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Functional significance of nuclear export and mRNA binding of meiotic regulator Spo5 in fission yeast.

Togashi N, Yamashita A, Sato M, Yamamoto M - BMC Microbiol. (2014)

Bottom Line: Among the four family members, namely Pcr1, Atf1, Atf21, and Atf31, only the mRNA encoding Pcr1 binds to Spo5.Spo5 is exported from the nucleus with mRNAs via the Rae1-dependent pathway.RRMs are necessary for this process and also for the function of Spo5 after the nuclear export.

View Article: PubMed Central - HTML - PubMed

Affiliation: Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan. yamamoto@nibb.ac.jp.

ABSTRACT

Background: Meiotic cells undergo two rounds of nuclear division and generate gametes. Previous studies have indicated that a number of transcription factors modulate the transcriptome in successive waves during meiosis and spore formation in fission yeast. However, the mechanisms underlying the post-transcriptional regulation in meiosis are not fully understood. The fission yeast spo5+ gene encodes a meiosis-specific RNA-binding protein, which is required for the progression of meiosis II and spore formation. However, the target RNA molecules of Spo5 are yet to be identified. Characterization of meiosis-specific RNA-binding proteins will provide insight into how post-transcriptional regulation influence gene expression during sexual differentiation.

Results: To assess the functional significance of RNA-recognition motifs (RRMs) of Spo5, we constructed a series of new spo5 truncated mutants and previously reported spo5 missense mutants. In addition, we isolated novel spo5 missense mutants. The phenotypic characteristics of these mutants indicated that the RRMs are essential for both the localization and function of the protein. Interestingly, Spo5 is exported from the nucleus to the cytoplasm via the Rae1-dependent mRNA export pathway, but is unlikely to be involved in global mRNA export. Furthermore, cytoplasmic localization of Spo5 is important for its function, which suggests the involvement of Spo5 in post-transcriptional regulation. We identified pcr1+ mRNA as one of the critical targets of Spo5. The pcr1+ gene encodes an activating transcription factor/cAMP response element binding (ATF/CREB) transcription factor family. Among the four family members, namely Pcr1, Atf1, Atf21, and Atf31, only the mRNA encoding Pcr1 binds to Spo5.

Conclusions: Spo5 is exported from the nucleus with mRNAs via the Rae1-dependent pathway. RRMs are necessary for this process and also for the function of Spo5 after the nuclear export. Spo5 appears to influence the activity of pcr1+ mRNA, and the mechanism of how Spo5 stimulates the mRNA to promote the progression of meiosis II and spore formation remains an intriguing question for future research.

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Spo5 localization to the cytoplasm facilitated by the mRNA export pathway does not involve global mRNA export. (A) Spo5–GFP accumulated in the nucleus in temperature-sensitive rae1-167 cells. Cells were incubated at 25°C for 6 h and then transferred to 36°C for 3 h. Cut11-4mRFP was used as a nuclear envelope marker. The Spo5–GFP signal was evident in cells undergoing meiotic prophase I through meiosis II. Scale bar, 5 μm. (B) To block RNA-polymerase II-dependent transcription of mRNAs, we used 1, 10-phenanthroline [49]. Wild-type diploid cells were transferred to MM-N to induce meiosis, and after 3.5 hours (Time 0), 1,10-phenanthroline was added to half of the culture at a final concentration of 500 ng/μL. Microscopic observation of Spo5-GFP was carried out after 2 hours. Scale bar, 5 μm. (C) Quantitative analysis of Spo5-GFP localization in cells treated with 1,10-phenanthroline. The number of cells examined is as follows: 0 h, n = 152; 2 h(-), n = 239; and 2 h(+), n = 207. (D) Cells expressing Spo5–GFP (-LMB) were treated with 100 ng/mL LMB and observed after 1 h (+LMB). Mei2-mCherry served as a positive control since it accumulates in the nucleus upon LMB addition. Scale bar, 5 μm. (E) Pabp–GFP accumulated in the nucleus in rae1-167 cells, whereas it did not do so in spo5∆ cells. Scale bar, 5 μm.
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Figure 2: Spo5 localization to the cytoplasm facilitated by the mRNA export pathway does not involve global mRNA export. (A) Spo5–GFP accumulated in the nucleus in temperature-sensitive rae1-167 cells. Cells were incubated at 25°C for 6 h and then transferred to 36°C for 3 h. Cut11-4mRFP was used as a nuclear envelope marker. The Spo5–GFP signal was evident in cells undergoing meiotic prophase I through meiosis II. Scale bar, 5 μm. (B) To block RNA-polymerase II-dependent transcription of mRNAs, we used 1, 10-phenanthroline [49]. Wild-type diploid cells were transferred to MM-N to induce meiosis, and after 3.5 hours (Time 0), 1,10-phenanthroline was added to half of the culture at a final concentration of 500 ng/μL. Microscopic observation of Spo5-GFP was carried out after 2 hours. Scale bar, 5 μm. (C) Quantitative analysis of Spo5-GFP localization in cells treated with 1,10-phenanthroline. The number of cells examined is as follows: 0 h, n = 152; 2 h(-), n = 239; and 2 h(+), n = 207. (D) Cells expressing Spo5–GFP (-LMB) were treated with 100 ng/mL LMB and observed after 1 h (+LMB). Mei2-mCherry served as a positive control since it accumulates in the nucleus upon LMB addition. Scale bar, 5 μm. (E) Pabp–GFP accumulated in the nucleus in rae1-167 cells, whereas it did not do so in spo5∆ cells. Scale bar, 5 μm.

Mentions: RRMs appear to be essential for the translocation of Spo5 from the nucleus to the cytoplasm. Therefore, we investigated whether Spo5 might be exported to the cytoplasm via the mRNA export machinery using a mutant of the mRNA export factor, Rae1. The Rae1-dependent pathway is conserved from the budding yeast [23] to humans [24]. The fission yeast rae1-167 mutant exhibited defective mRNA export at restrictive temperatures [25]. We induced meiosis in wild-type (WT) and rae1-167 cells at 25°C, the latter of which produced Spo5 protein in a comparable or slightly lower amount (Additional file 1: Figure S1), and transferred the cells to 36°C to inactivate Rae1-167. In WT cells, Spo5–GFP was mainly localized to the cytoplasm both at 25°C and 36°C (Figure 2A). By contrast, Spo5–GFP accumulated in the nucleus in rae1-167 cells at the restrictive temperature (Figure 2A). This suggests that Spo5 is likely to be exported to the cytoplasm via the mRNA export machinery. Consistent with this idea, Spo5 accumulated in the nucleus when mRNA synthesis was inhibited by the addition of 1,10-phenanthroline (Figure 2B,C). We also noticed that sporulation was markedly inefficient in rae1-167 cells (spo5-GFP rae1+, 83% vs. spo5-GFP rae1-167, 19%), indicating that Rae1 plays an important role in meiotic progression and sporulation.


Functional significance of nuclear export and mRNA binding of meiotic regulator Spo5 in fission yeast.

Togashi N, Yamashita A, Sato M, Yamamoto M - BMC Microbiol. (2014)

Spo5 localization to the cytoplasm facilitated by the mRNA export pathway does not involve global mRNA export. (A) Spo5–GFP accumulated in the nucleus in temperature-sensitive rae1-167 cells. Cells were incubated at 25°C for 6 h and then transferred to 36°C for 3 h. Cut11-4mRFP was used as a nuclear envelope marker. The Spo5–GFP signal was evident in cells undergoing meiotic prophase I through meiosis II. Scale bar, 5 μm. (B) To block RNA-polymerase II-dependent transcription of mRNAs, we used 1, 10-phenanthroline [49]. Wild-type diploid cells were transferred to MM-N to induce meiosis, and after 3.5 hours (Time 0), 1,10-phenanthroline was added to half of the culture at a final concentration of 500 ng/μL. Microscopic observation of Spo5-GFP was carried out after 2 hours. Scale bar, 5 μm. (C) Quantitative analysis of Spo5-GFP localization in cells treated with 1,10-phenanthroline. The number of cells examined is as follows: 0 h, n = 152; 2 h(-), n = 239; and 2 h(+), n = 207. (D) Cells expressing Spo5–GFP (-LMB) were treated with 100 ng/mL LMB and observed after 1 h (+LMB). Mei2-mCherry served as a positive control since it accumulates in the nucleus upon LMB addition. Scale bar, 5 μm. (E) Pabp–GFP accumulated in the nucleus in rae1-167 cells, whereas it did not do so in spo5∆ cells. Scale bar, 5 μm.
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Figure 2: Spo5 localization to the cytoplasm facilitated by the mRNA export pathway does not involve global mRNA export. (A) Spo5–GFP accumulated in the nucleus in temperature-sensitive rae1-167 cells. Cells were incubated at 25°C for 6 h and then transferred to 36°C for 3 h. Cut11-4mRFP was used as a nuclear envelope marker. The Spo5–GFP signal was evident in cells undergoing meiotic prophase I through meiosis II. Scale bar, 5 μm. (B) To block RNA-polymerase II-dependent transcription of mRNAs, we used 1, 10-phenanthroline [49]. Wild-type diploid cells were transferred to MM-N to induce meiosis, and after 3.5 hours (Time 0), 1,10-phenanthroline was added to half of the culture at a final concentration of 500 ng/μL. Microscopic observation of Spo5-GFP was carried out after 2 hours. Scale bar, 5 μm. (C) Quantitative analysis of Spo5-GFP localization in cells treated with 1,10-phenanthroline. The number of cells examined is as follows: 0 h, n = 152; 2 h(-), n = 239; and 2 h(+), n = 207. (D) Cells expressing Spo5–GFP (-LMB) were treated with 100 ng/mL LMB and observed after 1 h (+LMB). Mei2-mCherry served as a positive control since it accumulates in the nucleus upon LMB addition. Scale bar, 5 μm. (E) Pabp–GFP accumulated in the nucleus in rae1-167 cells, whereas it did not do so in spo5∆ cells. Scale bar, 5 μm.
Mentions: RRMs appear to be essential for the translocation of Spo5 from the nucleus to the cytoplasm. Therefore, we investigated whether Spo5 might be exported to the cytoplasm via the mRNA export machinery using a mutant of the mRNA export factor, Rae1. The Rae1-dependent pathway is conserved from the budding yeast [23] to humans [24]. The fission yeast rae1-167 mutant exhibited defective mRNA export at restrictive temperatures [25]. We induced meiosis in wild-type (WT) and rae1-167 cells at 25°C, the latter of which produced Spo5 protein in a comparable or slightly lower amount (Additional file 1: Figure S1), and transferred the cells to 36°C to inactivate Rae1-167. In WT cells, Spo5–GFP was mainly localized to the cytoplasm both at 25°C and 36°C (Figure 2A). By contrast, Spo5–GFP accumulated in the nucleus in rae1-167 cells at the restrictive temperature (Figure 2A). This suggests that Spo5 is likely to be exported to the cytoplasm via the mRNA export machinery. Consistent with this idea, Spo5 accumulated in the nucleus when mRNA synthesis was inhibited by the addition of 1,10-phenanthroline (Figure 2B,C). We also noticed that sporulation was markedly inefficient in rae1-167 cells (spo5-GFP rae1+, 83% vs. spo5-GFP rae1-167, 19%), indicating that Rae1 plays an important role in meiotic progression and sporulation.

Bottom Line: Among the four family members, namely Pcr1, Atf1, Atf21, and Atf31, only the mRNA encoding Pcr1 binds to Spo5.Spo5 is exported from the nucleus with mRNAs via the Rae1-dependent pathway.RRMs are necessary for this process and also for the function of Spo5 after the nuclear export.

View Article: PubMed Central - HTML - PubMed

Affiliation: Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan. yamamoto@nibb.ac.jp.

ABSTRACT

Background: Meiotic cells undergo two rounds of nuclear division and generate gametes. Previous studies have indicated that a number of transcription factors modulate the transcriptome in successive waves during meiosis and spore formation in fission yeast. However, the mechanisms underlying the post-transcriptional regulation in meiosis are not fully understood. The fission yeast spo5+ gene encodes a meiosis-specific RNA-binding protein, which is required for the progression of meiosis II and spore formation. However, the target RNA molecules of Spo5 are yet to be identified. Characterization of meiosis-specific RNA-binding proteins will provide insight into how post-transcriptional regulation influence gene expression during sexual differentiation.

Results: To assess the functional significance of RNA-recognition motifs (RRMs) of Spo5, we constructed a series of new spo5 truncated mutants and previously reported spo5 missense mutants. In addition, we isolated novel spo5 missense mutants. The phenotypic characteristics of these mutants indicated that the RRMs are essential for both the localization and function of the protein. Interestingly, Spo5 is exported from the nucleus to the cytoplasm via the Rae1-dependent mRNA export pathway, but is unlikely to be involved in global mRNA export. Furthermore, cytoplasmic localization of Spo5 is important for its function, which suggests the involvement of Spo5 in post-transcriptional regulation. We identified pcr1+ mRNA as one of the critical targets of Spo5. The pcr1+ gene encodes an activating transcription factor/cAMP response element binding (ATF/CREB) transcription factor family. Among the four family members, namely Pcr1, Atf1, Atf21, and Atf31, only the mRNA encoding Pcr1 binds to Spo5.

Conclusions: Spo5 is exported from the nucleus with mRNAs via the Rae1-dependent pathway. RRMs are necessary for this process and also for the function of Spo5 after the nuclear export. Spo5 appears to influence the activity of pcr1+ mRNA, and the mechanism of how Spo5 stimulates the mRNA to promote the progression of meiosis II and spore formation remains an intriguing question for future research.

Show MeSH
Related in: MedlinePlus