Kar4, the Yeast Homolog of METTL14, is Required for mRNA m A Methylation and Meiosis.

UNLABELLED

, the yeast homolog of the mammalian mRNA N A-methyltransferase complex component , is required for two disparate developmental programs in : mating and meiosis. To understand '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 Δ/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 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 in Δ/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 , a meiotic translational regulator. Overexpression of and together allowed sporulation of Δ/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.

AUTHOR SUMMARY

In yeast, is required for mating and meiosis. A genetic screen for function-specific mutations identified 25 alleles that map to different surfaces on a predicted structure of the Kar4 protein (Kar4p). The mating-specific alleles interfere with Kar4p's ability to interact with the transcription factor Ste12p, its known partner in mating. The meiosis-specific alleles revealed an independent function: Kar4p is required for entry into meiosis and initiation of S-phase. During meiosis, Kar4p interacts with all components of the mRNA methyltransferase complex and Δ/Δ mutants have greatly reduced levels of mRNA methylation. Thus, Kar4p is a member of the yeast methyltransferase complex. Overexpression of the meiotic transcriptional activator rescued the meiotic entry defect but did not lead to sporulation, implying that Kar4p has more than one meiotic function. Suppression by Ime1p overexpression led to arrest after premeiotic DNA synthesis, but before sporulation. Loss of Kar4's sporulation function can be suppressed by overexpression of a translation regulator, Rim4p. Overexpression of both and allowed sporulation in Δ/Δ cells.