N-methyladenosine RNA (mA) is a prevalent messenger RNA modification in vertebrates. Although its functions in the regulation of post-transcriptional gene expression are beginning to be unveiled, the precise roles of mA during development of complex organisms remain unclear. Here we carry out a comprehensive molecular and physiological characterization of the individual components of the methyltransferase complex, as well as of the YTH domain-containing nuclear reader protein in Drosophila melanogaster. We identify the member of the split ends protein family, Spenito, as a novel bona fide subunit of the methyltransferase complex. We further demonstrate important roles of this complex in neuronal functions and sex determination, and implicate the nuclear YT521-B protein as a main mA effector in these processes. Altogether, our work substantially extends our knowledge of mA biology, demonstrating the crucial functions of this modification in fundamental processes within the context of the whole animal.

The combination of Reverse Transcription (RT) and high-throughput sequencing has emerged as a powerful combination to detect modified nucleotides in RNA via analysis of either abortive RT-products or of the incorporation of mismatched dNTPs into cDNA. Here we simultaneously analyze both parameters in detail with respect to the occurrence of N-1-methyladenosine (m1A) in the template RNA. This naturally occurring modification is associated with structural effects, but it is also known as a mediator of antibiotic resistance in ribosomal RNA. In structural probing experiments with dimethylsulfate, m1A is routinely detected by RT-arrest. A specifically developed RNA-Seq protocol was tailored to the simultaneous analysis of RT-arrest and misincorporation patterns. By application to a variety of native and synthetic RNA preparations, we found a characteristic signature of m1A, which, in addition to an arrest rate, features misincorporation as a significant component. Detailed analysis suggests that the signature depends on RNA structure and on the nature of the nucleotide 3′ of m1A in the template RNA, meaning it is sequence dependent. The RT-signature of m1A was used for inspection and confirmation of suspected modification sites and resulted in the identification of hitherto unknown m1A residues in trypanosomal tRNA.

Repression of genes by Polycomb requires that PRC2 modifies their chromatin by trimethylating lysine 27 on histone H3 (H3K27me3). At transcriptionally active genes, di- and trimethylated H3K36 inhibit PRC2. Here, the cryo-EM structure of PRC2 on dinucleosomes reveals how binding of its catalytic subunit EZH2 to nucleosomal DNA orients the H3 N-terminus via an extended network of interactions to place H3K27 into the active site. Unmodified H3K36 occupies a critical position in the EZH2-DNA interface. Mutation of H3K36 to arginine or alanine inhibits H3K27 methylation by PRC2 on nucleosomes in vitro. Accordingly, Drosophila H3K36A and H3K36R mutants show reduced levels of H3K27me3 and defective Polycomb repression of HOX genes. The relay of interactions between EZH2, the nucleosomal DNA and the H3 N-terminus therefore creates the geometry that permits allosteric inhibition of PRC2 by methylated H3K36 in transcriptionally active chromatin.

In the sentence beginning 'The meQTL are enriched for functionally relevant characteristics', please provide a de inition for Hi-C.2. In the author list, there are two authors with the name Panos Deloukas with different af iliations. Please con irm whether this is the same individual and whether the two authors can be merged. 3.Please check your article carefully, coordinate with any co-authors and enter all inal edits clearly in the eproof, remembering to save frequently. Once corrections are submitted, we cannot routinely make further changes to the article. 4. Note that the eproof should be amended in only one browser window at any one time; otherwise changes will be overwritten. 5. Author surnames have been highlighted. Please check these carefully and adjust if the irst name or surname is marked up incorrectly. Note that changes here will affect indexing of your article in public repositories such as PubMed. Also, carefully check the spelling and numbering of all author names and af iliations, and the corresponding email address(es).6. You cannot alter accepted Supplementary Information iles except for critical changes to scienti ic content. If you do resupply any iles, please also provide a brief (but complete) list of changes. If these are not considered scienti ic changes, any altered Supplementary iles will not be used, only the originally accepted version will be published. If applicable, please ensure that any accession codes and datasets whoseDOIs or other identi iers are mentioned in the paper are scheduled for public release as soon as possible, we recommend within a few days of submitting your proof, and update the database record with publication details from this article once available. 8. Your paper has been copy edited. Please review every sentence to ensure that it conveys your intended meaning; if changes are required, please provide further clari ication rather than reverting to the original text. Please note that formatting (including hyphenation, Latin words,and any reference citations that might be mistaken for exponents) has been made consistent with our house style. 9. Please con irm or correct the city name inserted in af iliations 5, 10, 12, 13, 21, 22, 39 and 44. 10. In the sentence beginning 'Our meQTL replicate in data generated by the Illumina', please provide a de inition for EPIC and con irm whether the de inition provided for MeDIP-seq is correct. 11. In the legend for Fig. 2g, please provide a de inition for SAT. 12. In the sentence beginning 'We used summary data-based Mendelian', please con irm whether it is correct to say '0.05 ÷ n tests'. 13. In the sentence beginning 'We then tested the 4,811 eQTL genes', please provide a de inition for coloc PP4. 14. Please note, we reserve ‘signi icant' and its derivatives for statistical signi icance. This has been reworded where this was not the intended meaning (for example to important, notable, ...