Kar4 is Required for the Normal Pattern of Meiotic Gene Expression

Abstract

Kar4p, the yeast homolog of the mammalian methyltransferase subunit METTL14, is required for the initiation of meiosis and has at least two distinct functions in regulating the meiotic program. Cells lacking Kar4p can be driven to sporulate by co-overexpressing the master meiotic transcription factor, IME1, and the translational regulator, RIM4, suggesting that Kar4p functions at both the transcriptional and translational level to regulate meiosis. Using microarray analysis and RNA sequencing, we found that kar4{Delta}/{Delta} mutants have a largely wild type transcriptional profile with the exception of two groups of genes that show delayed and reduced expression: (1) a set of Ime1p-dependent early genes as well as IME1, and (2) a set of late genes dependent on the mid-meiotic transcription factor, Ndt80p. The early gene expression defect is rescued by overexpressing IME1, but the late defect is only suppressed by overexpression of both IME1 and RIM4. Mass spectrometry analysis identified several genes involved in meiotic recombination with strongly reduced protein levels, but with little to no reduction in transcript levels in kar4{Delta}/{Delta} after IME1 overexpression. The low levels of these proteins were rescued by overexpression of RIM4 and IME1, but not by the overexpression of IME1 alone. These data expand our understanding of the role of Kar4p in regulating meiosis and provide key insights into a potential mechanism of Kar4ps later meiotic function that is independent of mRNA methylation. Author SummaryKar4p is required at two stages during meiosis. Cells lacking Kar4p have a severe loss of mRNA methylation and arrest early in the meiotic program, failing to undergo either pre-meiotic DNA synthesis or meiotic recombination. The early block is rescued by overexpression of the meiotic transcription factor, IME1. The kar4{Delta}/{Delta} cells show delayed and reduced expression of a set of Ime1p-dependent genes expressed early in meiosis as well as a set of later genes that are largely Ndt80p-dependent. Overexpression of IME1 rescues the expression defect of these early genes and expedites the meiotic program in the wild type S288C strain background. However, IME1 overexpression is not sufficient to facilitate sporulation in kar4{Delta}/{Delta}. Completion of meiosis and sporulation requires the additional overexpression of a translational regulator, RIM4. Analysis of kar4{Delta}/{Delta}s proteome during meiosis with IME1 overexpression revealed that proteins important for meiotic recombination have reduced levels that cannot be explained by equivalent reductions in transcript abundance. IME1 overexpression by itself rescues the defect associated with a catalytic mutant of Ime4p, implying that the early defect reflects mRNA methylation. The residual defects in protein levels likely reflect the loss of a non-catalytic function of Kar4p, and the methylation complex, which requires overexpression of RIM4 to suppress.