mmi1 and rep2 mRNAs are novel RNA targets of the Mei2 RNA-binding protein during early meiosis in Schizosaccharomyces pombe

Abstract

The RNA-binding protein Mei2 is crucial for meiosis in Schizosaccharomyces pombe. In mei2 mutants, pre-meiotic S-phase is blocked, along with meiosis. Mei2 binds a long non-coding RNA (lncRNA) called meiRNA, which is a 'sponge RNA' for the meiotic inhibitor protein Mmi1. The interaction between Mei2, meiRNA and Mmi1 protein is essential for meiosis. But mei2 mutants have stronger and different phenotypes than meiRNA mutants, since mei2Δ arrests before pre-meiotic S, while the meiRNA mutant arrests after pre-meiotic S but before meiosis. This suggests Mei2 may bind additional RNAs. To identify novel RNA targets of Mei2, which might explain how Mei2 regulates pre-meiotic S, we used RNA immunoprecipitation and cross-linking immunoprecipitation. In addition to meiRNA, we found the mRNAs for mmi1 (which encodes Mmi1) and for the S-phase transcription factor rep2 There were also three other RNAs of uncertain relevance. We suggest that at meiotic initiation, Mei2 may sequester rep2 mRNA to help allow pre-meiotic S, and then may bind both meiRNA and mmi1 mRNA to inactivate Mmi1 at two levels, the protein level (as previously known), and also the mRNA level, allowing meiosis. We call Mei2-meiRNA a 'double sponge' (i.e. binding both an mRNA and its encoded protein).

Keywords: Mei2; Mmi1; Rep2; lncRNA; meiosis; pombe.

Figure 1.

meiRNA enrichment after Mei2-TAP immunoprecipitation. (a) meiRNA enrichment after native RNA immunoprecipitation (RIP) of Mei2 shown by qRT-PCR. The y-axis shows log2 fold enrichment. srp7 is shown as a specificity control. (b) meiRNA enrichment after UV cross-linking immunoprecipitation (CLIP) by qRT-PCR is shown. srp7 is shown as a specificity control. (c) Integrated Genome Browser view of the sme2 locus after RIP-CHIP. (d) Integrative Genomics Viewer snapshot of the sme2 locus after CLIP-Seq. (e) CLIP-Seq coverage reads at the sme2 locus. The y-axis scale is normalized for all samples in (c–e).

Figure 2.

RNA partners of Mei2 in early meiosis with known roles in meiosis. (a) Venn diagram showing number of significant targets obtained by Mei2-TAP RIP-CHIP and CLIP-Seq. 6 RNA targets of Mei2 were enriched in both experiments. (b) Probe intensities from RIP-CHIP for important Mei2 targets mmi1, rep2 and mug110. (c) CLIP-Seq coverage from Mei2-TAP and Msa1 at the mmi1, rep2, and mug110 loci. The y-axis scale is normalized for (b) and (c).

Figure 3.

Enrichment profile along RNA length of Mei2 and Msa1 targets. (a) Enrichment profile of significant targets of Mei2 from CLIP-Seq determined by Homer. Each column in the heatmap shows enrichment that was calculated for a 5-nucleotide bin. Each row of the heatmap shows enrichment from 5′ to 3′ along the length of one particular Mei2 RNA target, normalized by length. (b) Enrichment profiles along length of Msa1 CLIP-Seq targets.

Figure 4.

Mei2 titrates out inhibitors of meiosis. During early meiosis Mei2 binds to meiRNA, which binds Mmi1 protein, thus sequestering Mmi1. Mei2 also binds mmi1 mRNA, and probably prevents synthesis of new Mmi1 protein. Mei2 also binds rep2 mRNA, and probably prevents synthesis of new Rep2 protein. Titration of these three meiotic inhibitors (Mmi1, mmi1 mRNA, and rep2 mRNA) facilitate initiation of meiosis. The mechanism by which Mei2 forms a dot (here shown as a speculative Mei2–Mei2 protein–protein interaction) is unknown. Also unknown is whether the Mmi1 binding sites on the mmi1 mRNA participate in the titration of Mmi1 protein.