Abstract
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.
Highlights
When innate immune cells like macrophages sense pathogens, they undergo dramatic gene expression reprogramming and upregulate thousands of genes
Overall knockdown efficiency varied between factors, with only about 50% knockdown efficiency achieved for heterogenous nuclear ribonucleoproteins (hnRNPs) C, hnRNP K, hnRNP U, SRSF1 and SRSF7 and 70-90% knockdown achieved for SRSF2, SRSF6, SRSF9, hnRNP F and hnRNP M (Figure 1A)
Our study illustrates the diverse effects the SR/hnRNP family splicing factors have on the macrophage transcriptome both in uninfected macrophages and in cells infected with the gramnegative bacterial pathogen Salmonella Typhimurium
Summary
When innate immune cells like macrophages sense pathogens, they undergo dramatic gene expression reprogramming and upregulate thousands of genes. Early response genes, are readily induced upon activation of pathogen sensing cascades. Many of these transcripts reach maximal abundance as soon as 30 minutes post-pathogen sensing [6, 7]. The timing and induction of primary and secondary response genes relies on a number of tightly regulated mechanisms, including but not limited to, cooperative binding of transcription factors [8, 9], nucleosome occupancy and histone modification at promoters [10, 11], signal-dependent interactions between transcription factor subunits [12, 13], and selective interaction with the transcriptional elongation machinery [14]
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