Kar4, the Yeast Homolog of METTL14, is Required for mRNA m⁶A Methylation and Meiosis

Abstract

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.

Vir1p, the Yeast Homolog of Virilizer, is Required for mRNA m6A Methylation and Meiosis

“…shown to be when m 6 A levels peaked (Park, Sporer et al 2023) (Fig 2C and Fig 2D). This is consistent with work that has shown there is an early burst of IME1 expression that is m 6 A independent, but that sustained induction of IME1 requires mRNA methylation (Bushkin, Pincus et al 2019).…”

“…Interestingly, overexpression of IME1 alone rescues the meiotic defect of a catalytically dead mutant of Ime4p, as well as slz1Δ/Δ (Park, Sporer et al 2023). Suppression of the Ime4p catalytic mutant by Ime1p overexpression suggests that this "Mei" function is associated with m 6 A methylation.…”

“…Previous work from our lab identified function-specific alleles of Kar4p that are defective either in mating (Mat) or in one or both of its functions in meiosis (Mei and Spo). The Mei function involves Kar4p's role in mRNA methylation and the Spo function appears to involve a non-catalytic function that promotes the translation of transcripts during meiosis (Park, Sporer et al 2023, Park, Remillard et al 2023. To understand the full complement of proteins involved in mRNA methylation and Kar4p's Spo function, we conducted immunoprecipitation followed by mass spectrometry (IP-MS) to identify meiotic binding partners.…”

“…Looking specifically at genes with a 2-fold or greater defect at 4 hours in vir1Δ/Δ, which is when a gene expression defect becomes noticeable on the heat map, GO-term analysis to their rapid turnover (Bushkin, Pincus et al 2019). Strains lacking either Kar4p or Ime4p in which Rme1p levels are reduced or absent do progress through pre-meiotic DNA synthesis, but not do not complete meiosis and spore formation (Bushkin, Pincus et al 2019, Park, Sporer et al 2023. Suppression of Kar4p's Mei function by loss of Rme1p was due to the restoration of normal levels of IME1 expression (Park, Sporer et al 2023).…”

“…Strains lacking either Kar4p or Ime4p in which Rme1p levels are reduced or absent do progress through pre-meiotic DNA synthesis, but not do not complete meiosis and spore formation (Bushkin, Pincus et al 2019, Park, Sporer et al 2023. Suppression of Kar4p's Mei function by loss of Rme1p was due to the restoration of normal levels of IME1 expression (Park, Sporer et al 2023). Given that Vir1p also appears to be working in this same pathway, we asked if a vir1Δ/Δ rme1Δ/Δ double mutant could undergo premeiotic DNA synthesis.…”