The IME4 gene of the yeast Saccharomyces cerevisiae encodes an activator of meiotic development, but the basis for its activity is obscure. IME4 was originally identified by virtue of its interaction with the mating type pathway, and its function, as defined genetically, is to promote meiotic development. Indeed, overexpression of IME4 overrides the normal requirements for the a1 and alpha2 regulatory molecules, permitting sporulation to occur in inappropriate cell types. Homology to a human protein called MTA-70 suggests a surprising mode of action for the IME4 protein. MTA-70 is believed to be the catalytic subunit of a nuclear mRNA methyltransferase complex, whose biochemical activity is the transfer of methyl groups from S-adenosylmethionine (AdoMet) to a subset of adenosine bases in mammalian mRNAs. The present study examines whether the Ime4p also binds AdoMet, and whether the conserved catalytic residues are essential for IME4 function in vivo. In vivo labeling, HPLC analysis, and in vitro studies using transcription-competent nuclear extracts will determine whether adenosine base methylation occurs in yeast mRNAs, as this modification has not been observed previously in this organism. Although the homology to the vertebrate enzyme suggests a role for Ime4p in an aspect of mRNA metabolism, many other possibilities are plausible. Specifically, Ime4p could interact with tRNAs, rRNAs or snRNAs, all of which are known to contain methylated bases in yeast. This research will employ two- and three-hybrid systems to identify proteins and RNAs that interact with Ime4p, thereby distinguishing whether this protein is involved in mRNA metabolism as opposed to other RNA or even protein level functions. Taken together with the methylase homology, genetic work suggests that Ime4p mediates an aspect of RNA metabolism that is particularly important to sporulating yeast, or one that is exploited in the regulation of this pathway. Examination of genetic interactions with known nutritional or developmentally-responsive RNA pathways will determine whether IME4 is involved in any of these. Detailed phenotypic analysis of ime4 mutant cells will pinpoint precisely the roles of the wild type protein in promoting sporulation. Methylation is a ubiquitous feature of mammalian mRNAs, including viral RNAs, but its role is not known. The very early time of its occurrence, before splicing, permits this modification to influence virtually every later aspect of mRNA function. Hence, mRNA methylation could play a critical, but unappreciated role in gene expression. The yeast system potentially offers experimental advantages to understanding this issue.
