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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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RNA recognition motifs are essential for the localization and function of Spo5. (A) Localization analyses using truncated mutants of Spo5. Localization of wild-type and mutant proteins of Spo5-GFP (green) during the period between meiosis I and meiosis II was detected using the nuclear envelope marker Cut11-4mRFP (red). Numbers on the right indicate the frequency of cells displaying nuclear GFP signals. Schematic images depict the domains of the mutant proteins. Black boxes depict two RNA-recognition motifs, RRM1 and RRM2, respectively. Scale bar, 5 μm. (B) Sporulation efficiency of specific mutants used in (A), measured at 30°C (n > 500). Error bars indicate standard deviation. (C) Localization analyses using point mutants of Spo5. Localization of Spo5-GFP harboring specific point mutations was observed as in (A). Positions of mutation sites (×) are shown in schematic images. Scale bar, 5 μm. (D) Sporulation efficiency of the strains used in (C), measured at 30°C (n > 500). Error bars indicate standard deviation.
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Figure 1: RNA recognition motifs are essential for the localization and function of Spo5. (A) Localization analyses using truncated mutants of Spo5. Localization of wild-type and mutant proteins of Spo5-GFP (green) during the period between meiosis I and meiosis II was detected using the nuclear envelope marker Cut11-4mRFP (red). Numbers on the right indicate the frequency of cells displaying nuclear GFP signals. Schematic images depict the domains of the mutant proteins. Black boxes depict two RNA-recognition motifs, RRM1 and RRM2, respectively. Scale bar, 5 μm. (B) Sporulation efficiency of specific mutants used in (A), measured at 30°C (n > 500). Error bars indicate standard deviation. (C) Localization analyses using point mutants of Spo5. Localization of Spo5-GFP harboring specific point mutations was observed as in (A). Positions of mutation sites (×) are shown in schematic images. Scale bar, 5 μm. (D) Sporulation efficiency of the strains used in (C), measured at 30°C (n > 500). Error bars indicate standard deviation.

Mentions: First, we evaluated the mechanism underlying the subcellular localization of Spo5. A previous study suggested that the C-terminal half of Spo5 is required for its cytoplasmic localization [19]. As this region contains the RRMs, we examined whether these might be responsible for the localization of Spo5. We constructed a series of truncation mutants that lacked the RRM region (Figure 1A). Each mutant carried the mutated spo5 gene in place of the wild-type gene on the chromosome, and expressed the mutant protein from the authentic spo5 promoter. All truncated mutant proteins that lacked at least one RRM showed abnormal nuclear accumulation, as indicated by the fluorescent signals of fused green fluorescent protein (GFP) (Figure 1A). We confirmed that mutant proteins were produced in a comparable amount to wild-type Spo5, by examining the shortest mutant Spo5(1–296) and the RRM2-deletion mutant, although the protein level might be slightly lower in the latter case (Additional 1: Figure S1). The RRM truncation mutants were defective in sporulation (Figure 1B), suggesting that the existence of intact RRMs is correlated with the cytoplasmic localization and proper function of Spo5. While we could not exclude the possibility that some of the effect of the mutations on sporulation might be due to lower levels of the mutant proteins, the effect on sporulation was apparently more dramatic than the effect on protein levels, supporting the importance of the RRMs/cytoplasmic localization in Spo5 function.There was one exception, namely Spo5(1–456)-GFP, which has two RRMs but lacks the C-terminal region. Although it localized mostly to the cytoplasm, it was not functional, whereas Spo5(1–525)-GFP, which has two RRMs and an additional C-terminal portion, was functional (Figure 1A,B). This indicates that, in addition to intact RRMs, the C-terminal region adjacent to the second RRM is essential for the Spo5 function.


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)

RNA recognition motifs are essential for the localization and function of Spo5. (A) Localization analyses using truncated mutants of Spo5. Localization of wild-type and mutant proteins of Spo5-GFP (green) during the period between meiosis I and meiosis II was detected using the nuclear envelope marker Cut11-4mRFP (red). Numbers on the right indicate the frequency of cells displaying nuclear GFP signals. Schematic images depict the domains of the mutant proteins. Black boxes depict two RNA-recognition motifs, RRM1 and RRM2, respectively. Scale bar, 5 μm. (B) Sporulation efficiency of specific mutants used in (A), measured at 30°C (n > 500). Error bars indicate standard deviation. (C) Localization analyses using point mutants of Spo5. Localization of Spo5-GFP harboring specific point mutations was observed as in (A). Positions of mutation sites (×) are shown in schematic images. Scale bar, 5 μm. (D) Sporulation efficiency of the strains used in (C), measured at 30°C (n > 500). Error bars indicate standard deviation.
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Related In: Results  -  Collection

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Figure 1: RNA recognition motifs are essential for the localization and function of Spo5. (A) Localization analyses using truncated mutants of Spo5. Localization of wild-type and mutant proteins of Spo5-GFP (green) during the period between meiosis I and meiosis II was detected using the nuclear envelope marker Cut11-4mRFP (red). Numbers on the right indicate the frequency of cells displaying nuclear GFP signals. Schematic images depict the domains of the mutant proteins. Black boxes depict two RNA-recognition motifs, RRM1 and RRM2, respectively. Scale bar, 5 μm. (B) Sporulation efficiency of specific mutants used in (A), measured at 30°C (n > 500). Error bars indicate standard deviation. (C) Localization analyses using point mutants of Spo5. Localization of Spo5-GFP harboring specific point mutations was observed as in (A). Positions of mutation sites (×) are shown in schematic images. Scale bar, 5 μm. (D) Sporulation efficiency of the strains used in (C), measured at 30°C (n > 500). Error bars indicate standard deviation.
Mentions: First, we evaluated the mechanism underlying the subcellular localization of Spo5. A previous study suggested that the C-terminal half of Spo5 is required for its cytoplasmic localization [19]. As this region contains the RRMs, we examined whether these might be responsible for the localization of Spo5. We constructed a series of truncation mutants that lacked the RRM region (Figure 1A). Each mutant carried the mutated spo5 gene in place of the wild-type gene on the chromosome, and expressed the mutant protein from the authentic spo5 promoter. All truncated mutant proteins that lacked at least one RRM showed abnormal nuclear accumulation, as indicated by the fluorescent signals of fused green fluorescent protein (GFP) (Figure 1A). We confirmed that mutant proteins were produced in a comparable amount to wild-type Spo5, by examining the shortest mutant Spo5(1–296) and the RRM2-deletion mutant, although the protein level might be slightly lower in the latter case (Additional 1: Figure S1). The RRM truncation mutants were defective in sporulation (Figure 1B), suggesting that the existence of intact RRMs is correlated with the cytoplasmic localization and proper function of Spo5. While we could not exclude the possibility that some of the effect of the mutations on sporulation might be due to lower levels of the mutant proteins, the effect on sporulation was apparently more dramatic than the effect on protein levels, supporting the importance of the RRMs/cytoplasmic localization in Spo5 function.There was one exception, namely Spo5(1–456)-GFP, which has two RRMs but lacks the C-terminal region. Although it localized mostly to the cytoplasm, it was not functional, whereas Spo5(1–525)-GFP, which has two RRMs and an additional C-terminal portion, was functional (Figure 1A,B). This indicates that, in addition to intact RRMs, the C-terminal region adjacent to the second RRM is essential for the Spo5 function.

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