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).
Highlights
Mei2 is an RNA-binding protein required for initiating pre-meiotic DNA synthesis and meiosis in Schizosaccharomyces pombe
We wished to identify novel RNA targets of Mei2 in meiosis, and for that purpose it was important to know at what times in meiotic time-courses Mei2 was being highly expressed
We added a TAP-tag to the C-terminus of Mei2 in an h90 strain and allowed it to sporulate in nitrogen-deficient malt extract (ME) medium
Summary
Mei is an RNA-binding protein required for initiating pre-meiotic DNA synthesis and meiosis in Schizosaccharomyces pombe. Upon nitrogen starvation and meiotic initiation, the Mei2 – meiRNA complex sequesters Mmi protein in the nucleus, allowing the mRNAs targeted by Mmi to escape degradation and be translated in the cytoplasm, initiating meiosis [17]. The long, meiotic form of meiRNA has 23 of these Mmi motifs [23] These motifs bind and titrate Mmi to keep it localized to the sme locus in the nucleus during meiosis [19,23]. The mei2-644A mutant, bearing a mutation in the C-terminal RRM, is phenotypically similar to the mei2D allele, in that it is defective in pre-meiotic DNA synthesis (and meiosis) [1] This led Watanabe & Yamamoto [1] to suggest ‘Mei functions at least twice, prior to pre-meiotic DNA synthesis and prior to meiosis I, by coupling with different RNA partners’. The binding of rep mRNA could potentially explain why mei2D, but not sme2D, fails at pre-meiotic DNA synthesis
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