The fission yeast meiotic regulator Mei2p forms a dot structure in the horse-tail nucleus in association with the sme2 locus on chromosome II

Abstract

Fission yeast Mei2p is an RNA-binding protein essential for induction of both premeiotic DNA synthesis and first meiotic division. Mei2p forms a dot structure at an apparently fixed position in the horse-tail nucleus during meiotic prophase. This dot formation requires a meiosis-specific RNA species, meiRNA, which is indispensable for meiosis I, and the emergence of the dot is an indicator of the ability of the cell to perform meiosis I. Herein, we have sought the identity of this dot. Analyses using chromosome segregation in haploid meiosis, reciprocal translocation of chromosomes, and gene translocation have led us to conclude that the Mei2p dot is in association with the sme2 gene on the short arm of chromosome II, which encodes meiRNA. Transcripts of sme2, rather than the DNA sequence of the gene, seem to be the determinant of the localization of the Mei2p dot. However, evidence suggests that the dot may not be a simple reflection of the attachment of Mei2p to meiRNA undergoing transcription. We speculate that the Mei2p dot is a specialized structure, either to foster the assembly of Mei2p and meiRNA or to perform some unidentified function indispensable for meiosis I.

Figure 3.

A map of chromosomes indicating the sites where the LacO sequence was integrated. (A) LacO integration sites on the three chromosomes, which were used in the early stage of analysis to determine the Mei2p dot position. Bars indicate chromosomes and open circles indicate centromeres. This panel was drawn according to the following references (Hoheisel et al., 1993; Mizukami et al., 1993; Yamamoto et al., 1999) and the genome sequence information presented by Sanger Centre (Cambridge, United Kingdom). (B) A detailed chart of the vicinity of sme2 on the short arm of chromosome II. The centromere is rightward. Site D shown in A corresponds to the right end of this chart. New integration sites F, G, and H are shown. The genes shown in this chart are presented for references and are not LacO integration sites. Cosmids covering this area are also presented for references.

Figure 1.

Relative localization of the Mei2p dot, the SPB, and the nucleolus in the meiotic prophase nucleus. The Mei2p dot was visualized with anti-HA, the SPB, with anti-Sad1p, and the nucleolus, with GFP-tagged Gar2p. DNA was counterstained with Hoechst 33342. (A) Cells of JW215 (h⁹⁰ gar2-GFP) growing mitotically were live observed. (B) JW215 cells carrying pREP41-Mei2p-3HA were fixed under the meiotic conditions and stained for DNA, Mei2p-3HA, and SPB. (C) Cells of the taz1Δ strain JW329 (h90 taz1Δ gar2-GFP) were analyzed as in B. The merged images display DNA (blue), Gar2-GFP (green), Mei2p-3HA (red), and SPB (white). Bar, 10 μm.

Figure 2.

Haploid meiosis in which the first division is either reductional or equational. (A) Schematic illustration of haploid meiosis that is enabled either by the expression of the two mating-type genes (mat1-M and mat1-P) (top) or by the inactivation of Pat1 kinase (bottom). Chromosome segregation is reductional in the former, whereas it is equational in the latter (Yamamoto and Hiraoka, 2003). Bars indicate sister chromatids and open circles indicate centromeres. Surrounding large circles indicate nuclei. (B) A typical haploid cell that carried both mating-type genes (JW917). The arrowhead indicates the Mei2p-GFP dot at telophase of meiosis I. DNA counterstained with Hoechst 33342 is shown in blue. (C) A typical haploid cell that underwent pat1-driven meiosis (JW916). The arrowheads indicate the separated Mei2p-GFP dots. Horizontal edges of B and C, 10 μm.

Figure 4.

Location of the Mei2p dot (red) relative to a specific LacO marker (green) in a horse-tail nucleus undergoing zygotic meiosis. LacO was inserted at each genetic locus on chromosome II as indicated in the panel. DNA (blue) was counterstained with Hoechst33342. The LacO marker at cut3 seems to be split probably due to temporary separation of the homologous chromosomes. The LacO marker at either site D or site E was visualized also in the taz1Δ mutant. Bar, 10 μm.

Figure 5.

A schematic illustration of chromosome translocation. Red and green bars represent chromosome II and chromosome III, respectively. Open circles indicate centromeres. (A) Possible crossover when the inserted ade6-469 allele is closer to the centromere than the sme2 gene is. (B) Possible crossover when the sme2 gene is closer to the centromere than the inserted ade6-469 allele is.

Figure 6.

Position of the Mei2p dot in a strain with rearranged chromosomes. (A) A homothallic strain with arms of chromosomes II and III translocated at site G (JW935; left), and a homothallic strain with arms of chromosomes II and III translocated at site H (JW936; right). A zygote of each strain is shown. The red dot, indicated by an arrowhead, represents the Mei2p-CFP dot, and the green dot, indicated by an arrow, represents the SPB marked by Sad1p-GFP. (B) The Mei2p dot (red) was visualized in a zygote of JW940, in which the sme2 gene was translocated to the lys1 locus on chromosome I. The lys1 locus was marked with LacO and can be seen as a green dot. Bar, 10 μm.

Figure 7.

Mei2p dot formation in a strain with a sme2 allele that is transcriptionally inactive. (A) Nucleotide alterations introduced into the sme2-m allele. The putative TATA sequence, doubly underlined, was modified into the sequence shown below. The rightmost three nucleotides, underlined, correspond to the 5′ terminus of meiRNA. (B) The Mei2p dot was visualized in a zygote of either JW941 (left), which carried the sme2-m allele inserted at the lys1 locus in addition to the authentic sme2 gene, or JW937 (right), which carried the sme2⁺ allele inserted at the lys1 locus together with the authentic sme2 gene. The arrows indicate the Mei2p dot (green). DNA (blue) was counterstained with Hoechst33342. Bar, 10 μm. (C) Comparison of the level of meiRNA in JW231 (h90 wild type), JW456 (h90 sme2Δ), JW938 (h90 sme2Δ lys1::ura4⁺-sme2⁺), and in JW942 (h90 sme2Δ lys1::ura4⁺-sme2-m). Cells of these four strains were cultured in MM+N medium to the density of 8 × 10⁶ cells/ml and then transferred to MM-N medium. Samples were taken 0 and 4 h after the transfer. The amount of meiRNA was measured by Northern blotting. rRNA stained with ethidium bromide is shown as loading controls.