Yeast targets for mRNA methylation.

N(6)-Methyladenosine (m(6)A) is a modified base present in the mRNA of all higher eukaryotes and in Saccharomyces cerevisiae, where there is an increase in m(6)A levels during sporulation. The methyltransferase, Ime4, is responsible for this modification and has a role in the initiation of meiosis. However, neither the function, nor the extent of distribution of this nucleotide modification is established. We demonstrate that in S. cerevisiae, substantial levels of internal adenosine methylation are present in the GpA context in mRNA from sporulating cells, which is consistent with the preferred methylation consensus of higher eukaryotes. Based upon our quantification data, every second transcript could contain one m(6)A during meiosis. As methylation is distributed across all mRNA size ranges, it is likely that m(6)A is not limited to a small population of messages. We developed a new antibody based method for identifying m(6)A containing messages, and using this method the transcripts of three key, early regulators of meiosis, IME1, IME2 and IME4 itself, were identified as being methylated. The position of m(6)A in IME2 was narrowed down to a region in the 3'-end. Methylation of these and other targets suggests mechanisms by which IME4 could control developmental choices leading to meiosis.