Structural and Functional Characterization of the Human Enzyme Nocturnin

Abstract

The circadian protein Nocturnin (NOCT) belongs to the exonuclease, endonuclease and phosphatase (EEP) superfamily and is most similar to the CCR4-class of deadenylases that degrade the poly-adenosine poly(A) tails of mRNAs. NOCT-deficient mice are resistant to diet-induced obesity and exhibit increased bone density; however, the mechanisms by which NOCT regulates these processes remain unknown.

Based on homology, we predicted that NOCT adopts the α/β hydrolase fold that is characteristic of EEP deadenylases, has activity against poly(A) RNAs in vitro and regulates mRNA expression and half-lives in vivo. Since the NOCT null animals display alterations in lipid metabolism, we expected that we would identify target mRNAs of NOCT involved in lipid metabolism and adipogenesis.

To determine the structure of the NOCT catalytic domain, we solved a pair of crystal structures of NOCT, demonstrating that the structure is highly conserved with other EEP deadenylases. Purified recombinant NOCT lacked ribonuclease activity in vitro. However, in cell-based assays, we found that NOCT reduces translation and abundance of reporter mRNAs. Incomplete reversal of repression when the NOCT active site is mutated suggests a role for translational repression in addition to ribonuclease activity. NOCT-mediated repression of reporter mRNAs is additionally dependent upon the 3′ end, as reporters terminating with a 3′ MALAT1 structure cannot be repressed by NOCT. Together, these data suggest a model by which NOCT represses expression from its target mRNAs through RNA decay and translational repression.

We then examined the NOCT protein sequence for motifs indicating subcellular localization. Sequence features of the NOCT mRNA suggests that NOCT may be localized to the cytoplasm and the mitochondria. Additionally, the NOCT protein sequence has predicted motifs for mitochondrial localization. Consistent with these observations, we observed that the full-length NOCT is a preprotein that can be processed. Surprisingly, processing of NOCT appears to occur in a tissue-type specific manner, suggesting that NOCT is differentially located to the cytoplasm or mitochondria in different cell types.

To identify potential targets of NOCT we generated HEK293 cells with cytoplasmic overexpression of NOCT and performed RNA-Seq. A dataset of transcripts that change in abundance with NOCT overexpression was identified, demonstrating that NOCT is able to affect steady state levels of mRNAs. In addition to these potential NOCT targets, we further confirmed a report that NOCT has phosphatase activity against NADPH and NADP+, indicating that NOCT can hydrolyze non-mRNA substrates. These data suggest that NOCT may regulate metabolism through modulating the steady state levels of mRNAs as well as NADP(H).