer v.2.3. Results were filtered to exclude peptides with any missing values across replicates. Supplementary Table 5: hnRNP-C interactome derived from RBR-ID data. The non-crosslinked sample from the hnRNP-C RBR-ID was used to generate the hnRNP-C interactome. Normalized, log2-transformed data quantification for proteins identified in association with hnRNP-C. Data were generated from three biological replicates and two technical replicates (n = 6) per digestion condition. Fold changes were calculated in log2 and P values were calculated using unpaired, two-sided Student’s t-tests.

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Herrmann, C., Dybas, J.M., Liddle, J.C. et al. Adenovirus-mediated ubiquitination alters protein–RNA binding and aids viral RNA processing. Nat Microbiol 5, 1217–1231 (2020). https://doi.org/10.1038/s41564-020-0750-9

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