Jackson earned his B.S. from the University of North Carolina at Chapel Hill with majors in chemistry and biology. At UNC, Jackson worked with Bill Marzluff to study the translation of histone messenger mRNAs. Jackson’s interest in RNA biochemistry brought him to the lab of Daniel Schoenberg at The Ohio State University, where he is currently finishing his third year of graduate training. Jackson’s research uses a combination of cell biology and biochemistry to identify and understand how cytoplasmic enzymes catalyze “recapping” at the 5’ end of messenger RNAs.
RNA guanine-N7 methyltransferase (RNMT) is the component of the cytoplasmic
capping complex that catalyzes the methylation of recapped mRNAs
The Ohio State University, Columbus, Ohio
The N7-methylguanosine cap structure at the 5’ end of a messenger RNA (mRNA) is essential for recognition by cap-binding proteins that direct translation initiation. Accordingly, cyclic decapping and recapping of mRNAs in the cytoplasm is a mechanism that confers mammalian cells control of gene expression. In the current view of cytoplasmic (re)capping, three successive reactions are necessary to restore a mature cap structure onto a 5’ monophosphate end: monophosphate phosphorylation, guanylate transfer, and guanine-N7 methylation. The first two reactions are catalyzed respectively by a 5’ monophosphate kinase and capping enzyme (CE), which assemble on adjacent domains of the adapter protein Nck1 to form the cytoplasmic capping complex (cCC). The objective of this study was to identify the cytoplasmic enzyme responsible for guanine-N7 cap methylation and to determine whether it functions as a member of the cCC. Despite cap methyltransferase (RNMT) being well-characterized for its role in nuclear cap methylation, we demonstrate by immunofluorescence and subcellular fractionation that this enzyme also localizes to the cytoplasm in multiple cell types. Knockdown of RNMT reduces the cap methylation activity of cytoplasmic extracts, confirming that RNMT catalyzes the final reaction of cytoplasmic capping. Cytoplasmic RNMT co-elutes with CE and Nck1 by size exclusion chromatography, and co-precipitation experiments demonstrate that RNMT interacts with known components of the cCC. Using both recombinant and cellular proteins, we show that RNMT binds directly to CE and that this interaction is mediated by the C-terminal catalytic domain of RNMT and the extreme C-terminus of CE. We also show that the activity of cytoplasmic RNMT is stimulated by binding to its small protein cofactor RAM. Collectively, these results establish that RNMT is a component of the cCC and that it functions to complete the addition of the N7-methylguanosine cap necessary for a recapped mRNA to be translated.