(A) Enzymatic reactions involved in RNA 5′ capping. First, the RNA triphosphatase (TPase) hydrolyzes the 5′ triphosphate group (pppN−) to form a 5′ diphosphate (ppN−) (Reaction 1). Then, an RNA 5′ guanylyltransferase adds a GMP group to the 5′ diphosphate to form an unmethylated G-capped structure (GpppN−) through a reversible mechanism (Reaction 2). This is followed by an RNA cap guanosine N7-methyltransferase activity that adds a methyl group to the N7 position of the G cap and forms the Cap-0 structure (m7GpppN−) (Reaction 3). In metazoans, a fourth enzyme RNA cap 2′-O-methyltransferase (2′-OMTase) further adds a methyl group to the 2′-O position of the initiating nucleotide of the transcript, forming the Cap-1 structure (m7GpppNm−) (Reaction 4). (B) Catalytic domains of FCE. (Top panels) Individual domains of the AlphaFold2 prediction of FCE were aligned to published atomic structures. Insets show the alignment of highly conserved residues labeled with FCE residue numbering. (Bottom panels) Amino acid residues of the motifs were aligned with indicated enzymes. Cet1, Ceg1, and Abd1 are the TPase, GTase, and N7MTase of Saccharomyces cerevisiae, respectively. hRNMT is the human RNA cap N7MTase. MCE refers to Acanthamoeba polyphaga mimivirus capping enzyme. Conserved residues are highlighted in gray. FCE residues that are mutated in this study and the corresponding residues in orthologous enzymes are highlighted in black. (i) The predicted structure of FCE's TPase domain is aligned to the VCE TPase domain-ppRNA complex extracted from the CryoEM cotranscriptional capping complex (PDB ID 6RIE). The inset shows the alignment of the conserved ELE–ELE motif. (ii) The predicted structure of FCE's GTase domain is aligned to the X-ray crystal structure of Paramecium bursaria chlorella virus (PBCV-1) GTase with covalent GMP intermediate (PDB ID 1CKN). Inset shows the phosphamide covalent linkage between the catalytic Lys of PBCV-1 GTase and GMP. (iii) The predicted FCE N7MTase domain structure is aligned to the X-ray crystal structure of the N7MTase domain of VCE D1 subunit in complex with co-product SAH (PDB ID 4CKB). Inset shows the conserved SAM-binding site.
