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NF-κB mediates lipopolysaccharide-induced alternative pre-mRNA splicing of MyD88 in mouse macrophages.

ABSTRACT: Although a robust inflammatory response is needed to combat infection, this response must ultimately be terminated to prevent chronic inflammation. One mechanism that terminates inflammatory signaling is the production of alternative mRNA splice forms in the Toll-like receptor (TLR) signaling pathway. Whereas most genes in the TLR pathway encode positive mediators of inflammatory signaling, several, including that encoding the MyD88 signaling adaptor, also produce alternative spliced mRNA isoforms that encode dominant-negative inhibitors of the response. Production of these negatively acting alternatively spliced isoforms is induced by stimulation with the TLR4 agonist lipopolysaccharide (LPS); thus, this alternative pre-mRNA splicing represents a negative feedback loop that terminates TLR signaling and prevents chronic inflammation. In the current study, we investigated the mechanisms regulating the LPS-induced alternative pre-mRNA splicing of the MyD88 transcript in murine macrophages. We found that 1) the induction of the alternatively spliced MyD88 form is due to alternative pre-mRNA splicing and not caused by another RNA regulatory mechanism, 2) MyD88 splicing is regulated by both the MyD88- and TRIF-dependent arms of the TLR signaling pathway, 3) MyD88 splicing is regulated by the NF-κB transcription factor, and 4) NF-κB likely regulates MyD88 alternative pre-mRNA splicing per se rather than regulating splicing indirectly by altering MyD88 transcription. We conclude that alternative splicing of MyD88 may provide a sensitive mechanism that ensures robust termination of inflammation for tissue repair and restoration of normal tissue homeostasis once an infection is controlled.

SUBMITTER: Lee FF

PROVIDER: S-EPMC7196638 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundIn mammals, maternal gene products decay and zygotic genome activation (ZGA) during maternal to zygotic transition (MZT) is critical for the early embryogenesis. Y-box binding protein YBX1 plays vital roles in RNA stabilization and transcriptional regulation, but its roles remain to be elucidated during pre-implantation development.MethodsIn the present study, we re-analyzed transcriptional level of YBX1 in mice, human, bovine, and goat embryos using public RNA-seq datasets. We further performed siRNA microinjection to knock down the expression of YBX1, and RNA sequencing of the 8-cell stage embryos in the control and YBX1 knockdown group. To reveal the regulation mechanisms of YBX1, we conducted differentially expression analysis, alternative splicing (AS) analysis, enrichment analysis, and 5-EU staining using DESeq2, rMATs, clusterProfiler, and immunofluorescence technique, respectively.ResultsThe expression of YBX1 was increased during MZT in goat, bovine, human, and mice, but significantly decreased in YBX1 knockdown embryos compared with the controls, suggesting successfully knockdown of YBX1. The percentage of blastocyst was decreased, while embryos blocked at the 2- and 4-cell stage were increased in YBX1 knockdown embryos compared to the controls. Using RNA-seq, we identified 1623 up-regulated and 3531 down-regulated genes in the 8-cell stage YBX1 knockdown embryos. Of note, the down-regulated genes were enriched in regulation of RNA/mRNA stability and spliceosome, suggesting that YBX1 might medicate RNA stability and AS. To this end, we identified 3284 differential AS events and 1322 differentially expressed maternal mRNAs at the 8-cell stage YBX1 knockdown embryos. Meanwhile, the splicing factors and mRNA decay-related genes showed aberrant expression, and the transcriptional activity during ZGA in goat and mice was compromised when YBX1 was knocked down.ConclusionYBX1 serves an important role in maternal mRNA decay, alternative splicing, and the transcriptional activity required for early embryogenesis, which will broaden the current understanding of YBX1 functions during the stochastic reprogramming events.

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NF-κB mediates lipopolysaccharide-induced alternative pre-mRNA splicing of MyD88 in mouse macrophages.

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