Negative regulation of meiotic gene expression by the nuclear poly(a)-binding protein in fission yeast

Abstract

Meiosis is a cellular differentiation process in which hundreds of genes are temporally induced. Because the expression of meiotic genes during mitosis is detrimental to proliferation, meiotic genes must be negatively regulated in the mitotic cell cycle. Yet, little is known about mechanisms used by mitotic cells to repress meiosis-specific genes. Here we show that the poly(A)-binding protein Pab2, the fission yeast homolog of mammalian PABPN1, controls the expression of several meiotic transcripts during mitotic division. Our results from chromatin immunoprecipitation and promoter-swapping experiments indicate that Pab2 controls meiotic genes post-transcriptionally. Consistently, we show that the nuclear exosome complex cooperates with Pab2 in the negative regulation of meiotic genes. We also found that Pab2 plays a role in the RNA decay pathway orchestrated by Mmi1, a previously described factor that functions in the post-transcriptional elimination of meiotic transcripts. Our results support a model in which Mmi1 selectively targets meiotic transcripts for degradation via Pab2 and the exosome. Our findings have therefore uncovered a mode of gene regulation whereby a poly(A)-binding protein promotes RNA degradation in the nucleus to prevent untimely expression.

FIGURE 1.

Meiotic differentiation genes are up-regulated in pab2Δ cells. The expression levels of seven meiotic genes as well as of the adh1 and pgk1 housekeeping genes were analyzed using real-time quantitative PCR in the wild-type and pab2Δ strains. The expression levels are relative to the wild-type strain and normalized to the nda2 mRNA. The data and error bars represent the average and standard deviation from three independent experiments.

FIGURE 2.

Post-transcriptional control of meiotic differentiation genes by Pab2. A, ChIP assays using an RNA pol II-specific antibody were analyzed using real-time quantitative PCR to assess abundance of RNA pol II on two early meiotic genes, sme2 and ssm4. The -fold enrichment of RNA pol II is relative to an untranscribed region. The data and error bars represent the average and standard deviation from at least five independent experiments. B, schematic representation of the adh1P-sme2 construct. C, total RNA prepared from wild-type (lane 4), sme2Δ (lane 1), as well as sme2Δ and sme2Δ pab2Δ strains that express the meiRNA from the adh1P-sme2 chimeric construct (lanes 2 and 3, respectively) were analyzed for meiRNA and rmt1 expression levels by RT-PCR.

FIGURE 3.

Pab2 cooperates with the nuclear exosome in the negative regulation of meiotic genes. A, Northern blot analysis of total RNA prepared from various RNA degradation mutants in the presence (+) or absence (−) of Pab2. The meiRNA was detected using a sequence-specific probe. B, quantification of Northern blot experiments of meiRNA in various mutants of RNA degradation pathways. Expression levels are relative to the wild-type and normalized to the 5 S rRNA. The data and error bars represent the average and standard deviation from three independent experiments. C, real-time quantitative PCR analysis of spo5 and meu1 mRNAs in the same mutants described in A. The expression levels are relative to the wild-type strain and normalized to the nda2 mRNA. The data and error bars represent the average and standard deviation from three independent experiments.

FIGURE 4.

Pab2 contributes to the Mmi1-mediated pathway of meiotic gene regulation. Real-time quantitative PCR analysis of the indicated meiotic genes as well as the adh1 control gene in wild-type, pab2Δ, mmi1-ts3, and mmi1-ts3 pab2Δ strains at the permissive (25 °C) and non-permissive (36 °C) temperatures. The levels of transcripts are relative to the wild-type strain at each temperature and normalized to the srp7 RNA. The data and error bars represent the average and standard deviation from at least three independent experiments (*, p < 0.05; **, p < 0.01; and ***, p < 0.001; Student's t test).

FIGURE 5.

Regulation of meiotic mRNAs by Pab2 is direct and independent of the Mei2-meiRNA complex. A, real-time quantitative PCR analysis of the indicated meiotic genes in wild-type, pab2Δ, pab2Δ mei2Δ, and pab2Δ sme2Δ strains. The levels of transcripts are relative to the wild-type strain and normalized to the nda2 mRNA. The data and error bars represent the average and standard deviation from three independent experiments (*, p < 0.05; Student's t test). B, immunoprecipitation (IP) experiments showing that polyadenylated meiRNA, but not the cytoplasmic srp7 RNA, is enriched in Pab2-TAP precipitates. The meiRNA and srp7 cDNAs were synthesized using oligonucleotide d(T) and random primers, respectively. C, quantification using real-time quantitative PCR for the enrichment of meiotic RNAs in Pab2-TAP precipitates. The -fold changes are relative to an untagged control strain and normalized to the cytosolic srp7 RNA. The data and error bars represent the average and standard deviation from three independent experiments.

FIGURE 6.

Pab2 is a negative regulator of meiotic differentiation. A, sporulation analysis of wild-type, sme2Δ, and pab2Δ sme2Δ homothallic strains. The spots were stained for spores using iodine vapors and cells from each strain were visualized by microscopy. B, the cells were manually counted to assess sporulation percentage relative to a wild-type homothallic strain. The data and error bars represent the average and standard deviation from two independent experiments. In total, at least 600 cells were counted for each strain.