Fig 1.
mRNA methylation associated with different polysome fractions.
A cell extract from SK1 cells at 3 hours sporulation was fractionated by sucrose gradient centrifugation. The typical polysome profile shows the reduced translation associated with meiosis (a). mRNA extracted from different fractions was radiolabelled and the relative m6A content quantified. Input is the start mRNA, before fractionation. All other fractions are pooled fractions; fr1-3 is the subribosomal fractions; fr4-6 is the pooled monosomes; fr7-8 is the lower polysome, and fr9-10 is the higher polysome pooled fraction (Error bar shows SD; each measurement was the average of three biological replicates)(b).
Fig 2.
Cluster analysis of transcript polysome occupancy.
Total RNA from the different pooled sucrose gradient fractions were extracted and prepared for Affymetrix analysis. The input sample is the non-fractionated total RNA sample. The data for vegetative samples were ‘.cell’ files from Dominissini et al. [6] The raw chip data were analysed using Partek GS 6.0 software package. The gene list of significant genes (FDR<0.05, no fold change cut off was applied) was the base for hierarchic clustering using Partek GS 6.0. 12 different clusters were identified (labelled by different colour bars) (a). The gene lists for each different cluster were used to determine the enrichment for methylated transcripts using the yeast methylome from Schwartz et al. [17]. The statistical significance of the overlaps was determined using the online resource http://nemates.org/MA/progs/overlap_stats.html (b).
Fig 3.
Overlaps between the yeast methylome and the polysome fractions.
809 overlapping genes were identified between the yeast methylome dataset(1181 genes; all duplications were removed) [17] and the genelist for ribosome association (4094 significant genes) used for cluster analysis. The gene list overlaps were determined using the BioVenn web based tool].
Fig 4.
Polysome profiles and methylation levels after rapamycin treatment.
After 12 hours rapamycin exposure, (200ng/ml) the cell extract was fractionated using sucrose gradient centrifugation. The m6A levels from pooled polysome fraction 7–8 were determined using TLC method (grey colour). Equivalent samples from 3 hour sporulating cells from Fig 1 (black colour) are shown for comparison.
Fig 5.
Sporulation phenotype under rapamycin treatment in normal and methylation deficient strains.
SK1 and ime4Δ/ ime4Δ strains were treated with rapamycin, and at different time points samples were checked for ascus formation. The sporulation pattern of SK1 cells under rapamycin treatment looked similar to sporulation under starvation conditions. Sporulation was completed by the 5th day with a 91±5% sporulation efficiency. The ime4Δ/ ime4Δ strain showed some level of sporulation (22.5±12%), but with abnormal ascus formation (a). After 5 days rapamycin treatment the ime4Δ/ ime4Δ cells mainly produced elongated cells with only a few producing asci which were all abnormal. In contrast, the SK1 cells maintained a high efficiency of sporulation, although diad and asymmetric asci, with a void volume were common (b).