Resulting levels of NLuc RNA relative to the MS2-HT control were measured by northern blot analysis. ( A) Fusion proteins of MS2 coat proteins with WT NOCT or NOCT active site mutants were transfected into HEK293 cells along with the NLuc 4xMS2 poly(A) reporter. WT NOCT degrades poly(A) RNA and mutating active site residues stabilizes NLuc RNA. The most conserved residues are found in the active site and adjacent basic cleft. Neutral residues that are neither highly variable or conserved are white. The residues that are conserved are colored in violet, and the most variable residues are colored in teal. Residues are colored by conservation based on a multiple sequence alignment generated by ConSurf. ( G) A surface representation of the NOCT 1.48 Å resolution structure. The bound Mg 2+ ion is shown as a sphere. NOCT has a prominent basic cleft adjacent to the active site. ( F) A surface electrostatic rendering of the NOCT catalytic domain with surface potential contoured to ☓ kT/e. ( E) The active site of the 2.41 Å resolution structure and the adjacent basic residues are shown with the enzyme surface corresponding to the active site and basic patch superimposed. Hydrogen bond distance cutoffs were assigned an upper limit of 3.3 Å. Hydrogen bonds are depicted with dashed lines. A single Mg 2+ ion is bound in the active site, coordinated to Glu195. ( D) A detailed view of the active site of the 1.48 Å resolution structure. ![]() Two Mg 2+ ions are bound in the active site (one coordinated to Glu195 and the other to Asp324) and a sulphate anion is bound between Lys219 and Lys288 adjacent to the active site. ( C) A detailed view of the active site of the 2.41 Å resolution structure. ( B) Cartoon representation of the NOCT 120–431 1.48 Å resolution structure denoting the secondary structural elements of the NOCT catalytic domain colored using the PyMOL chainbow settings. ( A) A cartoon representation of the 1.48 Å resolution structure of the NOCT catalytic domain (residues 120–431). The crystal structure of NOCT reveals a conserved structure, active site and basic cleft. Together, these data demonstrate that NOCT is an exoribonuclease that can degrade mRNAs to inhibit protein expression, suggesting a molecular mechanism for its regulatory role in lipid metabolism and bone development. ![]() We also find the ability of NOCT to repress reporter mRNAs in cells depends upon the 3' end of the mRNA, as reporters terminating with a 3' MALAT1 structure cannot be repressed by NOCT. In contrast to the related deadenylase CNOT6L, purified recombinant NOCT lacks in vitro ribonuclease activity, suggesting that unidentified factors are necessary for enzymatic activity. ![]() The active site of NOCT is highly conserved with other exoribonucleases, and when directed to a transcript in cells, NOCT can reduce translation and abundance of that mRNA in a manner dependent on key active site residues. Here, we describe a pair of high-resolution crystal structures of the human NOCT catalytic domain. However, the mechanisms by which NOCT regulates these processes remain to be determined. NOCT-deficient mice are resistant to high-fat diet induced weight gain, and exhibit dysregulation of bone formation. The circadian protein Nocturnin (NOCT) belongs to the exonuclease, endonuclease and phosphatase superfamily and is most similar to the CCR4-class of deadenylases that degrade the poly-adenosine tails of mRNAs.
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