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.
The N7-methylguanosine cap is a hallmark of the 5' end of eukaryotic mRNAs and is required for gene expression. Loss of the cap was believed to lead irreversibly to decay. However, nearly a decade ago, it was discovered that mammalian cells contain enzymes in the cytoplasm that are capable of restoring caps onto uncapped RNAs. In this review, we summarize recent advances in our understanding of cytoplasmic RNA recapping and discuss the biochemistry of this process and its impact on regulating and diversifying the transcriptome. Although most studies focus on mammalian RNA recapping, we also highlight new observations for recapping in disparate eukaryotic organisms, with the trypanosome recapping system appearing to be a fascinating example of convergent evolution. We conclude with emerging insights into the biological significance of RNA recapping and prospects for the future of this evolving area of study. RNA Processing > RNA Editing and Modification Translation > Translation Regulation RNA Processing > Capping and 5' End Modifications RNA Turnover and Surveillance > Regulation of RNA Stability.
Cap homeostasis is the cyclical process of decapping and recapping that maintains the translation and stability of a subset of the transcriptome. Previous work showed levels of some recapping targets decline following transient expression of an inactive form of RNMT (ΔN-RNMT), likely due to degradation of mRNAs with improperly methylated caps. The current study examined transcriptome-wide changes following inhibition of cytoplasmic cap methylation. This identified mRNAs with 5′-terminal oligopyrimidine (TOP) sequences as the largest single class of recapping targets. Cap end mapping of several TOP mRNAs identified recapping events at native 5′ ends and downstream of the TOP sequence of EIF3K and EIF3D. This provides the first direct evidence for downstream recapping. Inhibition of cytoplasmic cap methylation was also associated with mRNA abundance increases for a number of transcription, splicing, and 3′ processing factors. Previous work suggested a role for alternative polyadenylation in target selection, but this proved not to be the case. However, inhibition of cytoplasmic cap methylation resulted in a shift of upstream polyadenylation sites to annotated 3′ ends. Together, these results solidify cap homeostasis as a fundamental process of gene expression control and show cytoplasmic recapping can impact regulatory elements present at the ends of mRNA molecules.
The polycomb repressive complexes 1 and 2 (PRCs; PRC1 and PRC2) are conserved histone-modifying enzymes that often function cooperatively to repress gene expression. The PRCs are regulated by long noncoding RNAs (lncRNAs) in complex ways. On the one hand, specific lncRNAs cause the PRCs to engage with chromatin and repress gene expression over genomic regions that can span megabases. On the other hand, the PRCs bind RNA with seemingly little sequence specificity, and at least in the case of PRC2, direct RNA-binding has the effect of inhibiting the enzyme. Thus, some RNAs appear to promote PRC activity, while others may inhibit it. The reasons behind this apparent dichotomy are unclear. The most potent PRC-activating lncRNAs associate with chromatin and are predominantly unspliced or harbor unusually long exons.Emerging data imply that these lncRNAs promote PRC activity through internal RNA sequence elements that arise and disappear rapidly in evolutionary time. These sequence elements may function by interacting with common subsets of RNA-binding proteins that recruit or stabilize PRCs on chromatin.
The Xist lncRNA requires Repeat A, a conserved RNA element located in its 5′ end, to induce gene silencing during X-chromosome inactivation. Intriguingly, Repeat A is also required for production of Xist. While silencing by Repeat A requires the protein SPEN, how Repeat A promotes Xist production remains unclear. We report that in mouse embryonic stem cells, expression of a transgene comprising the first two kilobases of Xist (Xist-2kb) causes transcriptional readthrough of downstream polyadenylation sequences. Readthrough required Repeat A and the ∼750 nucleotides downstream, did not require SPEN, and was attenuated by splicing. Despite associating with SPEN and chromatin, Xist-2kb did not robustly silence transcription, whereas a 5.5-kb Xist transgene robustly silenced transcription and read through its polyadenylation sequence. Longer, spliced Xist transgenes also induced robust silencing yet terminated efficiently. Thus, in contexts examined here, Xist requires sequence elements beyond its first two kilobases to robustly silence transcription, and the 5′ end of Xist harbors SPEN-independent transcriptional antiterminator activity that can repress proximal cleavage and polyadenylation. In endogenous contexts, this antiterminator activity may help produce full-length Xist RNA while rendering the Xist locus resistant to silencing by the same repressive complexes that the lncRNA recruits to other genes.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.