RNA guanine-7 methyltransferase catalyzes the methylation of cytoplasmically recapped RNAs

Abstract

Cap homeostasis is a cyclical process of decapping and recapping that impacts a portion of the mRNA transcriptome. The metastable uncapped forms of recapping targets redistribute from polysomes to non-translating mRNPs, and recapping is all that is needed for their return to the translating pool. Previous work identified a cytoplasmic capping metabolon consisting of capping enzyme (CE) and a 5′-monophosphate kinase bound to adjacent domains of Nck1. The current study identifies the canonical cap methyltransferase (RNMT) as the enzyme responsible for guanine-N7 methylation of recapped mRNAs. RNMT binds directly to CE, and its presence in the cytoplasmic capping complex was demonstrated by pulldown assays, gel filtration and proximity-dependent biotinylation. The latter also identified the RNMT cofactor RAM, whose presence is required for cytoplasmic cap methyltransferase activity. These findings guided development of an inhibitor of cytoplasmic cap methylation whose action resulted in a selective decrease in levels of recapped mRNAs.

RNA Cap Methyltransferase Activity Assay

“…, against nucleolin and tubulin) can determine whether cross-contamination occurred during the fractionation procedure. For an example of expected Western blot results, see Figure 1C of Trotman et al , 2017.…”

“…The N7 methyl group is particularly critical for the mRNA life cycle, as it drastically increases the binding affinity of cap-binding proteins (Niedzwiecka et al , 2002) and protects mRNAs from cap-quality control surveillance mechanisms (Jiao et al , 2013). We recently reported that the mammalian RNA guanine-7 methyltransferase (RNMT) functions beyond its canonical role in nuclear co-transcriptional cap synthesis to participate in cytoplasmic RNA recapping (Trotman et al , 2017). We used the protocol presented here to demonstrate that the cap methyltransferase activity of cytoplasmic RNMT is unexpectedly robust relative to nuclear RNMT.…”

“…was funded by NIH T32 training grant GM08512 (to The Ohio State University) and by a pre-doctoral fellowship from The Ohio State University Center for RNA Biology. The protocol presented here was adapted from those in Pillutla et al , 1998, Otsuka et al , 2009, and Cowling, 2010 and was used for experiments in Trotman et al , 2017.…”

“… 1 The recombinant human capping enzyme we used to generate the radiolabeled GpppRNA was produced in-house as described in Trotman et al (2017). Despite this enzyme stock having robust capping activity with non-radiolabeled GTP, the production of radiolabeled GpppRNA was rather inefficient.…”

“…The protocol presented here simply describes an easy way to generate the same 32 nt substrate RNA used in the initial characterization of human RNMT (Pillutla et al , 1998) and in our recent study (Trotman et al , 2017). …”