Identification of Mum2 interacting proteins

(A). Scheme of MTCs in mammals, Drosophila, Arabidopsis and yeast. Colour indicates matching orthologues. (B) Volcano plots of Mum2-TEV-ProA compared to untagged control. Diploid cells harbouring Mum2 tagged with TEV-ProA (FW7873) or untagged control (FW1511) were induced to enter meiosis. Protein extracts were incubated with ProA coated paramagnetic beads. TEV protease was used to elute Mum2 from the beads. Significantly enriched proteins are labelled in blue and known subunits of the MIS complex are labelled in red. (C) Similar analysis as in B, except that during IP extracts were either untreated (left panel) or RNase treated (right panel). (D) m6A levels as determined by LC-MS in WT diploid cells, or diploid cells harbouring ime4Δ, mum2Δ, or slz1Δ (FW1511, FW7030, FW6535, and FW6504). In short cells were grown to saturation in rich medium (YPD), shifted to BYTA and grown for another 16 hours, then shifted to sporulation medium (SPO). Samples were taken at 4 hours in SPO. Total RNA was extracted and followed by two rounds of polyA RNA purification. Subsequently, RNA was digested into nucleosides and m6A was quantified my LC-MS. The mean of n=3 biological replicates are shown. (E) Same analysis as in D, except that multiple time points after shifting cells to SPO were taken (0, 2, 4, 6, 8, 10, 12 hours in SPO). WT, slz1Δ or ime4Δ diploid cells were used. The mean values of n=2 biological repeats are shown. (F) m6A levels in WT, ime4Δ and kar4Δ cells (FW1511, FW7030, FW8246). Samples were collected at 4 hours in SPO. The mean of n=3 biological replicates are shown.

Functional characterization of Mum2 interactors

(A) WT, ime4Δ, and ygl036wΔ cells (FW1511, FW7030, FW9307) were induced to enter meiosis. RNA was extracted, and polyA RNA was purified. m6A levels were determined by m6A-ELISA kit supplied by Epiquik. The means of n=3 biological replicates are shown. (B) m6A-seq2 analysis of strains and condition described in A. Two independent clones of ygl036wΔ were used for the analysis. Shown are the sample index score representing 1308 known of m6A sites. The mean of n=3 biological repeats are shown. (C) m6A levels WT, ime4Δ, and dyn2Δ cells (FW1511, FW7030, FW10442). RNA was extracted, and polyA RNA was purified. m6A levels were determined by a non-commercial m6A-ELISA assay (Ensinck et al., 2023). The means of n=3 biological replicates are shown of WT and ime4Δ, and n=6 for dyn2Δ. (D) m6A-seq2 analysis after Npl3, Pab1 and Slz1 depletion. Diploid cells containing PAB1-AID, NPL3-AID, SLZ1-AID (FW10388, FW10389, FW10386) were treated at 2 hours in SPO for 2 hours with DMSO or IAA and CuSO4 for rapid depletion. A control strain was included that only harboured the TIR1 ligase expressed from the CUP1 promoter (FW5737). Shown are the m6A-sample indices, quantifying the overall levels of enrichment over 1308 previously defined m6A sites. The mean of n=2 biological repeats are shown.

Ylg036w-Vir1 and Slz1 have orthologues in humans

(A) Pair-wise sequence comparison of Ylg036w against all human protein coding genes. Global similarity scores (using Needle) are displayed on the x-axis, whereas local similarity scores (using Matcher) are displayed on the y-axis. (B) Pairwise structural similarity of Ygl036w against all human protein coding genes, using TMalign algorithm. A histogram of the TMalign density scores is displayed, along with an indication of the score against VIRMA, the top hit in this analysis, and the second highest hit, FACD2. TM-scores were calculated and normalized using the query gene length, Ygl036w. (C) Overlay of Alpha-fold predicted structures of Ylg036c-Vir1 and human VIRMA. Human VIRMA is shown in blue and Yeast Ylg036c-Vir1 is shown in pink. Also indicated are the Vir motif and regions with strong sequence similarities. (D) Analysis as in A using Slz1 as a query.

Topological and structural properties of the yeast MT

(A) Ime1, Mum2, Vir1, Kar4, and Slz1 expression depends on the presence of MTC components. WT, mum2Δ, ime4Δ, slz1Δ, kar4Δ or vir1Δ were examined for Ime4-V5 (first panel, FW6057, FW8362, FW8264, FW8633, and FW9483), Mum2-V5 (second panel, FW6500, FW9394, FW6534, FW9396, and FW9398), Vir1-V5 (third panel, FW9666, FW9663, FW9668, FW9670, and FW9643), Kar4-V5 (fourth panel, FW8216, FW9484, FW8212, FW9482, and FW9481), or Slz1-V5 (fifth panel, FW6502, FW9479, FW9477, FW9480, and FW9478) during early meiosis. Samples were taken at the indicated time points. Western blots were probed with anti-V5 antibodies and anti-Hxk1 as a loading control. (B) Table of western blot quantifications of the 4-hour time point described in C. Each row represents the protein expression (Ime4-V5, Mum2-V5, Vir1-V5, Kar4-V5 and Slz1-V5) and each column the deletion mutant (mum2Δ, ime4Δ, slz1Δ, kar4Δ or vir1Δ). Highlighted in yellow are the MTC components affected in protein levels (<0.5), but not at the mRNA levels. (C) Diploid cells untagged or harbouring Mum2 tagged with TEV-ProA in WT, slz1Δ, or kar4Δ background (FW1511, FW7873, FW10158, and FW10159) were induced to enter meiosis. Protein extracts were incubated with ProA coated paramagnetic beads. TEV protease was used to elute Mum2 from the beads. Shown are the enrichments for Ime4, Mum2, Vir1, Kar4, Slz1, Pab1, and Npl3. Two independent experiments are shown for slz1Δ. nd= not detected. (D) Purification of the 5-subunit recombinant m6A writer complex. Schematic representation showing the baculovirus recombinant co-expression and purification of the yeast MTC with five subunits (Ime4, Mum2, Vir1, Kar4, and Slz1) in insect cells. Genes are depicted by rectangles, promoters by arrows and terminators by grey boxes. The star in magenta represents the Strep-tag in the N-terminal domain of Ime4. (E) SDS-PAGE analysis of the purified recombinant m6A writer complex after affinity. Identities of bands confirmed by MS are labelled.

m6A dependent and independent roles of the MTC in meiosis

(A) Onset of meiosis in WT, ime4Δ, mum2Δ, slz1Δ, kar4Δ, and vir1Δ (FW1511, FW7030, FW6535, FW6504, FW8246, and FW9307). Cells induced to enter meiosis. Samples were taken at the indicated time points for DAPI staining. Cells were fixed, stained with DAPI, and nulei were counted for at least 200 cells per biological repeat. Cells with two or more DAPI masses were considered to undergo meiosis. The mean and SEM of n=3 biological repeats are displayed. (B) Similar analysis as A, except that the WT, ime4Δ, and dyn2Δ were analysed (FW1511, FW7030, FW10442 and FW10443). For the analysis two independent dyn2Δ strains were used. The mean of n=2 biological repeats are displayed. (C) Principal component analysis (PCA) of gene expression measurements across diverse strains and timepoints, done using PCAtools 2.6.0 for R. Strains described in A and B were induced to enter synchronized meiosis and samples were taken at the indicated time points (0, 2, 4, 6, and 8 hours in SPO). For analysis n=4 biological repeats were used, except for ime4Δ_4h, ime4Δ_0h, and vir1Δ_8h which had an n = 3. (D) Median expression patterns in WT and MTC mutants of five predefined gene clusters representing early metabolic genes (C1, n=67), early meiotic genes (C2, n=42; C3, n=34), early to middle meiotic genes (C4, n=53), middle meiotic genes (C5, n=136) (Chu et al., 1998). (E) Median gene expression fold change from timepoint 0 of Mrb1/Pho92-bound transcripts during the time course across the different deletion mutants. Dataset of Mrb1/Pho92 iCLIP-based targets associating with RNA in an m6A-dependent manner identified in (Varier et al., 2022) was used for the analysis. For each transcript a Z-score was calculated with respected to the 0-hour timepoint. The mean of at least n=3 biological replicates shown, and error bars represent the standard error of the mean.

Kar4 functions during mating and meiosis differ

(A) Expression of Kar4, Ime4, Slz1, and Mum2, either untreated or treated with mating pheromone. MATa harbouring V5 tagged alleles of Kar4, Slz1, Ime4, or Mum2 (FW8199, FW6426, FW5898 and FW6428) cells were grown in rich medium (YPD) until exponential growth, and then treated with α-factor for 30 minutes. Samples were collected for western blot and probed with anti-V5 antibodies. Hxk1 was used as a loading control. (B) Kar4 expression in WT, ime4Δ or mum2Δ MATa cells (FW8199, FW9415, FW8574) untreated or treated α-factor. Samples were collected for western blot and probed with anti-V5 antibodies. Hxk1 was used as a loading control. (C) Chromatin immunoprecipitation (ChIP) of untagged control, Kar4-V5, Ime4-V5, Mum2-V5, and Slz1-V5 MATa cells (FW1509, FW8199, FW5898, FW6426, and FW6428). Cells were grown in YPD and were either not treated or treated with α factor for 2 hours. (D) Kar4 localization in cells in rich medium, either untreated or treated α-factor and in cells entering meiosis. Cells were induced to enter meiosis in SPO, and samples were taken at 0, 3, and 6 hours in SPO. Kar4 fused to mNeongreen (mNG) was used for the analysis. To determine nuclear Kar4-mNG signal, we used histone H2B fused to mCherry (H2B-mCh). For the analysis we used MATa and MATa/α cells (FW8615 and FW8646). Representative images are shown. (E) Quantification of nuclear over whole cell mean signal for Kar4-mNG of data in C. At least n=50 cells were quantified for the analysis. (F) Chromatin immunoprecipitation followed by deep-sequencing (ChIP-seq) of MATa Kar4-V5 (FW8199) untreated or treated with α factor, and MATa/α Kar4-V5 (FW8616) at 0 and 4 hours in SPO as well as untagged control cells (FW1509 and FW1511). Samples were crosslinked with formaldehyde, extracts were sonicated, and protein-DNA complexes were purified using anti-V5 agarose beads and reverse crosslinked. Purified DNA was subjected to deep sequencing. ChIP-seq signals for the GIC2 and FUS1 loci are shown. (G) Quantification of the number of ChIP-seq peaks. (H) Motif analysis of Kar4 binding sites identified in Kar4-V5 ChIP-seq of α factor treated cells. (I) Photo activatable crosslinking of Kar4. Control and Kar4 (FW1511 and FW8633) cell entering meiosis were incubated with 4thiouracil (4TU). Cells were UV-crosslinked, and Kar4 was immunoprecipitated from protein extracts. The Kar4-IP was treated with different concentration of RNase, and subsequently radioactively labelled p32. Kar4-RNA complexes were separated by SDS page. (J) Chemical RNA-protein interactome analysis for assessing Ime4 binding to RNA. For the analysis we used Ime4-V5 cells in control, slz1Δ, kar4Δ, mum2Δ backgrounds (FW6057, FW8362, FW8264, and FW8633). In short, cells entering meiosis (4h SPO) were crosslinked with formaldehyde, polyA mRNAs were pulled down from protein extracts with oligo-dT coated magnetic beads, washed, samples were eluted with RNase, and assessed by western blotting. Membranes were probed for anti-V5 to detect Ime4. As a positive control, membranes were probed with anti-Pab1.

Model of the yeast MTC

Comparison of the yeast and the human MTCs. The human MTC consists of MAC and MACOM subcomplexes, while the yeast MTC forms a single complex. The conserved subunits are colour indicated. Mum2, Vir1, and Ime4 mutually stabilize each other. Further indicated are the m6A-dependent and m6A-independent MTC requirements, and Kar4’s separate function in mating.