Poster abstracts
Poster number 25 submitted by Jackson Trotman
RNMT catalyzes cytoplasmic mRNA cap methylation and is a component of the cytoplasmic capping complex
Jackson B. Trotman (Biological Chemistry & Pharmacology, The Ohio State University), Chandrama Mukherjee (Biological Chemistry & Pharmacology, The Ohio State University), Daniel R. Schoenberg (Biological Chemistry & Pharmacology, The Ohio State University)
Abstract:
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. 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.
Keywords: mRNA metabolism, cytoplasmic capping, metabolon