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Protein cofactor competition regulates the action of a multifunctional RNA helicase in different pathways.

Heininger AU, Hackert P, Andreou AZ, Boon KL, Memet I, Prior M, Clancy A, Schmidt B, Urlaub H, Schleiff E, Sloan KE, Deckers M, Lührmann R, Enderlein J, Klostermeier D, Rehling P, Bohnsack MT - RNA Biol (2016)

Bottom Line: We identify the orphan G-patch protein Cmg1 (YLR271W) as a novel cofactor of Prp43 and show that it stimulates the RNA binding and ATPase activity of the helicase.Interestingly, Cmg1 localizes to the cytoplasm and to the intermembrane space of mitochondria and its overexpression promotes apoptosis.Furthermore, our data reveal that different G-patch protein cofactors compete for interaction with Prp43.

View Article: PubMed Central - PubMed

Affiliation: a Institute for Molecular Biology, Georg-August University , Goettingen , Germany.

ABSTRACT
A rapidly increasing number of RNA helicases are implicated in several distinct cellular processes, however, the modes of regulation of multifunctional RNA helicases and their recruitment to different target complexes have remained unknown. Here, we show that the distribution of the multifunctional DEAH-box RNA helicase Prp43 between its diverse cellular functions can be regulated by the interplay of its G-patch protein cofactors. We identify the orphan G-patch protein Cmg1 (YLR271W) as a novel cofactor of Prp43 and show that it stimulates the RNA binding and ATPase activity of the helicase. Interestingly, Cmg1 localizes to the cytoplasm and to the intermembrane space of mitochondria and its overexpression promotes apoptosis. Furthermore, our data reveal that different G-patch protein cofactors compete for interaction with Prp43. Changes in the expression levels of Prp43-interacting G-patch proteins modulate the cellular localization of Prp43 and G-patch protein overexpression causes accumulation of the helicase in the cytoplasm or nucleoplasm. Overexpression of several G-patch proteins also leads to defects in ribosome biogenesis that are consistent with withdrawal of the helicase from this pathway. Together, these findings suggest that the availability of cofactors and the sequestering of the helicase are means to regulate the activity of multifunctional RNA helicases and their distribution between different cellular processes.

No MeSH data available.


Related in: MedlinePlus

Cmg1 specifically interacts with the RNA helicase Prp43. (A) Proteins were retrieved on IgG sepharose from extracts with or without tagged Cmg1, separated by SDS PAGE and stained with Coomassie. Inputs are shown on the left. Prp43 was identified in the Cmg1 eluate by mass spectrometry and Western blotting (bottom panel). The asterisk marks a background band also present in the control. (B) Recombinant Protein A-tagged Spp2 (as negative control) or Cmg1 were immobilised on IgG sepharose and incubated with purified GFP-tagged Prp43. After washing, co-purified GFP-Prp43 was eluted, then inputs and eluates were separated by SDS PAGE and analyzed by Coomassie staining. (C) Yeast two-hybrid analysis of Prp43 (fused to the GAL4 activation domain; GAL4-AD) was performed with full length (FL), the N-terminal G-patch domain (amino acids 1–85) or the C-terminus (amino acids 86–274) of Cmg1 (fused to the GAL4 binding domain; GAL4-BD), and Swm2 and Tgs1 as controls. The strains were spotted on plates not selecting (left) or selecting (right) for a yeast two-hybrid (Y2H) interaction. The domain structure of Cmg1, containing the G-patch domain and the domain of unidentified function (DUF4187) is shown at the top. (D) Protein A-tagged full length Prp43 or N- (92–767) or C- (1–657) terminally truncated versions of Prp43 were immobilised on IgG sepharose and incubated with MBP-tagged full length Cmg1 or Cmg1 1–85. After washing the beads, proteins were eluted, separated by SDS PAGE and analyzed by Commassie staining. Cmg1 and Cmg1 1–85 proteins co-precipitated with the different forms of Prp43 are indicated by the asterisk and double asterisk, respectively. For inputs of the binding experiments see Figure S1A.
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f0001: Cmg1 specifically interacts with the RNA helicase Prp43. (A) Proteins were retrieved on IgG sepharose from extracts with or without tagged Cmg1, separated by SDS PAGE and stained with Coomassie. Inputs are shown on the left. Prp43 was identified in the Cmg1 eluate by mass spectrometry and Western blotting (bottom panel). The asterisk marks a background band also present in the control. (B) Recombinant Protein A-tagged Spp2 (as negative control) or Cmg1 were immobilised on IgG sepharose and incubated with purified GFP-tagged Prp43. After washing, co-purified GFP-Prp43 was eluted, then inputs and eluates were separated by SDS PAGE and analyzed by Coomassie staining. (C) Yeast two-hybrid analysis of Prp43 (fused to the GAL4 activation domain; GAL4-AD) was performed with full length (FL), the N-terminal G-patch domain (amino acids 1–85) or the C-terminus (amino acids 86–274) of Cmg1 (fused to the GAL4 binding domain; GAL4-BD), and Swm2 and Tgs1 as controls. The strains were spotted on plates not selecting (left) or selecting (right) for a yeast two-hybrid (Y2H) interaction. The domain structure of Cmg1, containing the G-patch domain and the domain of unidentified function (DUF4187) is shown at the top. (D) Protein A-tagged full length Prp43 or N- (92–767) or C- (1–657) terminally truncated versions of Prp43 were immobilised on IgG sepharose and incubated with MBP-tagged full length Cmg1 or Cmg1 1–85. After washing the beads, proteins were eluted, separated by SDS PAGE and analyzed by Commassie staining. Cmg1 and Cmg1 1–85 proteins co-precipitated with the different forms of Prp43 are indicated by the asterisk and double asterisk, respectively. For inputs of the binding experiments see Figure S1A.

Mentions: The G-patch proteins Spp382/Ntr1, Sqs1/Pfa1 and Pxr1/Gno1 have been shown to modulate the functions of the RNA helicase Prp43 in splicing and ribosome biogenesis, and Spp2 regulates the activity of Prp2 in splicing. However, the helicase interaction partner of the remaining G-patch protein YLR271W (here named Cmg1) has remained unknown. We therefore performed pulldown experiments using a yeast strain expressing TAP-tagged Cmg1 and with a wildtype control strain. Interestingly, Cmg1 significantly co-precipitated a protein of about 90 kDa, which was identified by mass spectrometry as the RNA helicase Prp43 (Fig. 1A). The enrichment of Prp43 with TAP-tagged Cmg1 was also confirmed by Western blot analysis using antibodies recognizing Prp43 (Fig. 1A, lower panel). To further analyze the interaction, we purified recombinant Cmg1, Spp2 and Prp43 and performed binding experiments in vitro. Prp43 was co-purified with immobilised Cmg1, but not Spp2, demonstrating a direct and specific interaction of the two proteins (Fig. 1B). Using the yeast two-hybrid approach, we observed an in vivo interaction between Prp43 and both full length Cmg1 and the Cmg1 G-patch domain alone (amino acids 1–85; Fig. 1C). In contrast, neither the C-terminal part of Cmg1 (amino acids 86–274) nor Swm2 showed any interaction with Prp43, indicating that the G-patch domain of Cmg1 is required and sufficient for Prp43 interaction. The C-terminal OB-fold of Prp43 has been suggested to serve as a binding platform for other G-patch proteins.27,29,37 To determine whether the same region of Prp43 mediates the interaction with Cmg1, N- or C-terminally truncated versions of Prp43 (Prp43 92–767 and Prp43 1–657) were expressed recombinantly, purified and used in binding assays with MBP-tagged versions of full length Cmg1 and the Cmg1 G-patch domain (Cmg1 1–85). Consistent with the yeast two-hybrid analysis, both full length Cmg1 and Cmg1 1–85 bound to full length Prp43, whereas only a weak interaction was observed with either of the Prp43 truncations (Fig. 1D, Fig. S1A). This indicates that both the N- and C-terminus of Prp43 are required for efficient interaction with Cmg1, which is consistent with the close proximity of these domains as revealed by the crystal structure of Prp43,38 For the full length G-patch protein this is in contrast to the interaction of Sqs1 with Prp43 where full length Sqs1 is still able to bind to Prp43 1–657.38 However, the Sqs1 G-patch domain alone behaves similar to the G-patch domain of Cmg1 and hardly binds to Prp43 1–657. Importantly, these data suggest that several G-patch proteins have overlapping interaction sites on the RNA helicase.Figure 1.


Protein cofactor competition regulates the action of a multifunctional RNA helicase in different pathways.

Heininger AU, Hackert P, Andreou AZ, Boon KL, Memet I, Prior M, Clancy A, Schmidt B, Urlaub H, Schleiff E, Sloan KE, Deckers M, Lührmann R, Enderlein J, Klostermeier D, Rehling P, Bohnsack MT - RNA Biol (2016)

Cmg1 specifically interacts with the RNA helicase Prp43. (A) Proteins were retrieved on IgG sepharose from extracts with or without tagged Cmg1, separated by SDS PAGE and stained with Coomassie. Inputs are shown on the left. Prp43 was identified in the Cmg1 eluate by mass spectrometry and Western blotting (bottom panel). The asterisk marks a background band also present in the control. (B) Recombinant Protein A-tagged Spp2 (as negative control) or Cmg1 were immobilised on IgG sepharose and incubated with purified GFP-tagged Prp43. After washing, co-purified GFP-Prp43 was eluted, then inputs and eluates were separated by SDS PAGE and analyzed by Coomassie staining. (C) Yeast two-hybrid analysis of Prp43 (fused to the GAL4 activation domain; GAL4-AD) was performed with full length (FL), the N-terminal G-patch domain (amino acids 1–85) or the C-terminus (amino acids 86–274) of Cmg1 (fused to the GAL4 binding domain; GAL4-BD), and Swm2 and Tgs1 as controls. The strains were spotted on plates not selecting (left) or selecting (right) for a yeast two-hybrid (Y2H) interaction. The domain structure of Cmg1, containing the G-patch domain and the domain of unidentified function (DUF4187) is shown at the top. (D) Protein A-tagged full length Prp43 or N- (92–767) or C- (1–657) terminally truncated versions of Prp43 were immobilised on IgG sepharose and incubated with MBP-tagged full length Cmg1 or Cmg1 1–85. After washing the beads, proteins were eluted, separated by SDS PAGE and analyzed by Commassie staining. Cmg1 and Cmg1 1–85 proteins co-precipitated with the different forms of Prp43 are indicated by the asterisk and double asterisk, respectively. For inputs of the binding experiments see Figure S1A.
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Related In: Results  -  Collection

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f0001: Cmg1 specifically interacts with the RNA helicase Prp43. (A) Proteins were retrieved on IgG sepharose from extracts with or without tagged Cmg1, separated by SDS PAGE and stained with Coomassie. Inputs are shown on the left. Prp43 was identified in the Cmg1 eluate by mass spectrometry and Western blotting (bottom panel). The asterisk marks a background band also present in the control. (B) Recombinant Protein A-tagged Spp2 (as negative control) or Cmg1 were immobilised on IgG sepharose and incubated with purified GFP-tagged Prp43. After washing, co-purified GFP-Prp43 was eluted, then inputs and eluates were separated by SDS PAGE and analyzed by Coomassie staining. (C) Yeast two-hybrid analysis of Prp43 (fused to the GAL4 activation domain; GAL4-AD) was performed with full length (FL), the N-terminal G-patch domain (amino acids 1–85) or the C-terminus (amino acids 86–274) of Cmg1 (fused to the GAL4 binding domain; GAL4-BD), and Swm2 and Tgs1 as controls. The strains were spotted on plates not selecting (left) or selecting (right) for a yeast two-hybrid (Y2H) interaction. The domain structure of Cmg1, containing the G-patch domain and the domain of unidentified function (DUF4187) is shown at the top. (D) Protein A-tagged full length Prp43 or N- (92–767) or C- (1–657) terminally truncated versions of Prp43 were immobilised on IgG sepharose and incubated with MBP-tagged full length Cmg1 or Cmg1 1–85. After washing the beads, proteins were eluted, separated by SDS PAGE and analyzed by Commassie staining. Cmg1 and Cmg1 1–85 proteins co-precipitated with the different forms of Prp43 are indicated by the asterisk and double asterisk, respectively. For inputs of the binding experiments see Figure S1A.
Mentions: The G-patch proteins Spp382/Ntr1, Sqs1/Pfa1 and Pxr1/Gno1 have been shown to modulate the functions of the RNA helicase Prp43 in splicing and ribosome biogenesis, and Spp2 regulates the activity of Prp2 in splicing. However, the helicase interaction partner of the remaining G-patch protein YLR271W (here named Cmg1) has remained unknown. We therefore performed pulldown experiments using a yeast strain expressing TAP-tagged Cmg1 and with a wildtype control strain. Interestingly, Cmg1 significantly co-precipitated a protein of about 90 kDa, which was identified by mass spectrometry as the RNA helicase Prp43 (Fig. 1A). The enrichment of Prp43 with TAP-tagged Cmg1 was also confirmed by Western blot analysis using antibodies recognizing Prp43 (Fig. 1A, lower panel). To further analyze the interaction, we purified recombinant Cmg1, Spp2 and Prp43 and performed binding experiments in vitro. Prp43 was co-purified with immobilised Cmg1, but not Spp2, demonstrating a direct and specific interaction of the two proteins (Fig. 1B). Using the yeast two-hybrid approach, we observed an in vivo interaction between Prp43 and both full length Cmg1 and the Cmg1 G-patch domain alone (amino acids 1–85; Fig. 1C). In contrast, neither the C-terminal part of Cmg1 (amino acids 86–274) nor Swm2 showed any interaction with Prp43, indicating that the G-patch domain of Cmg1 is required and sufficient for Prp43 interaction. The C-terminal OB-fold of Prp43 has been suggested to serve as a binding platform for other G-patch proteins.27,29,37 To determine whether the same region of Prp43 mediates the interaction with Cmg1, N- or C-terminally truncated versions of Prp43 (Prp43 92–767 and Prp43 1–657) were expressed recombinantly, purified and used in binding assays with MBP-tagged versions of full length Cmg1 and the Cmg1 G-patch domain (Cmg1 1–85). Consistent with the yeast two-hybrid analysis, both full length Cmg1 and Cmg1 1–85 bound to full length Prp43, whereas only a weak interaction was observed with either of the Prp43 truncations (Fig. 1D, Fig. S1A). This indicates that both the N- and C-terminus of Prp43 are required for efficient interaction with Cmg1, which is consistent with the close proximity of these domains as revealed by the crystal structure of Prp43,38 For the full length G-patch protein this is in contrast to the interaction of Sqs1 with Prp43 where full length Sqs1 is still able to bind to Prp43 1–657.38 However, the Sqs1 G-patch domain alone behaves similar to the G-patch domain of Cmg1 and hardly binds to Prp43 1–657. Importantly, these data suggest that several G-patch proteins have overlapping interaction sites on the RNA helicase.Figure 1.

Bottom Line: We identify the orphan G-patch protein Cmg1 (YLR271W) as a novel cofactor of Prp43 and show that it stimulates the RNA binding and ATPase activity of the helicase.Interestingly, Cmg1 localizes to the cytoplasm and to the intermembrane space of mitochondria and its overexpression promotes apoptosis.Furthermore, our data reveal that different G-patch protein cofactors compete for interaction with Prp43.

View Article: PubMed Central - PubMed

Affiliation: a Institute for Molecular Biology, Georg-August University , Goettingen , Germany.

ABSTRACT
A rapidly increasing number of RNA helicases are implicated in several distinct cellular processes, however, the modes of regulation of multifunctional RNA helicases and their recruitment to different target complexes have remained unknown. Here, we show that the distribution of the multifunctional DEAH-box RNA helicase Prp43 between its diverse cellular functions can be regulated by the interplay of its G-patch protein cofactors. We identify the orphan G-patch protein Cmg1 (YLR271W) as a novel cofactor of Prp43 and show that it stimulates the RNA binding and ATPase activity of the helicase. Interestingly, Cmg1 localizes to the cytoplasm and to the intermembrane space of mitochondria and its overexpression promotes apoptosis. Furthermore, our data reveal that different G-patch protein cofactors compete for interaction with Prp43. Changes in the expression levels of Prp43-interacting G-patch proteins modulate the cellular localization of Prp43 and G-patch protein overexpression causes accumulation of the helicase in the cytoplasm or nucleoplasm. Overexpression of several G-patch proteins also leads to defects in ribosome biogenesis that are consistent with withdrawal of the helicase from this pathway. Together, these findings suggest that the availability of cofactors and the sequestering of the helicase are means to regulate the activity of multifunctional RNA helicases and their distribution between different cellular processes.

No MeSH data available.


Related in: MedlinePlus