RBM39 shapes innate immunity by controlling expression of key factors of the interferon response

Teng-Feng Li ¹ Paul Rothhaar ¹ Arthur Lang ¹ Oliver Grünvogel ¹ Ombretta Colasanti ¹ Santa Mariela Olivera Ugarte ¹ Jannik Traut ¹ Antonio Piras ² Nelson Acosta-Rivero ³ Vladimir Gonçalves Magalhães ⁴ Emely Springer ⁵ Andreas Betz ¹ Hao-En Huang ¹ Jeongbin Park ⁶ Ruiyue Qiu ⁷ Gnimah Eva Gnouamozi ³ Ann-Kathrin Mehnert ⁸ Viet Loan Dao Thi ^8,9 Stephan Urban ^3,9 Martina Muckenthaler ⁷ Matthias Schlesner ^10,6 Dirk Wohlleber ⁵ Marco Binder ⁴ Ralf Bartenschlager ^3,4,9 Andreas Pichlmair ^11,2 Volker Lohmann ^1,9*

• ¹ Department of Infectious Diseases, Molecular Virology, Section Virus-Host-Interactions, Medical Faculty Heidelberg, Heidelberg University, Heidelberg, Germany
• ² Institute of Virology, Technical University of Munich, Munich, Bavaria, Germany
• ³ Unit of Molecular Virology, Department of Infectious Diseases, Heidelberg University Hospital, Heidelberg, Baden-Württemberg, Germany
• ⁴ Department of Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), Heidelberg, Baden-Württemberg, Germany
• ⁵ Institute of Molecular Immunology, University Hospital rechts der Isar, Technical University of Munich, München, Baden-Württemberg, Germany
• ⁶ Bioinformatics and Omics Data Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany
• ⁷ Department of Pediatric Oncology, Hematology and Immunology, Heidelberg University Hospital, Heidelberg, Baden-Württemberg, Germany
• ⁸ Department of Infectious Diseases, Virology, Medical Faculty Heidelberg, Heidelberg University, Heidelberg, Germany
• ⁹ German Center for Infection Research, partner site Heidelberg, Heidelberg, Baden-Württemberg, Germany
• ¹⁰ Biomedical Informatics, Data Mining and Data Analytics, Faculty of Applied Computer Science and Medical Faculty, University of Augsburg, Augsburg, Germany
• ¹¹ German Center for Infection Research (DZIF), Partner site Munich, Braunschweig, Bavaria, Germany

The contribution of innate immunity to clearance of viral infections of the liver, in particular sensing via Toll-like receptor 3 (TLR3) is incompletely understood. We aimed to identify factors contributing to the TLR3-response in hepatocytes via CRISPR/Cas9 screening.A genome-wide CRISPR/Cas9 screen on the TLR3 pathway was performed in two liver-derived cell lines, followed by siRNA knockdown validation. SiRNA knockdown and Indisulam treatment were used to study the role of RNA-binding motif protein 39 (RBM39) in innate immunity upon poly(I:C) or cytokine treatment and viral infections. Transcriptome, proteome and alternative splicing was studied via RNA sequencing and mass spectrometry upon depletion of RBM39. Our CRISPR/Cas9 screen identified RBM39, which is highly expressed in hepatocytes, as an important regulator of the TLR3 pathway. Knockdown of RBM39 or the treatment with Indisulam, an aryl sulfonamide drug targeting RBM39 for proteasomal degradation, strongly reduced the induction of interferon-stimulated genes (ISGs) in response to double-stranded RNA (dsRNA) or viral infections. RNA sequencing (seq) and mass spectrometry identified that transcription and/or splicing of the key pathway components IRF3, RIG-I, and MDA5 were affected by RBM39 depletion, along with multiple other cellular processes identified previously. RBM39 knockdown further restrained type I and type III IFN pathways, by reducing expression of individual receptor subunits and STAT1/2. The function of RBM39 was furthermore not restricted to hepatocytes.We identified RBM39 as a regulatory factor of cell intrinsic innate immune signaling. Depletion of RBM39 impaired TLR3, RIG-I/MDA5, and IFN responses by affecting basal expression of key pathway components.