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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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Binding of Spo5 protein to pcr1+ mRNA. (A) Results of an EMSA assay indicating formation of the Spo5–pcr1+ RNA complex. Recombinant GST and GST–Spo5C (the C-terminal part of Spo5, aas 192–567) proteins were incubated with pcr1+ RNA including the coding region and both 5′- and 3′-UTRs (lanes 3 and 4) or control GFP RNA (lanes 1 and 2). ‘G’ indicates GST (50 ng), and ‘SpC’ indicates GST-Spo5C (20 ng). The red arrowhead indicates shifted RNA. (B) Spo5–GFP and pcr1+ mRNA form complexes in vivo. A cell extract was prepared from a diploid Spo5–GFP strain undergoing meiosis and treated with anti-GFP and the control anti-HA. Reverse transcription-polymerase chain reaction (RT-PCR) assay using the pull-downs was performed to detect pcr1+ and other ATF/CREB factors, atf1+, atf21+, and atf31+ mRNA. (C)pcr1+ RNA complexes with Spo5C more efficiently than atf21+ RNA does. atf21+ RNA carried the coding region and both 5′- and 3′-UTRs, similarly to pcr1+ RNA. The red arrowhead indicates shifted RNA. (D) Spo5C binds to pcr1+ RNA more efficiently than the mutant form Spo5C(FAFA), suggesting the involvement of the two phenylalanine residues in RNA binding. The red arrowhead indicates shifted RNA.
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Figure 5: Binding of Spo5 protein to pcr1+ mRNA. (A) Results of an EMSA assay indicating formation of the Spo5–pcr1+ RNA complex. Recombinant GST and GST–Spo5C (the C-terminal part of Spo5, aas 192–567) proteins were incubated with pcr1+ RNA including the coding region and both 5′- and 3′-UTRs (lanes 3 and 4) or control GFP RNA (lanes 1 and 2). ‘G’ indicates GST (50 ng), and ‘SpC’ indicates GST-Spo5C (20 ng). The red arrowhead indicates shifted RNA. (B) Spo5–GFP and pcr1+ mRNA form complexes in vivo. A cell extract was prepared from a diploid Spo5–GFP strain undergoing meiosis and treated with anti-GFP and the control anti-HA. Reverse transcription-polymerase chain reaction (RT-PCR) assay using the pull-downs was performed to detect pcr1+ and other ATF/CREB factors, atf1+, atf21+, and atf31+ mRNA. (C)pcr1+ RNA complexes with Spo5C more efficiently than atf21+ RNA does. atf21+ RNA carried the coding region and both 5′- and 3′-UTRs, similarly to pcr1+ RNA. The red arrowhead indicates shifted RNA. (D) Spo5C binds to pcr1+ RNA more efficiently than the mutant form Spo5C(FAFA), suggesting the involvement of the two phenylalanine residues in RNA binding. The red arrowhead indicates shifted RNA.

Mentions: To test the possibility that Spo5 may bind to pcr1+ mRNA to modulate its expression/function during meiosis, we performed an electrophoresis mobility shift assay (EMSA) using recombinant Spo5 protein and pcr1+ RNA transcribed in vitro. As shown in Figure 5A, the C-terminal part of Spo5, including the two RRMs (Spo5C, aas 192–567), fused to glutathione S-transferase (GST) (GST-Spo5C), associated with the pcr1+ RNA (red arrowhead), but not with the control GFP RNA. To confirm direct interaction of Spo5 and pcr1+ RNA in vivo, we performed an RNA-immunoprecipitation assay using the spo5-GFP diploid strain. Immunoprecipitation by anti-GFP antibody indicated that pcr1+ mRNA formed a complex with Spo5-GFP in vivo, but that the mRNAs of other ATF/CREB factors did not (Figure 5B). To further confirm the specificity of binding, we compared binding of Spo5C to pcr1+ RNA and atf21+ RNA transcribed similarly in vitro. While Spo5C bound to atf21+ RNA to some extent (Figure 5C, lanes 7 and 8), probably because of its weak non-specific affinity for RNA, which we noticed previously [21], it was clear that Spo5C could bind to pcr1+ RNA more strongly than atf21+ RNA, as 50 ng of the protein was enough to shift pcr1+ RNA (Figure 5C, lane 2). We also confirmed that Spo5C carrying the F341A and F427A mutations, designated Spo5C(FAFA), lost the binding ability to pcr1+ RNA (Figure 5D, compare lanes 2 vs. 7, and lanes 3 vs. 8), demonstrating the importance of the RRMs for the binding. These observations indicate that pcr1+ mRNA is one of the critical targets of Spo5. We have shown that cdc13+ mRNA also binds to Spo5 [21]. A previous report suggested that a long 3′ UTR of cdc13+, cdc25+ and ste9+ mRNA might determine its stability [38]. The pcr1+ mRNA also carries a relatively long 3′ untranslated region (UTR), but our observation does not support the idea that Spo5 stabilizes pcr1+ mRNA (Additional file 4: Figure S4A). Thus, it is an interesting hypothesis to be confirmed that Spo5 may control certain activity of pcr1+ mRNA through binding to its long 3′ UTR.


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)

Binding of Spo5 protein to pcr1+ mRNA. (A) Results of an EMSA assay indicating formation of the Spo5–pcr1+ RNA complex. Recombinant GST and GST–Spo5C (the C-terminal part of Spo5, aas 192–567) proteins were incubated with pcr1+ RNA including the coding region and both 5′- and 3′-UTRs (lanes 3 and 4) or control GFP RNA (lanes 1 and 2). ‘G’ indicates GST (50 ng), and ‘SpC’ indicates GST-Spo5C (20 ng). The red arrowhead indicates shifted RNA. (B) Spo5–GFP and pcr1+ mRNA form complexes in vivo. A cell extract was prepared from a diploid Spo5–GFP strain undergoing meiosis and treated with anti-GFP and the control anti-HA. Reverse transcription-polymerase chain reaction (RT-PCR) assay using the pull-downs was performed to detect pcr1+ and other ATF/CREB factors, atf1+, atf21+, and atf31+ mRNA. (C)pcr1+ RNA complexes with Spo5C more efficiently than atf21+ RNA does. atf21+ RNA carried the coding region and both 5′- and 3′-UTRs, similarly to pcr1+ RNA. The red arrowhead indicates shifted RNA. (D) Spo5C binds to pcr1+ RNA more efficiently than the mutant form Spo5C(FAFA), suggesting the involvement of the two phenylalanine residues in RNA binding. The red arrowhead indicates shifted RNA.
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Related In: Results  -  Collection

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Figure 5: Binding of Spo5 protein to pcr1+ mRNA. (A) Results of an EMSA assay indicating formation of the Spo5–pcr1+ RNA complex. Recombinant GST and GST–Spo5C (the C-terminal part of Spo5, aas 192–567) proteins were incubated with pcr1+ RNA including the coding region and both 5′- and 3′-UTRs (lanes 3 and 4) or control GFP RNA (lanes 1 and 2). ‘G’ indicates GST (50 ng), and ‘SpC’ indicates GST-Spo5C (20 ng). The red arrowhead indicates shifted RNA. (B) Spo5–GFP and pcr1+ mRNA form complexes in vivo. A cell extract was prepared from a diploid Spo5–GFP strain undergoing meiosis and treated with anti-GFP and the control anti-HA. Reverse transcription-polymerase chain reaction (RT-PCR) assay using the pull-downs was performed to detect pcr1+ and other ATF/CREB factors, atf1+, atf21+, and atf31+ mRNA. (C)pcr1+ RNA complexes with Spo5C more efficiently than atf21+ RNA does. atf21+ RNA carried the coding region and both 5′- and 3′-UTRs, similarly to pcr1+ RNA. The red arrowhead indicates shifted RNA. (D) Spo5C binds to pcr1+ RNA more efficiently than the mutant form Spo5C(FAFA), suggesting the involvement of the two phenylalanine residues in RNA binding. The red arrowhead indicates shifted RNA.
Mentions: To test the possibility that Spo5 may bind to pcr1+ mRNA to modulate its expression/function during meiosis, we performed an electrophoresis mobility shift assay (EMSA) using recombinant Spo5 protein and pcr1+ RNA transcribed in vitro. As shown in Figure 5A, the C-terminal part of Spo5, including the two RRMs (Spo5C, aas 192–567), fused to glutathione S-transferase (GST) (GST-Spo5C), associated with the pcr1+ RNA (red arrowhead), but not with the control GFP RNA. To confirm direct interaction of Spo5 and pcr1+ RNA in vivo, we performed an RNA-immunoprecipitation assay using the spo5-GFP diploid strain. Immunoprecipitation by anti-GFP antibody indicated that pcr1+ mRNA formed a complex with Spo5-GFP in vivo, but that the mRNAs of other ATF/CREB factors did not (Figure 5B). To further confirm the specificity of binding, we compared binding of Spo5C to pcr1+ RNA and atf21+ RNA transcribed similarly in vitro. While Spo5C bound to atf21+ RNA to some extent (Figure 5C, lanes 7 and 8), probably because of its weak non-specific affinity for RNA, which we noticed previously [21], it was clear that Spo5C could bind to pcr1+ RNA more strongly than atf21+ RNA, as 50 ng of the protein was enough to shift pcr1+ RNA (Figure 5C, lane 2). We also confirmed that Spo5C carrying the F341A and F427A mutations, designated Spo5C(FAFA), lost the binding ability to pcr1+ RNA (Figure 5D, compare lanes 2 vs. 7, and lanes 3 vs. 8), demonstrating the importance of the RRMs for the binding. These observations indicate that pcr1+ mRNA is one of the critical targets of Spo5. We have shown that cdc13+ mRNA also binds to Spo5 [21]. A previous report suggested that a long 3′ UTR of cdc13+, cdc25+ and ste9+ mRNA might determine its stability [38]. The pcr1+ mRNA also carries a relatively long 3′ untranslated region (UTR), but our observation does not support the idea that Spo5 stabilizes pcr1+ mRNA (Additional file 4: Figure S4A). Thus, it is an interesting hypothesis to be confirmed that Spo5 may control certain activity of pcr1+ mRNA through binding to its long 3′ UTR.

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