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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.

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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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Nuclear export is important for the function of Spo5. (A) Spo5 fused with an NLS sequence derived from SV40 large T antigen and GFP (Spo5–NLS–GFP) accumulated in the nucleus during meiosis. Scale bar, 5 μm. (B) Addition of the NLS caused deficient sporulation. Differential interference contrast (DIC) images are shown. Scale bar, 5 μm. (C) Quantitative representation of the reduction in sporulation efficiency (light blue) and the frequency of four-spore asci (magenta) (n > 500). Error bars indicate standard deviation. (D) Nuclear accumulation of Spo5(RRM1∆ )–GFP was suppressed by the fusion of the NES sequence. Scale bar, 5 μm. (E) Addition of NES did not suppress the sporulation defects of Spo5(RRM1∆ ) (n > 500). Error bars indicate standard deviation.
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Figure 3: Nuclear export is important for the function of Spo5. (A) Spo5 fused with an NLS sequence derived from SV40 large T antigen and GFP (Spo5–NLS–GFP) accumulated in the nucleus during meiosis. Scale bar, 5 μm. (B) Addition of the NLS caused deficient sporulation. Differential interference contrast (DIC) images are shown. Scale bar, 5 μm. (C) Quantitative representation of the reduction in sporulation efficiency (light blue) and the frequency of four-spore asci (magenta) (n > 500). Error bars indicate standard deviation. (D) Nuclear accumulation of Spo5(RRM1∆ )–GFP was suppressed by the fusion of the NES sequence. Scale bar, 5 μm. (E) Addition of NES did not suppress the sporulation defects of Spo5(RRM1∆ ) (n > 500). Error bars indicate standard deviation.

Mentions: First, we used an external nuclear localization signal (NLS) to alter the localization of Spo5 artificially from the cytoplasm to the nucleus. Although the addition of NLS appeared to make the protein less stable (Additional file 1: Figure S1), cells expressing Spo5–NLS–GFP accumulated the fusion proteins detectably in the nucleus, indicating that the NLS sequence was functional (Figure 3A). The sporulation efficiency of these cells was significantly reduced, and they produced abnormally shaped spores or asci with less than four spores (Figure 3B,C). While the destabilization of Spo5–NLS was likely to contribute largely to the sporulation defect, enforced nuclear migration of Spo5 also appeared to abolish its function to promote meiosis and sporulation, suggesting that the nuclear export of Spo5 is important for its optimal function. The viability of the spores generated in spo5-NLS-GFP cells was comparable to that of the control (spo5-GFP, 50% vs. spo5-NLS-GFP 40%), implying that the loss of Spo5 function did not affect the germination potential.


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)

Nuclear export is important for the function of Spo5. (A) Spo5 fused with an NLS sequence derived from SV40 large T antigen and GFP (Spo5–NLS–GFP) accumulated in the nucleus during meiosis. Scale bar, 5 μm. (B) Addition of the NLS caused deficient sporulation. Differential interference contrast (DIC) images are shown. Scale bar, 5 μm. (C) Quantitative representation of the reduction in sporulation efficiency (light blue) and the frequency of four-spore asci (magenta) (n > 500). Error bars indicate standard deviation. (D) Nuclear accumulation of Spo5(RRM1∆ )–GFP was suppressed by the fusion of the NES sequence. Scale bar, 5 μm. (E) Addition of NES did not suppress the sporulation defects of Spo5(RRM1∆ ) (n > 500). Error bars indicate standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4109790&req=5

Figure 3: Nuclear export is important for the function of Spo5. (A) Spo5 fused with an NLS sequence derived from SV40 large T antigen and GFP (Spo5–NLS–GFP) accumulated in the nucleus during meiosis. Scale bar, 5 μm. (B) Addition of the NLS caused deficient sporulation. Differential interference contrast (DIC) images are shown. Scale bar, 5 μm. (C) Quantitative representation of the reduction in sporulation efficiency (light blue) and the frequency of four-spore asci (magenta) (n > 500). Error bars indicate standard deviation. (D) Nuclear accumulation of Spo5(RRM1∆ )–GFP was suppressed by the fusion of the NES sequence. Scale bar, 5 μm. (E) Addition of NES did not suppress the sporulation defects of Spo5(RRM1∆ ) (n > 500). Error bars indicate standard deviation.
Mentions: First, we used an external nuclear localization signal (NLS) to alter the localization of Spo5 artificially from the cytoplasm to the nucleus. Although the addition of NLS appeared to make the protein less stable (Additional file 1: Figure S1), cells expressing Spo5–NLS–GFP accumulated the fusion proteins detectably in the nucleus, indicating that the NLS sequence was functional (Figure 3A). The sporulation efficiency of these cells was significantly reduced, and they produced abnormally shaped spores or asci with less than four spores (Figure 3B,C). While the destabilization of Spo5–NLS was likely to contribute largely to the sporulation defect, enforced nuclear migration of Spo5 also appeared to abolish its function to promote meiosis and sporulation, suggesting that the nuclear export of Spo5 is important for its optimal function. The viability of the spores generated in spo5-NLS-GFP cells was comparable to that of the control (spo5-GFP, 50% vs. spo5-NLS-GFP 40%), implying that the loss of Spo5 function did not affect the germination potential.

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