We adapted UV CLIP (cross-linking immunoprecipitation) to accurately locate tens of thousands of m 6 A residues in mammalian mRNA with single-nucleotide resolution. More than 70% of these residues are present in the 3 ′ -most (last) exons, with a very sharp rise (sixfold) within 150-400 nucleotides of the start of the last exon. Two-thirds of last exon m 6 A and >40% of all m 6 A in mRNA are present in 3 ′ untranslated regions (UTRs); contrary to earlier suggestions, there is no preference for location of m 6 A sites around stop codons. Moreover, m 6 A is significantly higher in noncoding last exons than in next-to-last exons harboring stop codons. We found that m 6 A density peaks early in the 3 ′ UTR and that, among transcripts with alternative polyA (APA) usage in both the brain and the liver, brain transcripts preferentially use distal polyA sites, as reported, and also show higher proximal m 6 A density in the last exons. Furthermore, when we reduced m6A methylation by knocking down components of the methylase complex and then examined 661 transcripts with proximal m6A peaks in last exons, we identified a set of 111 transcripts with altered (approximately two-thirds increased proximal) APA use. Taken together, these observations suggest a role of m 6 A modification in regulating proximal alternative polyA choice.

Understanding the biologic role of N 6 -methyladenosine (m 6 A) RNA modifications in mRNA requires an understanding of when and where in the life of a pre-mRNA transcript the modifications are made. We found that HeLa cell chromatin-associated nascent pre-mRNA (CA-RNA) contains many unspliced introns and m 6 A in exons but very rarely in introns. The m 6 A methylation is essentially completed upon the release of mRNA into the nucleoplasm. Furthermore, the content and location of each m 6 A modification in steady-state cytoplasmic mRNA are largely indistinguishable from those in the newly synthesized CA-RNA or nucleoplasmic mRNA. This result suggests that quantitatively little methylation or demethylation occurs in cytoplasmic mRNA. In addition, only ∼10% of m 6 As in CA-RNA are within 50 nucleotides of 5 ′ or 3 ′ splice sites, and the vast majority of exons harboring m 6 A in wild-type mouse stem cells is spliced the same in cells lacking the major m 6 A methyltransferase Mettl3. Both HeLa and mouse embryonic stem cell mRNAs harboring m 6 As have shorter half-lives, and thousands of these mRNAs have increased half-lives (twofold or more) in Mettl3 knockout cells compared with wild type. In summary, m 6 A is added to exons before or soon after exon definition in nascent pre-mRNA, and while m 6 A is not required for most splicing, its addition in the nascent transcript is a determinant of cytoplasmic mRNA stability. Studying nascent RNA synthesis in cultured cells using very brief pulse labeling with radioactive nucleosides allowed a number of advances in understanding premRNA synthesis and processing in the era before rapid nucleic acid sequencing. Examples include polyA addition on pre-mRNA before completion of mRNA processing and cytoplasmic entry (Darnell et al. 1971;Edmonds et al. 1971) and locating the first known boundaries of eukaryotic polymerase II transcription units through studying labeled nascent adenovirus transcripts (Bachenheimer and Darnell 1975;Evans et al. 1977;Weber et al. 1977).These early experiments were joined by a cell fractionation technique originated by Wuarin and Schibler (1994) that uses a 1 M urea solution to liberate a "chromatin" fraction from nuclei. This fraction provides a stringent purification of growing nascent pre-mRNA chains, isolated as a chromatin-associated RNA fraction (referred to as CA-RNA), along with RNA polymerase II plus all nuclear DNA and associated histones. Using specific labeled DNA probes, Wuarin and Schibler (1994) demonstrated removal in liver cell nuclei of some, but not all, introns from two specific nascent pre-mRNAs: a transcription factor pre-mRNA involved in circadian rhythm and the HMG coA reductase pre-mRNA. Recently, Pandya-Jones and Black (2009) adapted this procedure to study the extent and order of intron removal in cultured human carcinoma cell nuclei, again showing that many, but not all, introns are removed in CA-RNA.

We describe a comprehensive quantitative measure of the splicing impact of a complete set of RNA 6-mer sequences by deep sequencing successfully spliced transcripts. All 4096 6-mers were substituted at five positions within two different internal exons in a 3-exon minigene, and millions of successfully spliced transcripts were sequenced after transfection of human cells. The results allowed the assignment of a relative splicing strength score to each mutant molecule. The effect of 6-mers on splicing often depended on their location; much of this context effect could be ascribed to the creation of different overlapping sequences at each site. Taking these overlaps into account, the splicing effect of each 6-mer could be quantified, and 6-mers could be designated as enhancers (ESEseqs) and silencers (ESSseqs), with an ESRseq score indicating their strength. Some 6-mers exhibited positional bias relative to the two splice sites. The distribution and conservation of these ESRseqs in and around human exons supported their classification. Predicted RNA secondary structure effects were also seen: Effective enhancers, silencers and 39 splice sites tend to be single stranded, and effective 59 splice sites tend to be double stranded. 6-mers that may form positive or negative synergy with another were also identified. Chromatin structure may also influence the splicing enhancement observed, as a good correspondence was found between splicing performance and the predicted nucleosome occupancy scores of 6-mers. This approach may prove of general use in defining nucleic acid regulatory motifs, substitute for functional SELEX in most cases, and provide insights about splicing mechanisms.[Supplemental material is available for this article.]The transfer of genetic information from DNA to protein in living things is accomplished with accuracy, precision, and fidelity. These qualities characterize pre-mRNA splicing as much as transcription and translation (Fox-Walsh and Hertel 2009). The accurate identification of splice sites in long metazoan transcripts depends not only on the splice-site sequences that are substrates for the splicing reaction, but also on short RNA stretches known as exonic and intronic splicing enhancers (ESEs and ISEs) and silencers (ESSs and ISSs). These so-called splicing regulatory motifs are manifold and,