m6A modification of a 3' UTR site reduces RME1 mRNA levels to promote meiosis

Abstract

Despite the vast number of modification sites mapped within mRNAs, known examples of consequential mRNA modifications remain rare. Here, we provide multiple lines of evidence to show that Ime4p, an N6-methyladenosine (m⁶A) methyltransferase required for meiosis in yeast, acts by methylating a site in the 3' UTR of the mRNA encoding Rme1p, a transcriptional repressor of meiosis. Consistent with this mechanism, genetic analyses reveal that IME4 functions upstream of RME1. Transcriptome-wide, RME1 is the primary message that displays both increased methylation and reduced expression in an Ime4p-dependent manner. In yeast strains for which IME4 is dispensable for meiosis, a natural polymorphism in the RME1 promoter reduces RME1 transcription, obviating the requirement for methylation. Mutation of a single m⁶A site in the RME1 3' UTR increases Rme1p repressor production and reduces meiotic efficiency. These results reveal the molecular and physiological consequences of a modification in the 3' UTR of an mRNA.

Fig. 1

RME1 alleles in this study. IME4 downregulates Rme1p through its m⁶A activity. a The three RME1 alleles are in the SK288C strain background. RME1-SK1A has a single A insertion 308 nucleotide upstream of the open reading frame (ORF) relative to rme1-S288C. b Quantitative reverse transcription PCR (RT-qPCR) quantification of RME1-SK1A and rme1-S288C expression during meiosis. Means and individual values from three biological replicates. Source data are provided in a Source Data file. c m⁶A immunoprecipitation (IP) followed by RT-qPCR using RME1 5′ untranslated region (UTR) primers or primers that span the end of the ORF and the 3′ UTR on mRNA isolated from IME4 and ime4-cat homozygous cells, both in rme1-S288C homozygous cells. Transcript levels were first normalized to PGK1 as an internal control, and then the fold enrichment of IP/input was calculated. The residual enrichment of RME1 transcripts in ime4-cat/ime4-cat cells relative to IME4/IME4 cells probably results from non-specific binding to the anti-m⁶A antibody^,. ACT1 serves as a non-methylated control. Means, individual values, and s.d. from three biological replicates. *p = 0.022, **p = 0.019, two-tailed t test. Source data are provided in a Source Data file. d Western blot using anti-FLAG antibody showing Rme1p expression from the rme1-S288C allele in strains homozygous for the indicated allelic backgrounds incubated in SPO media for 5 h. A non-specific (NS) band present in all lanes serves as a loading control. Source data are provided in a Source Data file

Fig. 2

IME4 m⁶A activity reduces RME1 mRNA levels to enable meiotic initiation. a Polysome profiles (absorbance at 254 nm vs. distance from the top of the tube) of mitotic cells during logarithmic growth and 2 h into meiosis. The locations of the 40S, 60S, monosome, and polysome peaks are indicated. Source data are provided in a Source Data file. b Polysome profiles of meiotic IME4/IME4 and ime4-cat/ime4-cat cells, both in rme1-S288C/rme-S288C. The inset is a magnification of the polysome area with the number of ribosomes in each peak. The highlighted area marks the pooled fractions used for RNA-seq of polysome-associated mRNA. Source data are provided in a Source Data file. c RNA-seq quantifications of RME1 transcript from input mRNA prior to gradient fractionation (total mRNA) and pooled polysome fractions (polysomal RNA) from IME4/IME4 and ime4-cat/ime4-cat meiotic cells. Individual values, means, and s.d. from three biological replicates. Two-way analysis of variance p values are indicated. Source data are provided in a Source Data file. d Rank order plot of mRNA levels in total RNA from ime4-cat/ime4-cat and IME4/IME4 meiotic cells. Means from three biological replicates. Source data are provided in a Source Data file. e Left: Quantitative reverse transcription PCR (RT-qPCR) quantification of IME1 expression in RME1-Δ/RME1-Δ during meiosis in IME4/IME4 and ime4-Δ/ime4-Δ cells. Middle: RT-qPCR quantification of IME1 expression in rme1-S288C/rme1-S288C during meiosis in IME4/IME4 and ime4-Δ/ime4-Δ cells. Right: RT-qPCR quantification of IME1 expression in rme1-S288C/rme1-S288C during meiosis in IME4/IME4 and ime4-cat/ime4-cat cells. Means and individual values from three biological replicates. Source data are provided in a Source Data file. f As in e above but with measurements of IME2 expression. Means and individual values from three biological replicates. Source data are provided in a Source Data file

Fig. 3

IME4 m⁶A is required for meiotic DNA replication, and a non-m⁶A function is required for meiotic divisions. a Flow cytometric analysis of DNA content in IME4 and ime4 mutants (rows) in three color-coded RME1 allele backgrounds over a meiotic time course (columns). A quantification of the 24-h time point is on the right of each row. Percentage of cells with 4N includes both sporulated and non-sporulated cells. Means, individual values, and s.d. from two to five experiments. Source data are provided in a Source Data file. b Meiotic nuclear divisions in IME4 and ime4 mutants in three color-coded RME1 allele backgrounds as assayed by DAPI staining of nuclei after 24 h in SPO medium. The percentage of cells with two or more nuclei is indicated on the horizontal axis. Means, individual values, and s.d. from three experiments. At least 200 cells were counted per strain per experiment. Source data are provided in a Source Data file. c Sporulation after 48 h in SPO media in IME4 and ime4 mutants in three color-coded RME1 allele backgrounds as scored by light microscopy. Means, individual values, and s.d. from three experiments. At least 200 cells were counted per strain per experiment. Source data are provided in a Source Data file. d A model depicting the various points along the S. cerevisiae meiotic program in which different IME4 functions are needed in different RME1 backgrounds. “RME1 on” denotes the highly expressed rme1-S288C allele, while “RME1 off” denotes either the hypomorph RME1-SK1A or RME1-Δ. Arrows represent an ability to progress through the various landmarks that appear at the top, while vertical red lines indicate an inability to progress further. e A model for the functions of IME4 in meiosis: IME4 promotes meiotic DNA replication by repression of RME1, which represses the master activator of meiosis, IME1. IME1 is also activated by nutritional signals. IME4 has a second function downstream of IME1 in promoting meiotic divisions (dashed line)

Fig. 4

RME1 mRNA harbors an m⁶A site at its 3′ untranslated region (UTR). a m⁶A-seq data from meiotic IME4 homozygotes and ime4-cat homozygotes. Data from three biological replicates are presented as immunoprecipitation (IP) to input ratio in IME4, divided by the same ratio in ime4-cat. m⁶A sites enriched >1.75-fold in IME4 relative to ime4-cat cells that have a p value of <0.01 in a two-tailed t test are highlighted in orange. b RNA-seq data from meiotic IME4 homozygotes and ime4-cat homozygotes cells. Data from three biological replicates are presented as normalized counts in ime4-cat, divided by normalized counts in IME4. mRNAs increased >1.75-fold in ime4-cat/ime4-cat relative to IME4/IME4 cells (p value <0.01, two-tailed t test) are highlighted in purple. mRNAs decreased >1.75-fold in ime4-cat/ime4-cat relative to IME4/IME4 cells (p value < 0.01, two-tailed t test) are highlighted in blue. c Venn diagram showing the overlap between the m⁶A-seq data and the RNA-seq data. d RME1 transcript with its 5′ and 3′ UTRs. IGV genome browser views of reads enriched following IP with anti-m⁶A antibodies in IME4/IME4 and ime4-cat/ime4-cat cells are shown for the entire transcript (top, autoscale read counts on the left of each track), and in a 100-bp window of the 3′ UTR (bottom, reads in this window were normalized to input controls). Fold enrichment of IP/input for reads in the window are shown on the left with means and s.d. from three biological replicates. *p = 0.0006, two-tailed t test. Source data are provided in a Source Data file. e PCR amplification of cDNA prepared from MazF-digested meiotic mRNA from IME4/IME4 and ime4-cat/ime4-cat cells. Three biological replicates. P1 and P2 primer locations relative to the probed methylation site are indicated. The MazF ACA site within the methylation site is highlighted in yellow, and the probed nucleotide is marked with an asterisk. Primers P3 and P4, which do not flank an ACA site, were used as control. Source data are provided in a Source Data file

Fig. 5

RME1 methylation mutants are defective in meiotic DNA replication. a rme1-S288C and rme1-10 have the same promoter and coding sequence, but they differ in their 3′ untranslated region: rme1-10 has a single A to T substitution of the +129 methylated A (marked with an m over it) in the methylation consensus motif (yellow). b RNA-seq quantifications of RME1 mRNA. RME1 mRNA from input RNA prior to gradient fractionation (total mRNA) and polysome-associated RNA (polysomal RNA) from IME4/IME4 rme1-S288C/rme1-S288C and IME4/IME4 rme1-10/rme1-10 cells incubated in SPO media for 5 h. Individual values, means, and s.d. from three biological replicates. Two-way analysis of variance p values are indicated. Because this experiment was performed together with the one shown in Fig. 2c, the results for RME1 mRNA in the IME4 background in Fig. 2c are re-plotted in this panel for the rme1-S288C background. Source data are provided in a Source Data file. c RNA-seq data from rme1-S288C homozygotes and rme1-10 homozygotes incubated for 5 h in SPO media. Data from three biological replicates are presented as normalized counts in rme1-10, divided by normalized counts in rme1-S288C. d Flow cytometric analysis of DNA content over a meiotic time course in IME4/IME4 rme1-10/rme1-10 compared to IME4/IME4 rme1-S288C/rme1-S288C and ime4-cat/ime4-cat rme1-S288C/rme1-S288C. Cells were incubated for 24 h at 30 °C or 37 °C, as indicated. Means, individual values, and s.d. from three biological replicates. *p = 0.011, **p = 0.034, two-tailed t test. Source data are provided in a Source Data file