Cytidine modifications in mRNA processing and function

Division Of Basic Sciences - Nci

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Abstract

Modified cytosine residues in DNA show asymmetric distribution within gene bodies, wherein 5mC and its oxidized derivatives 5-hydroxymethylcytosine (5hmC) and 5-carboxylcytosine (5caC) are enriched at exons of actively transcribed genes, while intronless genes and alternative exons that are excluded from spliced mRNA are globally hypomethylated. As pre-mRNA splicing occurs co-transcriptionally, the association between intragenic DNA methylation and regions that splice in mRNA suggested a potential functional connection. Research in my group examined this relationship bidirectionally: we asked whether splicing shapes the intragenic methylome and/or whether DNA methylation influences pre-mRNA splicing decisions. To address the former, we generated isogenic cell lines with stably integrated minigene reporters and determined that the ability to gain and maintain DNA methylation occurs independently of splicing (Nanan et al., Nucleic Acids Res, 2017). In contrast, we readily detected a role for DNA methylation in modulating co-transcriptional pre-mRNA splicing. We first established such a connection in our description of a CTCF/5mC splicing switch: intragenic CTCF transiently obstructs RNA polymerase II (pol II) elongation and facilitates spliceosome assembly at weak upstream exons, while overlapping 5mC leads to CTCF eviction and associated alternative exon exclusion (Shukla et al., Nature, 2011). These findings were the first to show a role for DNA methylation and for a DNA binding protein in the regulation of pre-mRNA splicing and provided experimental support for the kinetic regulation of splicing. We further established that coordinated exchange between CTCF and 5mC in regulated alternative pre-mRNA splicing is achieved through active oxidation of 5mC to 5caC, which promotes CTCF binding and related exon inclusion (Marina et al., EMBO J, 2016; Nanan et al., iScience, 2019). These studies provide proof-of-concept that modification of a single nucleotide in DNA can directly lead to proteome expansion through modulating alternative pre-mRNA splicing decisions. Significant questions remain, including the mechanisms supporting asymmetric distribution of methylation within gene bodies and impact of frequent DNMT and TET protein mutations on alternative pre-mRNA splicing in cancer cells. Recent technological advances have further catalogued an expansive repertoire of chemical modifications in RNA. To date, >170 modifications have been identified in eukaryotic RNA, a handful of which are found in mRNA. mRNA modifications have been documented to influence essentially every aspect of mRNA metabolism, including stability, translation, and splicing. Based on our expertise in RNA biology and epigenetics, we extended our research scope to include modifications in mRNA with a specific focus on cytidine. In so doing, we identified N4-acetylcytidine (ac4C) as a novel mRNA modification that is uniquely catalyzed by the enzyme N-acetyltransferase 10 (NAT10) (Arango et al., Cell, 2018). ac4C is the sole acetylation event to be described in eukaryotic RNA, and both NAT10 and ac4C are universally conserved across all domains of life. Through generating tools for transcriptome-wide mapping of ac4C, we found acetylation to be enriched in 5' untranslated regions (5'UTRs) and coding sequences (CDS) of substrate mRNAs, with the majority of sites occurring in the latter. Surprisingly, functional analysis of ac4C function revealed location-specific impacts on mRNA translation: while CDS ac4C promoted mRNA translation elongation, 5'UTR ac4C inhibited translation initiation (Arango et al., Molecular Cell, 2022). As NAT10 is amplified in a variety of human cancers, these findings highlight potential physiological relevance in the regulation of cell division. Our recent studies have focused on determining the mechanisms and functions of RNA acetylation, with a focus on therapeutic applications. To deepen our knowledge of the endogenous functions of ac4C, we have developed enhanced methods for its mapping in the cellular acetylome (Relier et al., RNA, 2024). We next aim to characterize the regulation of NAT10-catalyzed acetylation in normal development and disease. In addition, we are evaluating the utility of ac4C in next-generation mRNA-based medicines.