Controlling infectious disease without inducing unwanted inflammatory disease requires proper regulation of the innate immune response. Thus, innate immunity needs to be activated when needed during an infection, but must be limited to prevent damage. To accomplish this, negative regulators of innate immunity limit the response. Here we investigate one such negative regulator encoded by an alternative splice form of MyD88. MyD88 mRNA exists in two alternative splice forms: MyD88L, a long form that encodes a protein that activates innate immunity by transducing Toll-like receptor (TLR) signals; and a short form that encodes a different protein, MyD88S, that inhibits the response. We find that MyD88S levels regulate the extent of inflammatory cytokine production in murine macrophages. MyD88S mRNA levels are regulated by the SF3A and SF3B mRNA splicing complexes, and these mRNA splicing complexes function with TLR signaling to regulate MyD88S production. Thus, the SF3A mRNA splicing complex controls production of a negative regulator of TLR signaling that limits the extent of innate immune activation.

The innate immune response plays a key role in fighting infection by activating inflammation and stimulating the adaptive immune response. However, chronic activation of innate immunity can contribute to the pathogenesis of many diseases with an inflammatory component. Thus, various negatively acting factors turn off innate immunity subsequent to its activation to ensure that inflammation is self-limiting and to prevent inflammatory disease. These negatively acting pathways include the production of inhibitory acting alternate proteins encoded by alternative mRNA splice forms of genes in Toll-like receptor (TLR) signaling pathways. We previously found that the SF3a mRNA splicing complex was required for a robust innate immune response; SF3a acts to promote inflammation in part by inhibiting the production of a negatively acting splice form of the TLR signaling adaptor MyD88. Here we inhibit SF3a1 using RNAi and subsequently perform an RNAseq study to identify the full complement of genes and splicing events regulated by SF3a in murine macrophages. Surprisingly, in macrophages, SF3a has significant preference for mRNA splicing events within innate immune signaling pathways compared with other biological pathways, thereby affecting the splicing of specific genes in the TLR signaling pathway to modulate the innate immune response.

Myelodysplastic syndrome-associated spliceosome gene mutations enhance innate immune signaling Genes encoding spliceosome components including SF3B1, U2AF1, and SRSF2, are frequently somatically mutated in myelodysplastic syndromes (MDS), other hematologic malignancies, and solid tumors. 1 Typically these are mutually exclusive, heterozygous, missense, hotspot mutations that result in neomorphic or gain-offunction splicing phenotypes. These mutations alter splicing of many genes; however, overlap among the different splicing factors is limited. Thus, a common mechanism by which spliceosome mutations contribute to disease is suspected but has remained elusive. We previously demonstrated that inhibiting any of five different spliceosome genes (SF3B1, SF3A1, SF3A2, SF3A3, EFTUD2) in mouse or human macrophages reduces inflammatory cytokine production induced by multiple Toll-like receptor (TLR) agonists including the TLR4 agonist lipopolysaccharide (LPS). 2-5 Although these genes encode essential spliceosome components, partial gene knockdown (approx. 80%) reduces LPS-induced inflammatory cytokine production without affecting viability or phagocytosis. 2-5 Hence, innate immunity may be particularly sensitive to spliceosome perturbation. These observations suggest that MDS-associated spliceosome mutations might enhance innate immunity,

The extent of the innate immune response is regulated by many positively and negatively acting signaling proteins. This allows for proper activation of innate immunity to fight infection while ensuring that the response is limited to prevent unwanted complications. Thus mutations in innate immune regulators can lead to immune dysfunction or to inflammatory diseases such as arthritis or atherosclerosis. To identify novel innate immune regulators that could affect infectious or inflammatory disease, we have taken a comparative genomics RNAi screening approach in which we inhibit orthologous genes in the nematode Caenorhabditis elegans and murine macrophages, expecting that genes with evolutionarily conserved function also will regulate innate immunity in humans. Here we report the results of an RNAi screen of approximately half of the C. elegans genome, which led to the identification of many candidate genes that regulate innate immunity in C. elegans and mouse macrophages. One of these novel conserved regulators of innate immunity is the mRNA splicing regulator Eftud2, which we show controls the alternate splicing of the MyD88 innate immunity signaling adaptor to modulate the extent of the innate immune response.