Kar4, the Yeast Homolog of METTL14, is Required for mRNA m 6 A Methylation and Meiosis

Abstract

KAR4 , the yeast homolog of the mammalian mRNA N ⁶ A-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 kar4 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 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 kar4 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 RIM4 , a meiotic translational regulator. Overexpression of IME1 and RIM4 together allowed sporulation of kar4 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, KAR4 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 kar4 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 IME1 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 IME1 and RIM4 allowed sporulation in kar4 Δ/Δ cells.

Fig 1.. Kar4p is required early in meiosis.

(A) Fluorescence microscopy of the spindle pole body (Spc42-mCherry) and microtubules (GFP-Tub1) across a time course of meiosis (12, 24, and 48 hours post transfer into sporulation media) in wild type and kar4Δ/Δ. Graphs are the quantification of the number of cells in different meiotic stages (Monopolar Spindle, Meiosis I, Meiosis II, and Spores). Experiments were run in three biological replicates for each strain and at least 100 cells were counted for each replicate. Error bars represent standard deviation. (B) Flow cytometry analysis of DNA content in wild type and kar4Δ/Δ across the same meiotic time course of the microscopy. DNA was stained with propidium iodide.

Fig 2.. Kar4p has distinct functions in mating and meiosis.

(A) Schematic of the screen used to identify separation of function mutants of KAR4. (B) Limited mating assay to determine the ability of the KAR4 alleles to facilitate mating. (Left) Resulting growth on media selective for diploids after a limited mating assay between a strain carrying the indicated KAR4 allele and kar4Δ. (Right) Growth of strains carrying the indicated KAR4 allele mated to kar4Δ on YPD (non-selective rich media) overnight. (C) Spot assay to assess the ability of the different KAR4 alleles to initiate meiotic recombination after 24 hours of exposure to meiosis inducing conditions. (Left) Selection for successful recombination events by selecting for His+ Can^R recombinants. (Right) Growth of all strains on YPD. Spots are 10-fold serial dilutions of 1 OD of each sample.

Fig 3.. Kar4p engages in a function specific interaction with Ste12p and Ime4p.

(A) A one-hybrid assay to determine the ability of the different Kar4p alleles fused to the Gal Binding Domain to interact with Ste12p upon exposure to alpha-factor. (Left) Growth on non-selective media both with and without 3 μM alpha-factor. (Right) Growth on SC-His media to select for alleles that can maintain an interaction with Ste12p and drive expression of the HIS3 reporter gene. (B) A two-hybrid assay was used to determine the ability of the different Kar4p alleles to interact with Ime4p fused to the Gal Activating Domain after 24 hours of exposure to meiosis inducing conditions. (Left) Growth on non-selective media. (Right) Growth on both SC-HIS and SC-ADE to select for the alleles that can maintain an interaction with Ime4p and drive expression of the two reporter genes HIS3 and ADE2. Spots are 10-fold serial dilutions of 1 OD of each sample.

Fig 4.. Kar4p is required for mRNA m⁶A methylation.

(A) mRNA m⁶A levels measured using an ELISA like assay from EpiGenTek. The indicated mutations were made in the SK1 strain background and samples were harvested after four hours of exposure to meiosis inducing conditions. Experiments were run in three biological replicates for each strain and error bars represent standard deviation. (B) Western Blot of 3xFLAG-Rme1p in wild type and kar4Δ/Δ across a time course of meiosis. Kar2p is used as a loading control. (C) Western blot of 3xMYC-Ime4p after four hours in meiosis inducing media with either 100 μM cycloheximide or an equivalent amount of DMSO in both wild type and kar4Δ/Δ. (Top) 3xMYC-Ime4p levels with DMSO. (Middle) 3xMYC-Ime4p levels with cycloheximide. (Bottom) Quantification of three biological replicates of the cycloheximide chase experiment. The strains used are in the SK1 background. Kar2p is used as a loading control. “*” indicates a non-specific band.

Fig 5.. Kar4p interacts with the MIS complex components Mum2p and Slz1p.

(A) Western blots of total protein and co-IPs between Mum2p-4MYC and the alleles of Kar4p-3HA. (Top) Total protein samples from the extracts that were used for the Co-IPs. Kar2p is used as a loading control. Alleles proficient for Kar4p’s meiotic function (Mei⁺) are in blue and alleles in red are not (Mei⁻). (Bottom) Co-IPs where Mum2p-4MYC was purified and the co-purification of Kar4p-3HA was assayed. (B) Western blots of total protein and Co-IPs between Slz1p-3HA and Kar4p-9MYC. (Left) Total protein samples from the extracts that were used for the co-IPs. “*” indicates a non-specific band. (Right) Co-IPs where Slz1p-3HA was purified and the co-purification of Kar4p-13MYC was assayed. “‡” indicates the heavy chain of IgG from the anti-HA magnetic beads used for the Co-IP. (C) Western blots of total protein and Co-IPs between Kar4p-9MYC and Slz1p-3HA. (Left) Total protein samples from the extracts that were used for the Co-IPs. (Right) Co-IPs where Kar4p-9MYC was purified and the co-purification of Slz1–3HA was assayed.

Fig 6.. IME1 overexpression partially suppresses the kar4Δ/Δ meiotic defect.

(A) Growth of kar4Δ/Δ cells on SC-HIS+canavanine to screen for recombination after exposure to meiosis-inducing conditions carrying either KAR4, IME1, or IME2 cloned onto high-copy number 2μ plasmids. (B) Fluorescence microscopy of the spindle pole body (Spc42-mcherry) and microtubules (GFP-Tub1) across a time course of meiosis (12, 24, and 48 hours post movement into sporulation media) in wild type and kar4Δ/Δ with IME1 overexpressed from an estradiol-inducible promoter. Graphs are the quantification of the number of cells in different meiotic stages (Monopolar Spindle, Meiosis I, Meiosis II, and Spores). Experiments were run in three biological replicates for each strain and at least 100 cells were counted for each replicate. 1 μM of estradiol was used to induce expression. Error bars represent standard deviation. (C) Flow cytometry analysis of DNA content in wild type and kar4Δ/Δ with IME1 overexpressed across the same meiotic time course of the microscopy. DNA was stained with propidium iodide. (D) Western blot showing GFP-Ime1p levels across a time course of meiosis in wild type, kar4Δ/Δ, and kar4Δrme1Δ/ kar4Δrme1Δ. Kar2p is used as a loading control. (E) qPCR measuring IME1 transcript levels in wild type, kar4Δ/Δ, and kar4Δrme1Δ/ kar4Δrme1Δ. Data were normalized to levels of PGK1 expression. (F) Flow cytometry analysis of DNA content in wild type and kar4Δ/Δ carrying either the wild type allele of RME1, the hypomorphic −308A allele of RME1, or a deletion of RME1. DNA was stained with propidium iodide.

Fig 7.. Co-overexpression of IME1 and RIM4 permits sporulation in kar4Δ/Δ.

Sporulation of wild type, kar4Δ/Δ, mum2Δ/Δ, ime4Δ/Δ, slz1Δ/Δ, and ime4-cat/ime4Δ with either endogenous expression of IME1 and RIM4, overexpression of RIM4, overexpression of IME1, or overexpression of both IME1 and RIM4. 1 μM of estradiol was used to induce expression. All dyads, triads, and tetrads were counted across three biological replicates for each strain. At least 100 cells were counted after 48 hours post addition of estradiol. Error bars represent standard error.