The structure of human Nocturnin reveals a conserved ribonuclease domain that represses target transcript translation and abundance in cells

Abstract

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. NOCT-deficient mice are resistant to high-fat diet induced weight gain, and exhibit dysregulation of bone formation. However, the mechanisms by which NOCT regulates these processes remain to be determined. Here, we describe a pair of high-resolution crystal structures of the human NOCT catalytic domain. 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. In contrast to the related deadenylase CNOT6L, purified recombinant NOCT lacks in vitro ribonuclease activity, suggesting that unidentified factors are necessary for enzymatic activity. 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. 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.

Figure 1.

The crystal structure of NOCT reveals a conserved structure, active site and basic cleft. (A) A cartoon representation of the 1.48 Å resolution structure of the NOCT catalytic domain (residues 120–431). (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. (C) A detailed view of the active site of the 2.41 Å resolution structure. Two Mg²⁺ 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. (D) A detailed view of the active site of the 1.48 Å resolution structure. A single Mg²⁺ ion is bound in the active site, coordinated to Glu195. Hydrogen bonds are depicted with dashed lines. Hydrogen bond distance cutoffs were assigned an upper limit of 3.3 Å. (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. (F) A surface electrostatic rendering of the NOCT catalytic domain with surface potential contoured to ±3 kT/e. NOCT has a prominent basic cleft adjacent to the active site. The bound Mg²⁺ ion is shown as a sphere. (G) A surface representation of the NOCT 1.48 Å resolution structure. Residues are colored by conservation based on a multiple sequence alignment generated by ConSurf. The residues that are conserved are colored in violet, and the most variable residues are colored in teal. Neutral residues that are neither highly variable or conserved are white. The most conserved residues are found in the active site and adjacent basic cleft.

Figure 2.

NOCT is not active against poly(A)20 substrates in vitro. Recombinant NOCT_64–431, CNOT6L_158–555 and CNOT6L_158–555 E240A were incubated with 5′ Cy3 labeled poly(A)₂₀ RNA substrate. A representative gel is shown here. Where indicated, the chelator EDTA was added to stop the ribonuclease reaction.

Figure 3.

NOCT translationally represses a reporter mRNA in a cell-based tethered function assay. The effect of NOCT or control CNOT7 on normalized expression of NLuc from the NLuc 4xMS2BS mRNA p(A) reporter was measured in HEK293 cells, and log2 fold change in reporter activity is plotted relative to negative control MS2-HaloTag. Tethered effector proteins included MS2 coat protein fusions to WT NOCT_1–431, NOCT_64–431, CNOT7 and HaloTag. Error bars show 95% credible intervals; any comparisons where the 95% credible intervals do not overlap zero are considered significant and are denoted by a single star. Two stars denote a 95% posterior probability that the difference measured is >1.3-fold.

Figure 4.

NOCT repression of protein expression depends on conserved active site residues and the identity of the mRNA 3′ end. (A) Mutation of NOCT the active site reduced but did not eliminate translational repression of the NLuc 4xMS2BS p(A) reporter. Tethered effectors fused to MS2 coat protein are listed on the bottom of the graph. Log₂ fold change in reporter protein expression calculated relative to negative control MS2-HaloTag (MS2-HT) is plotted for each tethered effector. (B) As in (A), but with activity shown relative to WT NOCT. (C) Western blot detection of tethered effectors for three replicate samples using anti-V5 monoclonal antibody (WT and mutant MS2-NOCT effectors) or anti-HaloTag monoclonal antibody (MS2-HT and MS2-HT-CNOT7). (D) Tethered function assays (as in A and B) comparing repression of NLuc targets with the indicated 3′ ends by MS2-tethered effectors relative to that of MS2-HT. Error bars and significance stars are as in Figure 3.

Figure 5.

WT NOCT degrades poly(A) RNA and mutating active site residues stabilizes NLuc RNA. (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. Resulting levels of NLuc RNA relative to the MS2-HT control were measured by northern blot analysis. WT NOCT reduced the steady state levels of the NLuc reporter RNA. Mutating the NOCT active site residues to alanine appeared to reduce degredation; however, the width of the credible intervals precludes a firm conclusion as to whether or not there is residual activity relative to HT. (B) As in panel (A), but with data shown relative to WT NOCT. The data suggest that all tested mutations reduce activity relative to WT, although only the H414A and N326A mutations showed strong evidence of loss of activity. (C) As in A and B, showing mRNA levels of the indicated reporter. MS2-NOCT reduced the steady state levels of NLuc 4xMS2BS p(A) but not 4xMS2BS MALAT1. Statistical significance is denoted as previously described in Figure 3.