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.

Our understanding of the expanded genetic alphabet has been growing rapidly over the last two decades, and many of these developments came more than 80 years after the original discovery of a modified guanine in tuberculosis DNA. These new understandings, leading to the field of epigenetics, have led to exciting new fundamental and applied knowledge and to the development of novel classes of drugs exploiting this new biology. The number of methyl modifications to RNA is about seven times greater than those found on DNA, and our ability to interrogate these enigmatic nucleobases has lagged significantly until recent years as an explosion in technologies and understanding has revealed the roles and regulation of RNA methylation in several fundamental and disease-associated biological processes. Here, we outline how the technology has evolved and which strategies are commonly used in the modern epitranscriptomics revolution and give a foundation in the understanding and application of the rich variety of these methods to novel biological questions.

• Characterization of global RNA-seq biases specific to library preparation protocols • Mathematical framework to reverse engineer enzyme reactions that cause bias • Insights from reverse engineering allow optimization of RNA-seq protocols • Lowered incubation temperatures during library preparation improve sensitivity

Streptococcus uberis is a common cause of intramammary infection and mastitis in dairy cattle. Unlike other mammary pathogens, S. uberis evades detection by mammary epithelial cells, and the host–pathogen interactions during early colonisation are poorly understood. Intramammary challenge of dairy cows with S. uberis (strain 0140J) or isogenic mutants lacking the surface-anchored serine protease, SUB1154, demonstrated that virulence was dependent on the presence and correct location of this protein. Unlike the wild-type strain, the mutant lacking SUB1154 failed to elicit IL-1β from ex vivo CD14+ cells obtained from milk (bovine mammary macrophages, BMM), but this response was reinstated by complementation with recombinant SUB1154; the protein in isolation elicited no response. Production of IL-1β was ablated in the presence of various inhibitors, indicating dependency on internalisation and activation of NLRP3 and caspase-1, consistent with inflammasome activation. Similar transcriptomic changes were detected in ex vivo BMM in response to the wild-type or the SUB1154 deletion mutant, consistent with S. uberis priming BMM, enabling the SUB1154 protein to activate inflammasome maturation in a transcriptionally independent manner. These data can be reconciled in a novel model of pathogenesis in which, paradoxically, early colonisation is dependent on the innate response to the initial infection.

N6-methyladenosine (m⁶A) in mRNA regulates almost every stage in the mRNA life cycle, and the development of methodologies for the high-throughput detection of methylated sites in mRNA using m⁶A-specific methylated RNA immunoprecipitation with next-generation sequencing (MeRIPSeq) or m⁶A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) have revolutionized the m⁶A research field. Both of these methods are based on immunoprecipitation of fragmented mRNA. However, it is well documented that antibodies often have nonspecific activities, thus verification of identified m⁶A sites using an antibody-independent method would be highly desirable. We mapped and quantified the m⁶A site in the chicken β-actin zipcode based on the data from chicken embryo MeRIPSeq results and ourRNA-EpimodificationDetection andBase-Recognition(RedBaron) antibody-independent assay. We also demonstrated that methylation of this site in the β-actin zipcode enhances ZBP1 binding in vitro, while methylation of a nearby adenosine abolishes binding. This suggests that m⁶A may play a role in regulating localized translation of β-actin mRNA, and the ability of m⁶A to enhance or inhibit a reader protein's RNA binding highlights the importance of m⁶A detection at nucleotide resolution.