New mechanistic insights into Prp22-mediated exon ligation and mRNA release

Abstract

The DExD/H-box RNA helicase Prp22 catalyzes messenger RNA (mRNA) release from the spliceosome, and has also been implicated in proofreading the 3' splice site (3'SS), preventing exon ligation of mutant pre-mRNAs through an ATP-dependent mechanism. However, here we reveal an unexpected role for Prp22 in promoting exon ligation of both wild-type and mutant pre-mRNAs by stabilizing Slu7's association with the spliceosome prior to exon ligation. Notably, ATP binding, rather than hydrolysis, by Prp22 inhibits exon ligation of 3'SS mutant pre-mRNA. Following exon ligation, Prp22-mediated ATP hydrolysis facilitates the dissociation of both Slu7 and mRNA from the spliceosome. Remarkably, Prp22 and Cwc22, which bind the 3'- and 5'-exons respectively, remain associated with the released mRNA, whereas Slu7 and Fyv6 dissociate independently. We propose that Prp22 facilitates exon ligation by stabilizing Slu7 binding, with binding of ATP by Prp22 potentially destabilizing that interaction, thereby weakening contacts between the 5'-exon and the 3'SS to inhibit exon ligation. After exon ligation, Prp22-driven ATP hydrolysis induces a conformational change in Prp8 that disrupts its interdomain interactions, enabling mRNA release through the domain interfaces, with Prp22 and Cwc22 remaining associated with the released mRNA.

Graphical Abstract

Figure 1.

The architecture of Prp8, Slu7, Prp18, Prp22, Cwc22, and mRNA in the spliceosome P complex. The circle indicates the catalytic center of the spliceosome.

Figure 2.

Prp22 promotes aberrant 3′SS selection in splicing of 3′SS-mutated of ACT1 pre-mRNA. (A) Sequence of the BS-3′SS regions of ACT1 and ACAC pre-mRNA. The BS is boxed, the BP and conserved intronic 3′SS dinucleotides are in bold, and nucleotide changes at the 3′SS in ACAC are underlined. (B) Schematic of the experimental flow chart. (C) Splicing was performed with ACAC pre-mRNA for 20 min. Following the addition (lanes 6–10) or not (lanes 1–5) of 10 mM glucose, the reaction mixtures were further incubated for the indicated time frames. (D) Splicing was performed with ACAC pre-mRNA for 20 min in mock-treated (lane 1) or Prp22-depleted (lane 4) extracts. Following the addition (lanes 3 and 6) or not (lanes 2 and 5) of 10 mM glucose, the reaction mixtures were further incubated for 60 min.

Figure 3.

Prp22 does not promote release of Slu7 from the spliceosome before exon ligation. (A) Splicing was performed with ACAC pre-mRNA for 20 min and spliceosomes were precipitated with anti-Ntc20 antibody. Spliceosome were further incubated for 0–60 min without (lanes 1–5) or with (lanes 6–10) the addition of 2 mM ATP. (B) Splicing was performed with ACAC pre-mRNA in Slu7-V5 extracts, and spliceosomes were isolated by precipitation with anti-Ntc20 (lanes 1–5), anti-Prp22 (lanes 6–10), or anti-V5 (lanes 11–15) antibody. The spliceosomes were then incubated in the presence or absence of ATP, after which the supernatant and pellet fractions were separated. T, total; P, pellet; S, supernatant.

Figure 4.

Prp22 is associated with the mRNA upon mRNA release from the spliceosome, whereas both Slu7 and Fyv6 dissociate independently. (A) Western blotting and splicing reactions of extracts depleted of no protein (lane 1), Prp16 (lane 2), Slu7 (lane 3), or Prp22 (lane 4). (B) Schematic of the experimental flow chart. (C) Splicing was performed with wild-type ACT1 pre-mRNA using Prp22-depleted Slu7-V5 extracts. Following ATP depletion, recombinant Prp22 was added to the reaction mixtures, and the spliceosomes were precipitated with anti-Ntc20 (lanes 1–5), anti-Prp22 (lanes 6–10), or anti-V5 (lanes 11–15) antibody. The spliceosomes were then incubated in the absence or presence of ATP, and the supernatant and pellet fractions were separated. T, total; P, pellet; S, supernatant. (D) Western blotting of Fyv6-V5/Prp18-HA double-tagged extracts following incubation with PAS (lanes 1 and 4), anti-HA (lanes 2 and 5), or anti-V5 (lanes 3 and 6) antibody and separation of the supernatant and pellet fractions. Sup, supernatant; PAS, protein-A Sepharose. (E) Splicing was performed with wild-type ACT1 pre-mRNA using Prp22-depleted Fyv6-V5/Prp18-HA double-tagged extracts. Following ATP depletion, recombinant Prp22 was added to the reaction mixtures, and spliceosomes were precipitated with anti-Ntc20 (lanes 1–5), anti-V5 (lanes 6–10), or anti-HA (lanes 11–15) antibody. The spliceosomes were then incubated in the absence or presence of ATP, and the supernatant and pellet fractions were separated. T, total; P, pellet; S, supernatant.

Figure 5.

Cwc22 is released from the spliceosome in association with mRNA. (A) Crosslinking of Cwc22 to the mature mRNA. Splicing was performed using Cwc22-HA extracts in the presence of V5-tagged recombinant Prp22-S635A mutant protein, followed by UV_{254 nm} irradiation. After denaturation, the reaction mixtures were diluted tenfold with NET-2 buffer containing 300-mM NaCl and immunoprecipitated with anti-Ntc20, anti-Prp8, anti-Prp22, or anti-HA antibody. Denat, denaturation; C22, Cwc22. (B) Splicing was performed with ACAC pre-mRNA using Cwc22-HA extracts, followed by UV_{254 nm} irradiation. Reaction mixtures were treated as in panel (A) prior to immunoprecipitation. Denat, denaturation; C22, Cwc22. (C) Splicing was performed with wild-type ACT1 pre-mRNA in Prp22-depleted extracts. After ATP depletion, recombinant Prp22 was added to the reaction mixtures, and the spliceosomes were isolated by precipitation with anti-Ntc20 (lanes 1–5), anti-Prp22 (lanes 6–10), or anti-Cwc22 (lanes 11–15) antibody. The spliceosomes were then incubated in the presence or absence of ATP, after which the supernatant and pellet fractions were separated. T, total; P, pellet; S, supernatant.

Figure 6.

Prp22 stabilizes Slu7 on the spliceosome prior to but not after exon ligation. (A) Splicing was performed with ACAC pre-mRNA in Slu7-V5 extracts that had been mock-treated (lanes 1–4), depleted of Slu7 (lanes 5–12), or depleted of Prp22 (lanes 13–20), supplementing with recombinant Slu7 (lanes 9–12) or Prp22 (lanes 17–20). The reaction mixtures were then precipitated with anti-Ntc20, anti-V5 or anti-Prp22 antibody. α-20, anti-Ntc20 antibody; α-22, anti-Prp22 antibody. (B) Splicing was performed with ACAC (lane 1–8) or wild-type ACT1 pre-mRNA (lanes 9–12) in Slu7-V5 extracts that had been mock-treated (lanes 1–4) or depleted of Prp22 (lanes 5–12). The reaction mixtures were then precipitated with anti-Ntc20, anti-V5, or anti-Prp22 antibody. α-20, anti-Ntc20 antibody; α-22, anti-Prp22 antibody. (C) Bar graph summarizing quantification of the results from panel (B), displaying the percentages of RNA precipitated by anti-V5 or anti-Prp22 antibody relative to that precipitated by anti-Ntc20 antibody. The amounts of lariat-intron-exon 2 and lariat-intron were measured for ACAC and wild-type pre-mRNA, respectively. Data represent mean values from three experiments with standard deviation (SD) indicated.

Figure 7.

Prp22 accelerates the exon ligation reaction of wild-type pre-mRNA. (A) Splicing was performed with ACT1 pre-mRNA for 20 min in extracts depleted of Slu7 and Prp22 (lane 1). Following re-addition of Slu7–Prp18 without (lanes 3–6) or along with (lanes 7–10) Prp22, the reaction mixtures were further incubated for 1–10 min. (B) Graph of the exon ligation reaction kinetics from panel (A), calculated as the molar ratio of mRNA to the total of pre-mRNA, lariat-IVS-E2 and mRNA in each reaction. Data represent mean values from three independent experiments, with SD indicated.

Figure 8.

ATP hydrolysis is not required for inhibition of exon ligation. Splicing was performed with ACAC pre-mRNA for 20 min and spliceosomes were precipitated with anti-Ntc20 antibody. Spliceosome were further incubated for 20–60 min without (lanes 2–4) or with the addition of 2 mM ATP (lanes 5–7), ATPγS (lanes 8–10), AMP-PNP (lanes 11–13), or ADP (lanes 14–16).