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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.

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The interaction of Brr2 with RNA and the effect of Prp8-CTR on these interactions. (a) EMSA demonstrates that Hel308-II does not bind a 13nt ss or dsRNA with arbitrary sequence (GAGAUUUAUUUCG) in the absence or presence of ATP. UAP56, another DExD/H-box protein involved in splicing, is used as a positive control and binds both ss and dsRNA in the presence of ATP. (b) Hel308 (lanes 1–4) and full-length Brr2 (lanes 5–10) do not bind U4/U6 at indicated concentrations but the presence of Prp8-CTR generate a complex that binds U4/U6 better. Supershift experiments using anti-Brr2 and anti-Prp8 antibodies confirm the identity of the shifted bands (lanes 11–13). (c) Titration experiments indicate that Prp8-CTR binds U4/U6 with a Kd of 2.2±0.2 µM. Error bars and errors in Kd are s.d. d) Prp8-CTR binds ss or dsR13 very weakly.
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Figure 4: The interaction of Brr2 with RNA and the effect of Prp8-CTR on these interactions. (a) EMSA demonstrates that Hel308-II does not bind a 13nt ss or dsRNA with arbitrary sequence (GAGAUUUAUUUCG) in the absence or presence of ATP. UAP56, another DExD/H-box protein involved in splicing, is used as a positive control and binds both ss and dsRNA in the presence of ATP. (b) Hel308 (lanes 1–4) and full-length Brr2 (lanes 5–10) do not bind U4/U6 at indicated concentrations but the presence of Prp8-CTR generate a complex that binds U4/U6 better. Supershift experiments using anti-Brr2 and anti-Prp8 antibodies confirm the identity of the shifted bands (lanes 11–13). (c) Titration experiments indicate that Prp8-CTR binds U4/U6 with a Kd of 2.2±0.2 µM. Error bars and errors in Kd are s.d. d) Prp8-CTR binds ss or dsR13 very weakly.

Mentions: We next examined whether Hel308-II retains the ability to bind to RNA, even though it lacks ATPase and helicase activities. We demonstrated using Electrophoresis Mobility Shift Assay (EMSA) that purified Hel308-II does not bind a 13nt ss or ds RNA (GAGAUUUAUUUCG, arbitrary sequence, designated as ssR13 and dsR13) even at a concentration of 50 µM, in the absence or presence of ATP (Fig. 4a). Under similar conditions, the positive control UAP56 (a splicing factor and DEAD -box protein) clearly binds RNA at 10 µM concentration in the presence of ATP (ATP is known to increase the RNA binding affinity of many DEAD-box proteins including eIF4A 30,34) (Fig. 4a). Nor does Hel308-II have detectable binding to U4/U6 at 5 µM protein concentration in the presence or absence of ATP (Fig. 4b, lanes 1 and 2).


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)

The interaction of Brr2 with RNA and the effect of Prp8-CTR on these interactions. (a) EMSA demonstrates that Hel308-II does not bind a 13nt ss or dsRNA with arbitrary sequence (GAGAUUUAUUUCG) in the absence or presence of ATP. UAP56, another DExD/H-box protein involved in splicing, is used as a positive control and binds both ss and dsRNA in the presence of ATP. (b) Hel308 (lanes 1–4) and full-length Brr2 (lanes 5–10) do not bind U4/U6 at indicated concentrations but the presence of Prp8-CTR generate a complex that binds U4/U6 better. Supershift experiments using anti-Brr2 and anti-Prp8 antibodies confirm the identity of the shifted bands (lanes 11–13). (c) Titration experiments indicate that Prp8-CTR binds U4/U6 with a Kd of 2.2±0.2 µM. Error bars and errors in Kd are s.d. d) Prp8-CTR binds ss or dsR13 very weakly.
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Figure 4: The interaction of Brr2 with RNA and the effect of Prp8-CTR on these interactions. (a) EMSA demonstrates that Hel308-II does not bind a 13nt ss or dsRNA with arbitrary sequence (GAGAUUUAUUUCG) in the absence or presence of ATP. UAP56, another DExD/H-box protein involved in splicing, is used as a positive control and binds both ss and dsRNA in the presence of ATP. (b) Hel308 (lanes 1–4) and full-length Brr2 (lanes 5–10) do not bind U4/U6 at indicated concentrations but the presence of Prp8-CTR generate a complex that binds U4/U6 better. Supershift experiments using anti-Brr2 and anti-Prp8 antibodies confirm the identity of the shifted bands (lanes 11–13). (c) Titration experiments indicate that Prp8-CTR binds U4/U6 with a Kd of 2.2±0.2 µM. Error bars and errors in Kd are s.d. d) Prp8-CTR binds ss or dsR13 very weakly.
Mentions: We next examined whether Hel308-II retains the ability to bind to RNA, even though it lacks ATPase and helicase activities. We demonstrated using Electrophoresis Mobility Shift Assay (EMSA) that purified Hel308-II does not bind a 13nt ss or ds RNA (GAGAUUUAUUUCG, arbitrary sequence, designated as ssR13 and dsR13) even at a concentration of 50 µM, in the absence or presence of ATP (Fig. 4a). Under similar conditions, the positive control UAP56 (a splicing factor and DEAD -box protein) clearly binds RNA at 10 µM concentration in the presence of ATP (ATP is known to increase the RNA binding affinity of many DEAD-box proteins including eIF4A 30,34) (Fig. 4a). Nor does Hel308-II have detectable binding to U4/U6 at 5 µM protein concentration in the presence or absence of ATP (Fig. 4b, lanes 1 and 2).

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