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Global Transcriptomics Uncovers Distinct Contributions From Splicing Regulatory Proteins to the Macrophage Innate Immune Response

  • Wagner, Allison R.1
  • Scott, Haley M.1
  • West, Kelsi O.1
  • Vail, Krystal J.1, 2
  • Fitzsimons, Timothy C.1
  • Coleman, Aja K.1
  • Carter, Kaitlyn E.1
  • Watson, Robert O.1
  • Patrick, Kristin L.1
  • 1 Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas A&M Health, Bryan, TX , (United States)
  • 2 Department of Veterinary Pathobiology, Texas A&M University, College Station, TX , (United States)
Published Article
Frontiers in Immunology
Frontiers Media SA
Publication Date
Jul 09, 2021
DOI: 10.3389/fimmu.2021.656885
  • Immunology
  • Original Research


Pathogen sensing via pattern recognition receptors triggers massive reprogramming of macrophage gene expression. While the signaling cascades and transcription factors that activate these responses are well-known, the role of post-transcriptional RNA processing in modulating innate immune gene expression remains understudied. Given their crucial role in regulating pre-mRNA splicing and other RNA processing steps, we hypothesized that members of the SR/hnRNP protein families regulate innate immune gene expression in distinct ways. We analyzed steady state gene expression and alternatively spliced isoform production in ten SR/hnRNP knockdown RAW 264.7 macrophage-like cell lines following infection with the bacterial pathogen Salmonella enterica serovar Typhimurium (Salmonella). We identified thousands of transcripts whose abundance is increased or decreased by SR/hnRNP knockdown in macrophages. Notably, we observed that SR and hnRNP proteins influence expression of different genes in uninfected versus Salmonella-infected macrophages, suggesting functionalization of these proteins upon pathogen sensing. Likewise, we found that knockdown of SR/hnRNPs promoted differential isoform usage (DIU) for thousands of macrophage transcripts and that these alternative splicing changes were distinct in uninfected and Salmonella-infected macrophages. Finally, having observed a surprising degree of similarity between the differentially expressed genes (DEGs) and DIUs in hnRNP K and U knockdown macrophages, we found that hnRNP K and U knockdown macrophages are both more restrictive to Vesicular Stomatitis Virus (VSV), while hnRNP K knockdown macrophages are more permissive to Salmonella Typhimurium. Based on these findings, we conclude that many innate immune genes evolved to rely on one or more SR/hnRNPs to ensure the proper magnitude of their induction, supporting a model wherein pre-mRNA splicing is critical for regulating innate immune gene expression and controlling infection outcomes in macrophages ex vivo.

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