Nocturnin is a Molecular Link between the Circadian Clock and the Innate Immune System

Circadian clocks have been connected to numerous physiological functions in organisms. Nocturnin, a rhythmically expressed deadenylase first found in Xenopus retina is considered a means of circadian gene regulation. This study provides new evidence for a direct circadian regulation of the innate immune system through Nocturnin. Deadenylase assays further characterized Xenopus Nocturnin deadenylase activity and verified mouse Nocturnin deadenylase activity. The rhythmic expression of Nocturnin protein in mouse liver was also confirmed. Nocturnin was also acutely inducible by LPS, an endotoxin that initiates innate immune responses, in primary MEFs and hepatocytes. Meanwhile, Nocturnin responded to LPS differently in mouse liver when LPS was injected at different times of day. These data suggest a potential role of Nocturnin in innate immune responses, which have been shown to be regulated by the circadian clock through poorly understood mechanisms. Indeed, iNOS (inducible nitrix oxide synthase), an important innate immune response mediator, is rhythmically expressed in mouse liver on the basal level and during LPSinduced endotoxic shock. Nocturnin is necessary for the rhythmicity of iNOS mRNA under both conditions. Studies in primary MEF cultures revealed that Nocturnin stabilized iNOS mRNA likely through modulating the length of the poly(A) tail. This offers a possible mechanism for Nocturnin-regulated rhythmic expression of iNOS. Together, these data provide new evidence of a direct regulation of the immune system by the circadian clock on the molecular level through Nocturnin. ii Nocturnin does not contain an RNA-binding domain, and size exclusion chromatography showed that Nocturnin was in protein complexes. KSRP, a known important iNOS mRNA decay regulator, was identified as a potential Nocturnin-binding protein through a proteomic approach. However, investigation of Nocturnin-KSRP interaction and the localization of KSRP suggested that Nocturnin interacted with an unknown cytoplasmic protein instead of KSRP. Antibody mapping, siRNA knock-down, PCR, immunoprecipitation followed by Mass Spectrometry and western blotting revealed that the unknown protein was FBP3. This close family member of KSRP possibly recruits Nocturnin to iNOS mRNA and leads to the stabilization of the message. This also implicates an intriguing role of FBP3, whose in vivo function has never been identified before, during mRNA decay regulation.

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