![]() ![]() ![]() Multiple studies report that SRSF6 preferentially binds purine-rich exonic splicing enhancers, with a predicted consensus site in humans of USCGKM (where S represents G or C K represents U or G M represents A or C) ( Liu et al., 1998). SRSF6 is a 55 kDa protein that is essential for viability of Drosophilia melanogaster and Mus musculus ( Mason et al., 2020 Ring and Lis, 1994). One particularly striking phenotype we uncovered was that of Srsf6 KD macrophages, which express high basal levels of Ifnb1 and interferon stimulated genes (ISGs). This analysis revealed a remarkable degree of diversity in how individual SR proteins influence innate immune responses ( Wagner et al., 2021). To better define the contributions of SRSF proteins to innate immunity, we carried out a transcriptomics study of a panel of SRSF knockdown (KD) RAW 264.7 macrophage cell lines. SRSF3 has been shown to negatively regulate IL-1β release during Escherichia coli infection of THP-1 monocytes ( Moura-Alves et al., 2011) and SRSF2 promotes herpes simplex virus replication by binding to viral promoters and controlling splicing of viral transcripts ( Wang et al., 2016). SRSF1, the best studied SR protein, limits autoimmunity via a role in maintaining healthy regulatory T cells ( Katsuyama and Moulton, 2021). There are emerging roles for SR proteins in regulating immune homeostasis. Many connections have been made between SR proteins and cancer, with aberrant expression of SR proteins commonly observed in patients with multiple myeloma and acute myeloid leukemia ( Liu et al., 2022 Song et al., 2019 Wan et al., 2019). The SRs also function at other steps of the RNA life cycle including mRNA export, localization, decay, and translation ( Howard and Sanford, 2015). These proteins recognize and bind to exonic splicing enhancer sequences to define exon locations, thus directing the U snRNPs to cis-splicing signals in nearby introns. One major family of splicing regulators is the Serine/arginine rich, or SR proteins. Splicing regulatory proteins play a critical role in maintaining the fidelity of splicing while permitting the flexibility needed for alternative exon usage. Pre-mRNA splicing plays a key role in global regulation of the transcriptome and thus the proteome, with 92–94% of the human genome subject to alternative splicing ( Wang et al., 2008) and >80% of alternative splicing predicted to impact protein functionality ( Yura et al., 2006). While these transcripts are being synthesized by RNA polymerase II, they are subject to several critical co-transcriptional processing steps including 5’ capping, cleavage and polyadenylation, and pre-mRNA splicing, whereby introns are removed and exons are ligated together to generate mature RNAs ( Carpenter et al., 2014). When innate immune cells like macrophages sense pathogen or damage associated molecular patterns (PAMPs or DAMPs), they rapidly induce transcription of hundreds of genes encoding cytokines, chemokines, and antimicrobial mediators ( Hagai et al., 2018 Ramsey et al., 2008). This work defines BAX alternative splicing by SRSF6 as a critical node not only in mitochondrial homeostasis but also in the macrophage’s response to pathogens. Upon pathogen sensing, macrophages regulate SRSF6 expression to control the liberation of immunogenic mtDNA and adjust the threshold for entry into programmed cell death. Loss of SRSF6 promotes accumulation of BAX-κ, a variant that sensitizes macrophages to undergo cell death and triggers upregulation of interferon stimulated genes through cGAS sensing of cytosolic mitochondrial DNA. SRSF6-dependent orchestration of mitochondrial health is directed in large part by alternative splicing of the pro-apoptosis pore-forming protein BAX. Transcriptomic analysis of murine macrophage cell lines identified Serine/Arginine Rich Splicing factor 6 (SRSF6) as a gatekeeper of mitochondrial homeostasis. Despite the importance of pre-mRNA splicing in shaping the proteome, its role in balancing immune outcomes remains understudied. To mount a protective response to infection while preventing hyperinflammation, gene expression in innate immune cells must be tightly regulated.
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