Structural basis for substrate binding and catalytic mechanism of a human RNA:m5C methyltransferase NSun6

Abstract

5-methylcytosine (m5C) modifications of RNA are ubiquitous in nature and play important roles in many biological processes such as protein translational regulation, RNA processing and stress response. Aberrant expressions of RNA:m5C methyltransferases are closely associated with various human diseases including cancers. However, no structural information for RNA-bound RNA:m5C methyltransferase was available until now, hindering elucidation of the catalytic mechanism behind RNA:m5C methylation. Here, we have solved the structures of NSun6, a human tRNA:m5C methyltransferase, in the apo form and in complex with a full-length tRNA substrate. These structures show a non-canonical conformation of the bound tRNA, rendering the base moiety of the target cytosine accessible to the enzyme for methylation. Further biochemical assays reveal the critical, but distinct, roles of two conserved cysteine residues for the RNA:m5C methylation. Collectively, for the first time, we have solved the complex structure of a RNA:m5C methyltransferase and addressed the catalytic mechanism of the RNA:m5C methyltransferase family, which may allow for structure-based drug design toward RNA:m5C methyltransferase-related diseases.

Figure 1.

The proposed reaction scheme for RNA:m⁵C MTases. This scheme is modified from Foster et al., (22). In the first step, a Cys-thiol of the enzyme works as a nucleophile to attack the C6 atom of cytosine to form a covalent protein–RNA intermediate (intermediate 2). The C5 atom of intermediate 2 is activated for electrophilic substitution. This is followed by transfer of a methyl group from SAM to C5, generating a methylated covalent protein–RNA intermediate (intermediate 3). The final step involves a proton abstraction from C5 by a general base and β elimination of the enzyme.

Figure 2.

Overall structures. (A) Overall structure of apo hNSun6 in cartoon representation; residue numbers are indicated at the top; the MTase domain (residues 48–100 and 211–469) including both the RRM motif (deep blue, residues 48–100 and 211–220) and the catalytic core (light blue, residues 221–469), the PUA domain (pink, residues 113–201), the N-terminal extension (light green, residues 1–47) and linkers (cyan, Linker 1: residues 101–112, and Linker 2: residues 202–210) are highlighted. (B) Overall structure of hNSun6/tRNA/SFG complex in cartoon representation, tRNA is gold in color and SFG is depicted as a sphere. (C) The binding of tRNA (gold) to hNSun6 (surface representation). (D) The conformational changes of the PUA domain (pink) upon tRNA binding compared to that in apo hNSun6 (gray).

Figure 3.

Binding details of the tRNA acceptor with hNSun6 (A) Structural comparison of tRNA (right) in the hNSun6/tRNA/SFG complex compared to the typical structure of tRNA in free form (left, yeast tRNA^Phe, PDB ID: 4TNA). The 3΄-end of tRNA binds to a cleft formed between the MTase and PUA domains presented in cartoon (B) and surface representations (C). (D) CCA end (stick representation) binding to hNSun6 (surface representation). Recognition of C74 (E), C75 (F) and A76 (G) by hNSun6 is shown in detail. (H), (K) and (O) depict the MTase activities of hNSun6 and variants. (I) The relative position of U71 to hNSun6 and to SFG. (J) The recognition details of U73 (stick representation) and (L) the binding pocket (electrostatics surface representation) of U73. (M) and (N) show the binding details of C72 by hNSun6 from two different views.

Figure 4.

Binding between the D-stem region of tRNA and PUA domain The interactions between tRNA (gold) and the PUA domain. tRNA is depicted in cartoon representation, and the PUA domain is shown as an electrostatics surface representation (A) and a cartoon representation (B). (C) The MTase activities of hNSun6 and variants.

Figure 5.

Cofactor binding in hNSun6 (A) SFG binding details in the hNSun6/tRNA/SFG complex. SFG is shown (stick representation) with the carbon atom in salmon. (B) The MTase activities of hNSun6 and variants. (C) Representative ITC experiments of SAM titrated into WT hNSun6 protein solution. (D) The SAM binding affinity of hNSun6 and variants as measured by ITC.

Figure 6.

Insight into the role of active site residues of hNSun6 (A) A structural comparison of the two conserved Cys residues and methylation target nucleotide residue in the hNSun6/tRNA/SFG complex (middle) with those observed in RNA:m⁵U MTase (left; Escherichia coli RumA; PDB ID: 2BH2) and DNA:m⁵C MTase (right; M. HaeIII MTase from Haemophilus influenzae; PDB ID: 1DCT) from the same view. (B) Relative positions of the two conserved Cys residues relative to C72 and SFG in the hNSun6/tRNA/SFG complex. (C) and (D) display the sodium dodecyl sulphate-polyacrylamide gel electrophoresis of hNSun6 and its mutants in addition to their covalent complexes with RNA, as detected by western blot using an hNSun6 specific antibody. (E) and (F) show the MTase activities of hNSun6 and its variants. (G) The covalent protein–RNA complex formation of hNSun6-WT or -C326S at different time points in the presence or absence of SAM. In (C, D and G), the lower bands at ∼55 KDa indicate hNSun6 or its variants, while the upper bands at ∼80 KDa correspond to the protein–tRNA covalent complexes.