(2016) m6A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination. Nature, 540 (7632). pp. 301-304. ISSN 1476-4687 Access from the University of Nottingham repository: http://eprints.nottingham.ac.uk/39448/1/Nature_m6A_2016.pdf Copyright and reuse:The Nottingham ePrints service makes this work by researchers of the University of Nottingham available open access under the following conditions. This article is made available under the University of Nottingham End User licence and may be reused according to the conditions of the licence. For more details see: http://eprints.nottingham.ac.uk/end_user_agreement.pdf A note on versions:The version presented here may differ from the published version or from the version of record. If you wish to cite this item you are advised to consult the publisher's version. Please see the repository url above for details on accessing the published version and note that access may require a subscription. sex bias towards maleness. This is because m6A is required for female-specific AS of Sxl, 37 which determines female physiognomy, but also translationally represses male-specific 38 lethal2 (msl-2) to prevent dosage compensation normally occurring in males. We further 39show that the m6A reader protein YT521-B decodes m6A in the sex-specifically spliced 40 intron of Sxl, as its absence phenocopies dIME4 mutants. Loss of m6A also affects AS of 41 additional genes, predominantly in the 5'UTR, and has global impacts on the expression of 42 metabolic genes. Requirement of m6A and its reader YT521-B for female-specific Sxl AS 43 reveal that this hitherto enigmatic mRNA modification constitutes an ancient and specific 44 mechanism to adjust levels of gene expression. 45In mature mRNA the m6A modification is most prevalently found around the stop codon as well 46 as in 5'UTRs and in long exons in mammals, plants and yeast 2,3,6,7,15 . Since methylosome 47 components predominantly localize to the nucleus it has been speculated that m6A localized in 48Haussmann et al.3 pre-mRNA introns could have a role in AS regulation in addition to such a role when present in 49 long exons [9][10][11][12]16 . This prompted us to investigate whether m6A is required for Sxl AS, which 50 determines female sex and prevents dosage compensation in females 13 . We generated a null 51 allele of the Drosophila METTL3 methyltransferase homologue dIME4 by imprecise excision of 52 a P-element inserted in the promoter region. The excision ∆22-3 deletes most of the protein-53 coding region including the catalytic domain and is thus referred to as dIME4 null (Fig 1a). These 54 flies are viable and fertile, but flightless, and this phenotype can be rescued by a genomic 55 construct restoring dIME4 (Fig 1a and b). dIME4 shows increased expression in the brain, and 56 like in mammals and plants 17, localizes to the nucleus (Fig 1c,d). 57Following RNAse T1 digestion and 32 P end-labeling of RNA fragments we detected m6A after G 58 in polyA mRNA of adult flies at relatively low l...

Interest in mRNA methylation has exploded in recent years. The sudden interest in a 40 year old discovery was due in part to the finding of FTO’s (Fat Mass Obesity) N6-methyl-adenosine (m6A) deaminase activity, thus suggesting a link between obesity-associated diseases and the presence of m6A in mRNA. Another catalyst of the sudden rise in mRNA methylation research was the release of mRNA methylomes for human, mouse and Saccharomyces cerevisiae. However, the molecular function, or functions of this mRNA ‘epimark’ remain to be discovered. There is supportive evidence that m6A could be a mark for mRNA degradation due to its binding to YTH domain proteins, and consequently being chaperoned to P bodies. Nonetheless, only a subpopulation of the methylome was found binding to YTHDF2 in HeLa cells.The model organism Saccharomyces cerevisiae, has only one YTH domain protein (Pho92, Mrb1), which targets PHO4 transcripts for degradation under phosphate starvation. However, mRNA methylation is only found under meiosis inducing conditions, and PHO4 transcripts are apparently non-methylated. In this paper we set out to investigate if m6A could function alternatively to being a degradation mark in S. cerevisiae; we also sought to test whether it can be induced under non-standard sporulation conditions. We find a positive association between the presence of m6A and message translatability. We also find m6A induction following prolonged rapamycin treatment.

Cap-adjacent nucleotides of animal, protist and viral mRNAs can be O-methylated at the 2‘ position of the ribose (cOMe). The functions of cOMe in animals, however, remain largely unknown. Here we show that the two cap methyltransferases (CMTr1 and CMTr2) of Drosophila can methylate the ribose of the first nucleotide in mRNA. Double-mutant flies lack cOMe but are viable. Consistent with prominent neuronal expression, they have a reward learning defect that can be rescued by conditional expression in mushroom body neurons before training. Among CMTr targets are cell adhesion and signaling molecules. Many are relevant for learning, and are also targets of Fragile X Mental Retardation Protein (FMRP). Like FMRP, cOMe is required for localization of untranslated mRNAs to synapses and enhances binding of the cap binding complex in the nucleus. Hence, our study reveals a mechanism to co-transcriptionally prime mRNAs by cOMe for localized protein synthesis at synapses.

SummaryExperimental procedures for preparing RNA-seq and single-cell (sc) RNA-seq libraries are based on assumptions regarding their underlying enzymatic reactions. Here, we show that the fairness of these assumptions varies within libraries: coverage by sequencing reads along and between transcripts exhibits characteristic, protocol-dependent biases. To understand the mechanistic basis of this bias, we present an integrated modeling framework that infers the relationship between enzyme reactions during library preparation and the characteristic coverage patterns observed for different protocols. Analysis of new and existing (sc)RNA-seq data from six different library preparation protocols reveals that polymerase processivity is the mechanistic origin of coverage biases. We apply our framework to demonstrate that lowering incubation temperature increases processivity, yield, and (sc)RNA-seq sensitivity in all protocols. We also provide correction factors based on our model for increasing accuracy of transcript quantification in existing samples prepared at standard temperatures. In total, our findings improve our ability to accurately reflect in vivo transcript abundances in (sc)RNA-seq libraries.