Kar4, the Yeast Homolog of METTL14, is Required for mRNA m6A Methylation and Meiosis
Authors: Park Zachory M. , Sporer Abigail , Kraft Katherine , Lum Krystal , Blackman Edith , Belnap Ethan , Yellman Christopher , Rose Mark D. 
Published: January 2023

AbstractKAR4, the yeast homolog of the mammalian mRNA N6A-methyltransferase complex componentMETTL14, is required for two disparate developmental programs inSaccharomyces cerevisiae: mating and meiosis. To understandKAR4’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 ofkar4Δ/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 activatorIME1rescued 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 ofIME1inkar4Δ/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 ofRIM4, a meiotic translational regulator. Overexpression ofIME1andRIM4together allowed sporulation ofkar4Δ/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 SummaryIn yeast,KAR4is 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 andkar4Δ/Δ mutants have greatly reduced levels of mRNA methylation. Thus, Kar4p is a member of the yeast methyltransferase complex. Overexpression of the meiotic transcriptional activatorIME1rescued 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 bothIME1andRIM4allowed sporulation inkar4Δ/Δ cells.