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Regulation of toll-like receptor signaling by the SF3a mRNA splicing complex.

ABSTRACT: 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.

SUBMITTER: O'Connor BP

PROVIDER: S-EPMC4450051 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Regulation of toll-like receptor signaling by the SF3a mRNA splicing complex.

O'Connor Brian P BP Danhorn Thomas T De Arras Lesly L Flatley Brenna R BR Marcus Roland A RA Farias-Hesson Eveline E Leach Sonia M SM Alper Scott S

PLoS genetics 20150206 2

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 productio ...[more]

PMID: 25658809

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Project description:We previously reported that neuraminidase (NA) pretreatment of human PBMCs markedly increased their cytokine response to lipopolysaccharide (LPS). To study the mechanisms by which this occurs, we transfected HEK293T cells with plasmids encoding TLR4, CD14, and MD2 (three components of the LPS receptor complex), as well as a NFκB luciferase reporting system. Both TLR4 and MD2 encoded by the plasmids are α-2,6 sialylated. HEK293T cells transfected with TLR4/MD2/CD14 responded robustly to the addition of LPS; however, omission of the MD2 plasmid abrogated this response. Addition of culture supernatants from MD2 (sMD2)-transfected HEK293T cells, but not recombinant, non-glycosylated MD2 reconstituted this response. NA treatment of sMD2 enhanced the LPS response as did NA treatment of the TLR4/CD14-transfected cell supplemented with untreated sMD2, but optimal LPS-initiated responses were observed with NA-treated TLR4/CD14-transfected cells supplemented with NA-treated sMD2. We hypothesized that removal of negatively charged sialyl residues from glycans on the TLR4 complex would hasten the dimerization of TLR4 monomers required for signaling. Co-transfection of HEK293T cells with separate plasmids encoding either YFP- or FLAG-tagged TLR4, followed by treatment with NA and stimulation with LPS, led to an earlier and more robust time-dependent dimerization of TLR4 monomers on co-immunoprecipitation, compared to untreated cells. These findings were confirmed by fluorescence resonance energy transfer (FRET) analysis. Overexpression of human Neu1 increased LPS-initiated TLR4-mediated NFκB activation and a NA inhibitor suppressed its activation. We conclude that (1) sialyl residues on TLR4 modulate LPS responsiveness, perhaps by facilitating clustering of the homodimers, and that (2) sialic acid, and perhaps other glycosyl species, regulate MD2 activity required for LPS-mediated signaling. We speculate that endogenous sialidase activity mobilized during cell activation may play a role in this regulation.

Dataset Information

Regulation of toll-like receptor signaling by the SF3a mRNA splicing complex.

Publications

Regulation of toll-like receptor signaling by the SF3a mRNA splicing complex.

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