DEAH-box ATPase Prp16 has dual roles in remodeling of the spliceosome in catalytic steps

Abstract

The assembly of the spliceosome involves dynamic rearrangements of interactions between snRNAs, protein components, and the pre-mRNA substrate. DExD/H-box ATPases are required to mediate structural changes of the spliceosome, utilizing the energy of ATP hydrolysis. Two DExD/H-box ATPases are required for the catalytic steps of the splicing pathway, Prp2 for the first step and Prp16 for the second step, both belonging to the DEAH subgroup of the protein family. The detailed mechanism of their action was not well understood until recently, when Prp2 was shown to be required for the release of U2 components SF3a and SF3b, presumably to allow the binding of Cwc25 to promote the first transesterification reaction. We show here that Cwc25 and Yju2 are released after the reaction in Prp16- and ATP-dependent manners, possibly to allow for the binding of Prp22, Prp18, and Slu7 to promote the second catalytic reaction. The binding of Cwc25 to the spliceosome is destabilized by mutations at the branchpoint sequence, suggesting that Cwc25 may bind to the branch site. We also show that Prp16 has an ATP-independent role in the first catalytic step, in addition to its known role in the second step. In the absence of ATP, Prp16 stabilizes the binding of Cwc25 to the spliceosome formed with branchpoint mutated pre-mRNAs to facilitate their splicing. Our results uncovered novel functions of Prp16 in both catalytic steps, and provide mechanistic insights into splicing catalysis.

FIGURE 1.

Scheme for the spliceosome pathway, divided into spliceosome assembly, spliceosome activation, catalysis (reactions 1 and 2), and spliceosome disassembly steps. Yju2 is shown joining the spliceosome with NTC or in the first catalytic step. Prp16, Yju2, and Cwc25 are highlighted for they are the focus of this work.

FIGURE 2.

Yju2 and Cwc25 were accumulated on the spliceosome in the absence of Prp16 function. (A) Splicing was carried out using ACAC pre-mRNA in mock-treated (lanes 1–5) or Prp16-depleted extract (lanes 6–10), and the reaction mixtures were precipitated with antibodies against Ntc20, Prp16, Yju2, or Cwc25. RXN: 1/10 of the reaction mixture. (B,C) Western blotting of the spliceosome formed with biotinylated ACAC pre-mRNA and pulled-down with streptavidin Sepharose. (B) Splicing was carried out in mock-treated (lanes 1–4) or Prp16-depleted (lanes 5–8) extracts, +/− recombinant Prp16. (C) Splicing without (lanes 1,2) or with the addition of wild-type Prp16 (lanes 3,4) or prp16_D473A mutant protein (lanes 5,6). dPrp16: Prp16-depletion; Sub: substrate.

FIGURE 3.

Prp16 mediates the release of Yju2 and Cwc25. Splicing was carried out for 10 min with wild-type actin transcript in Prp16-depleted extracts, and ATP was depleted by incubation for 5 min upon the addition of 10 mM glucose. After addition or no addition of recombinant Prp16, the reaction mixtures were precipitated with antibody against Ntc20 (lanes 2–6), Yju2 (lanes 8–12,14–18) or Cwc25 (lanes 20–24,26–30). The precipitated spliceosome was reincubated in the presence or absence of ATP, and the pellet and supernatant fractions were separated. The amount of RNA was quantified by PhosphoImager and plotted in a bar graph with the sum of supernatant and pellet as 100%. The x-axis indicates lane numbers of the gel. R: 1/10 of the reaction mixture; SP: spliceosome; T: total precipitate; P: pellet; S: supernatant.

FIGURE 4.

Mutations at the branchpoint destabilized the association of Cwc25 with the spliceosome. (A) Splicing was carried out with ACAC (lanes 1–3), brG (lanes 4–6), or brC (lanes 7–9) pre-mRNA and the reaction mixtures were precipitated with anti-Ntc20 or anti-Prp22 antibody. R: 1/10 of the reaction mixture; α-20: anti-Ntc20 antibody; α-22: anti-Prp22 antibody. (B) Spliceosome formed with biotinylated ACAC, brG, or brC pre-mRNA was precipitated with streptavidin Sepharose followed by Western blotting. M: mock; d16: Prp16 depletion.

FIGURE 5.

Prp16 facilitated splicing of brG and brC pre-mRNA. (A) Splicing was carried out with wild-type (lanes 1,2), ACAC (lanes 3,4), brG (lanes 5,6), or brC (lanes 7,8) pre-mRNA in mock-treated (lanes 1,3,5,7) or Prp16-depleted (lanes 2,4,6,8) extracts. dPrp16: Prp16 depletion; M: mock; d: depletion. (B) Splicing was carried out with wild-type (lanes 1–4), brG (lanes 5–8), or brC (lanes 7,8) pre-mRNA in mock-treated (lanes 1,5,9) or Prp16-depleted (lanes 2–4,6–8,10–12) extracts without (lanes 2,6,10) or with the addition of wild-type (lanes 3,7,11) or D473A mutant (lanes 4,8,12) of Prp16.

FIGURE 6.

Cwc25-dependent association of Prp16 with the pre-catalytic spliceosome. (A) Splicing was carried out for 30 min with ACAC, brG, or brC pre-mRNA in Cwc25-HA extracts, and the reaction mixtures were separated into two aliquots. Glucose was added to one aliquot at 10 mM and incubated for 5 min (lanes 5–8,13–16,21–24). All mixtures were precipitated with anti-Ntc20, anti-HA, or anti-Prp16 antibody. (B) Splicing was carried out with brC pre-mRNA in mock-treated (lanes 1–6) or Cwc25-depleted (lanes 7–12) Prp16-HA extracts, and the reaction mixtures were separated into two aliquots. Glucose was added to one aliquot at 10 mM and incubated for 5 min (lanes 4–6,10–12). All mixtures were precipitated with anti-Ntc20 or anti-HA antibody. (C) Splicing was carried out with brC pre-mRNA in Cwc25-depleted, Prp16-HA extracts, and the reaction mixtures were separated into two aliquots. Glucose was added (lanes 7–12) or not added (lanes 1–6) to each aliquot at 10 mM and incubated for 5 min, and each mixture was separated into two aliquots. Recombinant Cwc25 was added (lanes 4–6,10–12) or not added (lanes 1–3,7–9) to each aliquot, and further incubated for 10 min. All mixtures were precipitated with anti-Ntc20 or anti-HA antibody. ΔATP: ATP depletion; RXN: 1/50 of reaction mixture; α-20: 1/5 of precipitates from anti-Ntc20 antibody; dCwc25: Cwc25 depletion.

FIGURE 7.

Schematic of the interplay between Prp16 and Cwc25 in the first catalytic step. After Prp2-mediated dissociation of SF3a/b, Yju2 and Cwc25 bind to the spliceosome to promote the first catalytic reaction. Prp16 binds to the spliceosome after the binding of Yju2 and Cwc25, and mediate their dissociation after the transesterification reaction if the reaction is fast, or before transesterification if the reaction is slow, as in the case of brC mutant. The D473A mutant of Prp16 can stabilize the association of Cwc25 with the spliceosome to promote the transesterification reaction.