Limits...
Structural evidence for consecutive Hel308-like modules in the spliceosomal ATPase Brr2.

Zhang L, Xu T, Maeder C, Bud LO, Shanks J, Nix J, Guthrie C, Pleiss JA, Zhao R - Nat. Struct. Mol. Biol. (2009)

Bottom Line: We demonstrate that Hel308-II interacts with Prp8 and Snu114 in vitro and in vivo.We further find that the C-terminal region of Prp8 (Prp8-CTR) facilitates the binding of the Brr2-Prp8-CTR complex to U4/U6.Our results have important implications for the mechanism and regulation of Brr2's activity in splicing.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, Colorado, USA.

ABSTRACT
Brr2 is a DExD/H-box helicase responsible for U4/U6 unwinding during spliceosomal activation. Brr2 contains two helicase-like domains, each of which is followed by a Sec63 domain with unknown function. We determined the crystal structure of the second Sec63 domain, which unexpectedly resembles domains 4 and 5 of DNA helicase Hel308. This, together with sequence similarities between Brr2's helicase-like domains and domains 1-3 of Hel308, led us to hypothesize that Brr2 contains two consecutive Hel308-like modules (Hel308-I and Hel308-II). Our structural model and mutagenesis data suggest that Brr2 shares a similar helicase mechanism with Hel308. We demonstrate that Hel308-II interacts with Prp8 and Snu114 in vitro and in vivo. We further find that the C-terminal region of Prp8 (Prp8-CTR) facilitates the binding of the Brr2-Prp8-CTR complex to U4/U6. Our results have important implications for the mechanism and regulation of Brr2's activity in splicing.

Show MeSH

Related in: MedlinePlus

Hel308-II interacts with Prp8 and Snu114 in vitro and in vivo. (a) GST pull down experiments demonstrate that the H2, S2, and Hel308-II domains interact with Prp8 and Snu114. (b) Co-immunoprecipitation experiments demonstrate that the S2-deletion strain has similar level of Brr2 but lower level of Prp8 and Snu114 than WT strains. Brr2 in yeast extract was pulled down using anti-polyoma antibody and probed in Western blot using anti-Brr2, anti-Prp8 and anti-Snu114 antibodies. Error bars indicate s.d.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2743687&req=5

Figure 5: Hel308-II interacts with Prp8 and Snu114 in vitro and in vivo. (a) GST pull down experiments demonstrate that the H2, S2, and Hel308-II domains interact with Prp8 and Snu114. (b) Co-immunoprecipitation experiments demonstrate that the S2-deletion strain has similar level of Brr2 but lower level of Prp8 and Snu114 than WT strains. Brr2 in yeast extract was pulled down using anti-polyoma antibody and probed in Western blot using anti-Brr2, anti-Prp8 and anti-Snu114 antibodies. Error bars indicate s.d.

Mentions: We then evaluated whether the Hel308-II module can potentially function as a protein interaction domain and mediate interactions with other spliceosomal proteins. We performed in vitro GST pull down experiments using GST-fused versions of several Brr2 domains, Prp8 domains, and Snu114 as baits, and HA-tagged Brr2 H2, S2, or Hel308-II domains as prey (Fig. 5a). These experiments demonstrate that the H2 and S2 domain of Brr2 interact with each other, consistent with the idea that the H2 and S2 domains come together to form a Hel308 module. Furthermore, the H2, S2, and Hel308-II domains all interact with both the N-terminal (residues 233–518) and C-terminal (Prp8-CTR, residues 1822–2395) regions of Prp8, as well as with Snu114. These interactions are specific as no interaction is detected with GST alone. Our results are consistent with previous observations using yeast two-hybrid analyses that the C-terminal region of yBrr2 (a construct containing the combination of residues 112–356 and 1184–2163) is responsible for the vast majority of interactions between Brr2 and many spliceosomal proteins including Prp8 (ref. 35). The interactions between the H2 domain of hBrr2 and hPrp8 as well as hSnu114 have also been observed previously using yeast two-hybrid analyses 36,37.


Structural evidence for consecutive Hel308-like modules in the spliceosomal ATPase Brr2.

Zhang L, Xu T, Maeder C, Bud LO, Shanks J, Nix J, Guthrie C, Pleiss JA, Zhao R - Nat. Struct. Mol. Biol. (2009)

Hel308-II interacts with Prp8 and Snu114 in vitro and in vivo. (a) GST pull down experiments demonstrate that the H2, S2, and Hel308-II domains interact with Prp8 and Snu114. (b) Co-immunoprecipitation experiments demonstrate that the S2-deletion strain has similar level of Brr2 but lower level of Prp8 and Snu114 than WT strains. Brr2 in yeast extract was pulled down using anti-polyoma antibody and probed in Western blot using anti-Brr2, anti-Prp8 and anti-Snu114 antibodies. Error bars indicate s.d.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2743687&req=5

Figure 5: Hel308-II interacts with Prp8 and Snu114 in vitro and in vivo. (a) GST pull down experiments demonstrate that the H2, S2, and Hel308-II domains interact with Prp8 and Snu114. (b) Co-immunoprecipitation experiments demonstrate that the S2-deletion strain has similar level of Brr2 but lower level of Prp8 and Snu114 than WT strains. Brr2 in yeast extract was pulled down using anti-polyoma antibody and probed in Western blot using anti-Brr2, anti-Prp8 and anti-Snu114 antibodies. Error bars indicate s.d.
Mentions: We then evaluated whether the Hel308-II module can potentially function as a protein interaction domain and mediate interactions with other spliceosomal proteins. We performed in vitro GST pull down experiments using GST-fused versions of several Brr2 domains, Prp8 domains, and Snu114 as baits, and HA-tagged Brr2 H2, S2, or Hel308-II domains as prey (Fig. 5a). These experiments demonstrate that the H2 and S2 domain of Brr2 interact with each other, consistent with the idea that the H2 and S2 domains come together to form a Hel308 module. Furthermore, the H2, S2, and Hel308-II domains all interact with both the N-terminal (residues 233–518) and C-terminal (Prp8-CTR, residues 1822–2395) regions of Prp8, as well as with Snu114. These interactions are specific as no interaction is detected with GST alone. Our results are consistent with previous observations using yeast two-hybrid analyses that the C-terminal region of yBrr2 (a construct containing the combination of residues 112–356 and 1184–2163) is responsible for the vast majority of interactions between Brr2 and many spliceosomal proteins including Prp8 (ref. 35). The interactions between the H2 domain of hBrr2 and hPrp8 as well as hSnu114 have also been observed previously using yeast two-hybrid analyses 36,37.

Bottom Line: We demonstrate that Hel308-II interacts with Prp8 and Snu114 in vitro and in vivo.We further find that the C-terminal region of Prp8 (Prp8-CTR) facilitates the binding of the Brr2-Prp8-CTR complex to U4/U6.Our results have important implications for the mechanism and regulation of Brr2's activity in splicing.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, Colorado, USA.

ABSTRACT
Brr2 is a DExD/H-box helicase responsible for U4/U6 unwinding during spliceosomal activation. Brr2 contains two helicase-like domains, each of which is followed by a Sec63 domain with unknown function. We determined the crystal structure of the second Sec63 domain, which unexpectedly resembles domains 4 and 5 of DNA helicase Hel308. This, together with sequence similarities between Brr2's helicase-like domains and domains 1-3 of Hel308, led us to hypothesize that Brr2 contains two consecutive Hel308-like modules (Hel308-I and Hel308-II). Our structural model and mutagenesis data suggest that Brr2 shares a similar helicase mechanism with Hel308. We demonstrate that Hel308-II interacts with Prp8 and Snu114 in vitro and in vivo. We further find that the C-terminal region of Prp8 (Prp8-CTR) facilitates the binding of the Brr2-Prp8-CTR complex to U4/U6. Our results have important implications for the mechanism and regulation of Brr2's activity in splicing.

Show MeSH
Related in: MedlinePlus