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KAR4, the yeast homolog of the mammalian mRNA N6A-methyltransferase complex component METTL14, is required for two disparate developmental programs in Saccharomyces cerevisiae: mating and meiosis. To understand KAR4's role in yeast mating and meiosis, we used a genetic screen to isolate 25 function-specific mutant alleles, which map to non-overlapping surfaces on a predicted structure of the Kar4 protein (Kar4p). Most of the mating-specific alleles (Mat-) abolish Kar4p's interaction with the transcription factor Ste12p, indicating that Kar4p's mating function is through Ste12p. In yeast, the mRNA methyltransferase complex was previously defined as comprising Ime4p (Kar4p's paralog and the homolog of mammalian METTL3), Mum2p (homolog of mammalian WTAP), and Slz1p (MIS), but not Kar4p. During meiosis, Kar4p interacts with Ime4p, Mum2p, and Slz1p. Moreover, cells lacking Kar4p have highly reduced levels of mRNA methylation during meiosis indicating that Kar4p is a key member of the methyltransferase complex, as it is in humans. Analysis of kar4Δ/Δ and 7 meiosis-specific alleles (Mei-) revealed that Kar4p is required early in meiosis, before initiation of S-phase and meiotic recombination. High copy expression of the meiotic transcriptional activator IME1 rescued the defect of these Mei- alleles. Surprisingly, Kar4p was also found to be required at a second step for the completion of meiosis and sporulation. Over-expression of IME1 in kar4Δ/Δ permits pre-meiotic S-phase, but most cells remained arrested with a monopolar spindle. Analysis of the function-specific mutants revealed that roughly half became blocked after premeiotic DNA synthesis and did not sporulate (Spo-). Loss of Kar4p's Spo function was suppressed by overexpression of RIM4, a meiotic translational regulator. Overexpression of IME1 and RIM4 together allowed sporulation of kar4Δ/Δ cells. Taken together, these data suggest that Kar4p regulates meiosis at multiple steps, presumably reflecting requirements for methylation in different stages of meiotic gene expression.

Kar4p, the yeast homolog of the mammalian methyltransferase subunit METTL14, is required for efficient mRNA m6A methylation, which regulates meiotic entry. Kar4p is also required for a second seemingly non-catalytic function during meiosis. Overexpression of the early meiotic transcription factor, IME1, can bypass the requirement for Kar4p in meiotic entry but the additional overexpression of the translational regulator, RIM4, is required to permit sporulation in kar4Δ/Δ. Using microarray analysis and RNA sequencing, we sought to determine the impact of removing Kar4p and consequently mRNA methylation on the early meiotic transcriptome in a strain background (S288c) that is sensitive to the loss of early meiotic regulators. We found that kar4Δ/Δ 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 likely the result of the loss of mRNA methylation and is rescued by overexpressing IME1, but the late defect is only suppressed by overexpression of both IME1 and RIM4. The requirement for RIM4 led us to predict that the non-catalytic function of Kar4p, like methyltransferase complex orthologs in other systems, may function at the level of translation. 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 kar4Δ/Δ 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 Kar4p's later meiotic function that is independent of mRNA methylation.