Identification and functional characterization of an mRNA Cap3 methyltransferase from Acanthamoeba castellani medusavirusi.

Eukaryotic mRNAs possess a characteristic 5'-end structure known as the cap. Cap0 (m7GpppN) is the simplest form, followed by Cap1 and Cap2, which contain additional O-methyl modifications at the 2'-hydroxyl positions of the ribose moieties of the first and second nucleotides, respectively. Higher-order cap structures, such as Cap3 and Cap4, have been identified in kinetoplastids and other organisms. Although the enzymes responsible for these modifications have been inferred through vivo studies, no enzyme with confirmed in vitro activity has been reported to date. In this study, we identified and functionally characterized a novel mRNA-dependent Cap3 methyltransferase from Acanthamoeba castellanii medusavirus, demonstrating in vitro Cap3 activity for the first time. Sequence analysis revealed homology to known cap methyltransferases. Enzyme kinetic analysis demonstrated an adequate Km value, indicating a moderate substrate affinity and suitability for practical applications. Furthermore, Cap3MTase enabled the preparation of novel Cap3-mRNAs, offering a valuable tool for mRNA modification and therapeutic development.