The First Structure of an RNA m5C Methyltransferase, Fmu, Provides Insight into Catalytic Mechanism and Specific Binding of RNA Substrate

Abstract

The crystal structure of E. coli Fmu, determined at 1.65 A resolution for the apoenzyme and 2.1 A resolution in complex with AdoMet, is the first representative of the 5-methylcytosine RNA methyltransferase family that includes the human nucleolar proliferation-associated protein p120. Fmu contains three subdomains which share structural homology to DNA m(5)C methyltransferases and two RNA binding protein families. In the binary complex, the AdoMet cofactor is positioned within the active site near a novel arrangement of two conserved cysteines that function in cytosine methylation. The site is surrounded by a positively charged cleft large enough to bind its unique target stem loop within 16S rRNA. Docking of this stem loop RNA into the structure followed by molecular mechanics shows that the Fmu structure is consistent with binding to the folded RNA substrate.

Structure of the human MTERF4–NSUN4 protein complex that regulates mitochondrial ribosome biogenesis

“…NSUN4 contains a typical RNA m5C MTase core domain and lacks variable N-or C-terminal extensions common for RNA recognition in other RNA m5C MTases (18,19,25) (Fig. 1A).…”

“…The SAM donor methyl points toward the open cleft where two loops comprising the conserved cysteine residues C258 and C310 are located. These cysteines are important for the methyl transfer mechanism from SAM to the incoming RNA (17,19). In this context, C310 in NSUN4 serves as a catalytic nucleophile, whereas C258 eases product release from the covalent enzyme-RNA intermediate.…”

“…A search for structural homologs to NSUN4 using Dali (26) revealed significant matches to other RNA m5C MTases (18,19,25), thus enabling a structural-based sequence alignment between NSUN4 and the E. coli structural homologs RsmF and RsmB, as well as RsmF from Thermus thermophilus (TTH) (Fig. S1B).…”

“…The crystal structures of the RNA m5C MTases RsmB and RsmF in prokaryotes (18,19) display a conserved MTase domain flanked by variable N-or C-terminal extensions proposed to function as RNA-binding modules. The methyl donor S-adenosyl-L-methionine (SAM) is bound at the base of a positively charged cleft in the MTase domain near two conserved cysteins important for the methylation mechanism.…”

Adenine methylation in eukaryotes: Apprehending the complex evolutionary history and functional potential of an epigenetic modification

“…Within this superfamily, nucleic acid MTases belong to two distinct clades, one including all nucleic acid C5 MTases 58 and the other uniting enzymes catalyzing methylation of both N 4 C and N 6 A, those modifying the N2 position of guanines in RNA, as well as certain protein MTases methylating the amide group of glutamine in proteins such as the ribosomal protein L3 and peptide release factors (HemK family) 59, 60, 61. Members of the latter clade are characterized by a [DNSH]PP[YFW] motif at the C‐terminus of conserved strand‐4 of the Rossmann domain 18, 20, 21, 62 (Fig.…”