RNA contains more than 100 distinct modifications that promote the functions of stable non-coding RNAs in translation and splicing. Recent technical advances have revealed widespread and sparse modification of messenger RNAs with N6-methyladenosine (m6A), 5-methylcytosine (m5C) and pseudouridine (Ψ). Here we discuss the rapidly evolving understanding of the location, regulation and function of these dynamic mRNA marks, collectively termed the epitranscriptome. We highlight differences among modifications and between species that could instruct ongoing efforts to understand how specific mRNAs target sites are selected and how their modification is regulated. Diverse molecular consequences of individual m6A modifications are beginning to be revealed but the effects of m5C and Ψ remain largely unknown. Future work linking molecular effects to organismal phenotypes will broaden our understanding of mRNA modifications as cell and developmental regulators.

Pseudouridine (Ψ) is a post-transcriptional RNA modification that alters RNA-RNA and RNAprotein interactions that affect gene expression. mRNA pseudouridylation was recently discovered as a widespread and conserved phenomenon, but the mechanisms responsible for selective, regulated pseudouridylation of specific sequences within mRNAs were unknown. Here, we have revealed new mRNA targets for five pseudouridine synthases and probed the determinants of mRNA target recognition by the predominant mRNA pseudouridylating enzyme, Pus1, by developing high-throughput kinetic analysis of pseudouridylation in vitro. Combining computational prediction and rational mutational analysis revealed an RNA structural motif that is both necessary and sufficient for mRNA pseudouridylation. Applying this structural context information predicted hundreds of additional mRNA targets, that we showed were pseudouridylated in vivo. These results demonstrate a structure-dependent mode of mRNA target recognition by a conserved pseudouridine synthase and implicate modulation of RNA structure as the likely mechanism to regulate mRNA pseudouridylation.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms

No abstract

Non-coding RNAs contain dozens of chemically distinct modifications, of which only a few have been identified in mRNAs. The recent discovery that certain tRNA modifying enzymes also target mRNAs suggests the potential for many additional mRNA modifications. Here, we show that conserved tRNA 2′-O-methyltransferases Trm3, 7,13 and 44, and rRNA 2′-O-methyltransferase Spb1, interact with specific mRNA sites in yeast by crosslinking immunoprecipitation and sequencing (CLIP-seq). We developed sequencing of methylation at two prime hydroxyls (MeTH-seq) for transcriptome-wide mapping of 2′-O-methyl ribose (Nm) with single-nucleotide resolution, and discover thousands of potential Nm sites in mRNAs. Genetic analysis identified hundreds of mRNA targets for the Spb1 methyltransferase, which can target both mRNA and noncoding RNA for environmentally regulated modification. Our work identifies Nm as a prevalent mRNA modification that is likely to be conserved and provides methods to investigate its distribution and regulation. KEYWORDS2′-O-methyltransferase, 2′-O-methylribose, CLIP-seq, MeTH-seq, mRNA modifications HIGHLIGHTS • MeTH-seq identifies 2′-O-methylribose genome-wide at single-nucleotide resolution • Five conserved methyltransferases interact with yeast mRNA

Eukaryotic messenger RNAs are extensively decorated with modified nucleotides and the resulting epitranscriptome plays important regulatory roles in cells 1. Pseudouridine (Ψ) is a modified nucleotide that is prevalent in human mRNAs and can be dynamically regulated 2–5. However, it is unclear when in their life cycle RNAs become pseudouridylated and what the endogenous functions of mRNA pseudouridylation are. To determine if pseudouridine is added co-transcriptionally, we conducted pseudouridine profiling 2 on chromatin-associated RNA to reveal thousands of intronic pseudouridines in nascent pre-mRNA at locations that are significantly associated with alternatively spliced exons, enriched near splice sites, and overlap hundreds of binding sites for regulatory RNA binding proteins. Multiple distinct pseudouridine synthases with tissue-specific expression pseudouridylate pre-mRNA sites, and genetic manipulation of the predominant pre-mRNA modifying pseudouridine synthases PUS1, PUS7 and RPUSD4 induced widespread changes in alternative splicing in cells, supporting a role for pre-mRNA pseudouridylation in alternative splicing regulation. Consistently, we find that individual pseudouridines identified in cells are sufficient to directly affect splicing in vitro. Together with previously observed effects of artificial pseudouridylation on RNA-RNA6–8 and RNA-protein 9–11 interactions that are relevant for splicing, our results demonstrate widespread co-transcriptional pre-mRNA pseudouridylation and establish the enormous potential for this RNA modification to control human gene expression.