Meiotic long non-coding meiRNA accumulates as a dot at its genetic locus facilitated by Mmi1 and plays as a decoy to lure Mmi1

Abstract

Long non-coding RNAs (lncRNAs) play key roles in the formation of nuclear bodies. In the fission yeast Schizosaccharomyces pombe, a lncRNA species termed meiRNA forms a nuclear dot structure at its own genetic locus, the sme2 locus, with its protein-binding partner Mei2. This dot structure, called Mei2 dot, promotes the progression of meiosis by suppressing Mmi1, a crucial factor involved in the selective elimination of meiosis-specific transcripts. The meiRNA itself is a target of Mmi1-mediated elimination and is supposed to function as a decoy to lure Mmi1. However, detailed mechanisms underlying the formation of Mei2 dot and inactivation of Mmi1 remain ambiguous. Here, we show that the localization of meiRNA, at its genetic locus sme2, depends on its association with Mmi1. We also demonstrate that one of the multiple Mmi1 foci in mitotic cells localizes to the sme2 locus. Furthermore, the overexpression of meiRNA promotes the accumulation of Mmi1 to the sme2 locus even in the absence of Mei2 and reduces the activity of Mmi1. These findings indicate that the retention of meiRNA at its genetic locus is facilitated by Mmi1, which then attracts scattered Mmi1 to inhibit its function.

Keywords: RNA localization; Schizosaccharomyces pombe; long non-coding RNA; meiosis; nuclear body.

Figure 1.

The 3′ region of meiRNA-L carrying DSR motifs binds with Mmi1, and the 5′ region binds with Mei2. (a) The location of the hexanucleotide DSR motifs within the sme2 gene. The UUAAAC, UCAAAC, UGAAAC and UAAAAC sequences are indicated by an orange, a yellow, a green and a blue arrowhead, respectively. Bold numbers indicate 3′-ends of meiRNA. The wavy lines indicate two isoforms of the meiRNA transcript. RNA probes used in the EMSA are shown at the top of the illustration. (b) EMSA for interaction between Mmi1 and meiRNA variants. Bacterially expressed and purified GST-Mmi1 (either 20 or 50 nM) or GST portion alone (3 µM) was incubated with DIG-labelled probes of meiRNA variants (0.1 nM) transcribed in vitro. The GFP transcript was used as a negative control. The relative abundances of free probes are indicated. (c) EMSA to detect the interaction between Mei2 and meiRNA variants. Bacterially expressed and purified GST-Mei2 (either 40 or 80 nM) or GST portion alone (3 µM) was incubated with DIG-labelled probes of meiRNA variants (0.2 nM) transcribed in vitro. The GFP transcript was used as a negative control. The relative abundances of free probes are indicated.

Figure 2.

The DSR motifs within meiRNA are required for dot formation. (a) Schematic illustration of the meiRNA visualizing system and two sme2 mutants: sme2–5′ and sme2–3′. In the sme2–5′ mutant, the terminator region of the nmt1 gene (Tnmt) is introduced 508 bp downstream from the transcription start site of sme2. In the sme2–3′ mutant, sme2(1–508) is removed. The MS2 loop was inserted in the indicated site. (b) Localization of meiRNA with the MS2 system. Wild-type (JS1) cells expressing MS2-loop-tagged meiRNA and CFP-Mmi1 from the respective endogenous promoters, and MS2-YFP and 4mCherry-LacI-NLS from the weakened adh1 promoter were examined by fluorescence microscopy under meiotic conditions. To mark the sme2 locus, 32 copies of the lacO repeat were inserted about 2 kb upstream of sme2. In the merged image, green indicates meiRNA (MS2-YFP), red indicates the sme2 locus (4mCherry-LacI-NLS) and blue indicates CFP-Mmi1. The dotted lines indicate the shape of the cell. Scale bar, 5 µm. Frequency of meiotic prophase cells containing meiRNA dot is indicated (n > 30). (c) Localization of meiRNA in the sme2 mutants. Wild-type (JS2), sme2–5′ (JS3) and sme2–3′ (JS4) cells expressing MS2-loop-tagged meiRNA variants, Mei2-mCherry and CFP-Mmi1 from the respective endogenous promoters and MS2-YFP from the weakened adh1 promoter were examined under meiotic conditions. In the merged images, green indicates meiRNA (MS2-YFP), red indicates Mei2-mCherry and blue indicates CFP-Mmi1. Scale bar, 5 µm; n > 30, (d) Localization of meiRNA, Mei2 and Mmi1 in sme2-DSRless cells. In sme2-DSRless mutants, all hexanucleotide DSR motifs were disrupted by point mutations from UNAAAC to UNAAGC. The sme2-DSRless cells (JS6) expressing MS2-loop-tagged DSR-less meiRNA, MS2-YFP, Mei2-mCherry and CFP-Mmi1 were examined under meiotic conditions. In the merged image, green indicates meiRNA-DSR-less (MS2-YFP), red indicates Mei2-mCherry and blue indicates CFP-Mmi1. Scale bar, 5 µm; n > 30. (e) Localization of meiRNA in mmi1Δ cells. mei4Δ (JS36) and mmi1Δ mei4Δ (JS37) cells expressing MS2-loop-tagged meiRNA from its endogenous promoter and MS2-GFP from the expression vector pREP81 were examined under meiotic conditions. Scale bar, 5 µm; n > 20.

Figure 3.

Mmi1 is attracted to the sme2 locus through the DSR motifs within meiRNA under mitotic conditions. (a) Localization of Mmi1 and Red1 in a mitotic nucleus. Wild-type cells (JS8) expressing Red1-YFP and CFP-Mmi1 from the respective endogenous promoters and 4mCherry-LacI-NLS from the weak adh1 promoter were examined under mitotic conditions. The lacO repeats were inserted upstream of sme2. Images of the nuclear region are shown. In the merged image, green indicates Red1-YFP, red indicates the sme2 locus and blue indicates CFP-Mmi1. Scale bar, 2 µm. Frequency of cells in which both Mmi1 and Red1 localized at the sme2 locus is indicated (n > 100). (b) Localization of Mmi1 and Red1 in sme2-m cells. sme2-m cells (JS9) expressing Red1-mCherry, GFP-LacI and CFP-Mmi1 were examined under mitotic conditions. The lacO repeats were inserted upstream of sme2. Images of the nuclear region are shown. In the merged image, green indicates Red1-mCherry, red indicates the sme2 locus and blue indicates CFP-Mmi1. Scale bar, 2 µm; n > 100. (c) Localization of Mmi1 and Red1 in sme2-DSRless cells. sme2-DSRless (JS10) cells expressing Red1-YFP, 4mCherry-LacI-NLS and CFP-Mmi1 were examined under mitotic conditions. The lacO repeats were inserted upstream of sme2. Images of the nuclear region are shown. In the merged image, green indicates Red1-YFP, red indicates the sme2 locus and blue indicates CFP-Mmi1. Scale bar, 2 µm; n > 100. (d) ChIP analysis of Red1 accumulation on the mei4 locus and the 5′ or 3′ region of the sme2 locus in wild-type (JS12), sme2-m (JS13) and sme2-DSRless (JS14) cells under mitotic conditions, relative to the act1 locus. Results represent the mean ± s.d. from three reactions. (e) Localization of Mmi1 and the mei4 locus. Wild-type (JS52) cells expressing CFP-Mmi1 and 4mCherry-LacI-NLS were examined under mitotic conditions. The lacO repeats were inserted downstream of mei4. Images of the nuclear region are shown. In the merged image, green indicates CFP-Mmi1 and red indicates the mei4 locus. Scale bar, 2 µm; n > 100.

Figure 4.

Overexpression of meiRNA causes the convergence of scattered Mmi1 foci. (a) Localization of overexpressed meiRNA in mitotically growing cells. Cells (JS16) expressing MS2-loop-tagged meiRNA from the strong nmt1 promoter, MS2-GFP, and 4mCherry-LacI-NLS from the weakened adh1 promoter were examined under mitotic conditions. The lacO repeats were inserted upstream of sme2. In the merged image, green indicates meiRNA (MS2-GFP), and red indicates the sme2 locus. Scale bar, 5 µm. Frequency of cells containing meiRNA dot is indicated (n > 100). (b) Localization of mitotic Mmi1 in cells overexpressing meiRNA. sme2⁺ (JS17) and Pnmt1-sme2 (JS18) cells expressing MS2-loop-tagged meiRNA, MS2-GFP and CFP-Mmi1 were examined under mitotic conditions. In the merged images, green indicates meiRNA (MS2-GFP), and red indicates CFP-Mmi1. Scale bar, 5 µm. (c) Percentages of cells containing 1, 2, 3 or 4 and more Mmi1 dots when sme2 variants were overexpressed. More than 100 cells were counted in the sme2⁺ (JS17), Pnmt1-sme2 (JS18), Pnmt1-sme2–5′ (JS19) and Pnmt1-sme2–3′ (JS20) strains under mitotic conditions. (d) Localization of mitotic Mmi1 in cells overexpressing sme2–5′ or sme2–3′. Pnmt1-sme2–5′ (JS19) and Pnmt1-sme2–3′ (JS20) cells expressing CFP-Mmi1 were examined under mitotic conditions. Scale bar, 5 µm.

Figure 5.

Overexpression of meiRNA reduces the Mmi1 activity. (a) Expression of DSR-containing mei4 and ssm4 mRNAs was examined by northern blot analysis in sme2-overexpressing cells. Wild-type (JS33) and Pnmt1-sme2 (JS34) cells were grown in MM media at 30°C. mmi1-ts3 (JV579) and mmi1-ts6 (JV582) cells were incubated in YE media at 25°C and then shifted to 37°C for 2 h. The rRNAs stained with ethidium bromide are shown in the bottom panel as loading controls. (b) Expression of mei4 and ssm4 mRNAs was examined by northern blot analysis in sme2-overexpressing mmi1 mutant cells. mmi1-ts6 (JV582) and mmi1-ts6 Pnmt1-sme2 (JS35) cells were incubated in MM media at 25°C, 28°C and 30°C, respectively. The rRNAs stained with ethidium bromide are shown as loading controls. (c) Sequence-specific competition of meiRNA for the binding between Mmi1 and the DSR region of mei4 mRNA. GST-Mmi1 (30 nM) or GST portion alone (3 µM) was mixed with DIG-labelled mei4DSR transcripts (0.5 nM) and non-labelled competitor meiRNA variants, mei4DSR or GFP transcripts (5 or 50 nM). The relative abundances of free probes are indicated.

Figure 6.

DSR transcripts form a dot. (a) Localization of overexpressed ura4 and ssm4 RNA in mitotically growing cells. Pnmt1-MS2loop-ura4 (JS31) and Pnmt1-MS2loop-ssm4 (JS32) cells expressing MS2-GFP from the expression vector pREP81 were examined under mitotic conditions. Scale bar, 5 µm. Frequencies of cells containing RNA dot are indicated (n > 50). (b) Localization of mitotic Mmi1 in cells overexpressing ura4 and ssm4. Pnmt1-MS2loop-ura4 (JS50) and Pnmt1-MS2loop-ssm4 (JS51) cells expressing CFP-Mmi1 were examined under mitotic conditions. Scale bar, 5 µm. (c) Percentages of cells containing 1, 2, 3 or 4 and more Mmi1 dots when ura4 or ssm4 were overexpressed. More than 100 cells were counted in the Pnmt1-MS2loop-ura4 (JS50) and Pnmt1-MS2loop-ssm4 (JS51) strains under mitotic conditions.