Dynamic interactions of Ntr1-Ntr2 with Prp43 and with U5 govern the recruitment of Prp43 to mediate spliceosome disassembly

Abstract

The Saccharomyces cerevisiae splicing factors Ntr1 (also known as Spp382) and Ntr2 form a stable complex and can further associate with DExD/H-box RNA helicase Prp43 to form a functional complex, termed the NTR complex, which catalyzes spliceosome disassembly. We show that Prp43 interacts with Ntr1-Ntr2 in a dynamic manner. The Ntr1-Ntr2 complex can also bind to the spliceosome first, before recruiting Prp43 to catalyze disassembly. Binding of Ntr1-Ntr2 or Prp43 does not require ATP, but disassembly of the spliceosome requires hydrolysis of ATP. The NTR complex also dynamically interacts with U5 snRNP. Ntr2 interacts with U5 component Brr2 and is essential for both interactions of NTR with U5 and with the spliceosome. Ntr2 alone can also bind to U5 and to the spliceosome, suggesting a role of Ntr2 in mediating the binding of NTR to the spliceosome through its interaction with U5. Our results demonstrate that dynamic interactions of NTR with U5, through the interaction of Ntr2 with Brr2, and interactions of Ntr1 and Prp43 govern the recruitment of Prp43 to the spliceosome to mediate spliceosome disassembly.

FIG. 1.

ATP-independent binding of NTR complex to the spliceosome. α-Ntr1, anti-Ntr1 antibody. (A) Scheme of assaying binding of NTR complex to the spliceosome. (B) Splicing reactions were carried out in wild-type (lanes 1 to 5) or Ntr1-depleted extracts (lanes 6 to 15) at 25°C for 30 min, then ATP (lanes 6 to 10) or glucose (lanes 11 to 15) was added to a final concentration of 2 mM or 2%, respectively, and the mixtures were further incubated for 10 min. Affinity-purified NTR complex was then added to the reaction mixtures and incubated for a further 10 minutes. The reaction mixtures were then precipitated with anti-Ntc20, anti-Ntr1, anti-Ntr2, or anti-V5 antibody, and RNA was isolated and analyzed by electrophoresis on 8% polyacrylamide-8 M urea gels. dNTR, Ntr1-depleted extracts; RXN, reaction. (C) Precipitates described for panel B (lanes 2, 7, 12, and 17) were incubated at 25°C for 5 min in the presence (lanes 5, 6, 10, 11, 15, 16, 20, and 21) or absence (lanes 3, 4, 8, 9, 13, 14, 18, and 19) of ATP, and supernatant and pellet fractions were collected. RNA was isolated and analyzed by electrophoresis on 8% polyacrylamide-8 M urea gels. R, reaction; T, total precipitate; P, pellet; S, supernatant. (D) Supernatant fractions shown in lanes 11 and 21 in panel C were sedimented on 10 to 30% glycerol gradients.

FIG. 2.

Ntr1 and Ntr2 could bind to the spliceosome independently of Prp43. (A) Splicing reactions were carried out in mock-depleted (lane 1 to 5) or Prp43-depleted Prp43-V5 extracts (lanes 6 to 10) and then precipitated with anti-Ntc20 (α-Ntc20), anti-Ntr1 (α-Ntr1), anti-Ntr2 (α-Ntr2), or anti-V5 (α-V5) antibody. RNA was isolated and analyzed by electrophoresis on 8% polyacrylaminde-8 M urea gels. dPrp43, Prp43-depleted extracts; RXN, reaction. (B) Precipitates with anti-Ntr1 antibody shown in lane 8 of panel A were incubated with (lanes 7, 8, 9, and 10) or without (lanes 3 to 6) Prp43 or with the D215A mutant of Prp43 (lanes 11 and 12) in the presence (lanes 5, 6, and 9 to 12) or absence (lanes 3, 4, 7, and 8) of ATP. Supernatant and pellet fractions were collected, and RNA was isolated and analyzed by electrophoresis on 8% polyacrylamide-8 M urea gels. Percentages of supernatant and pellet fractions are also presented in a bar graph with the sum of supernatant and pellet as 100%. R, reaction; T, total precipitate; P, pellet; S, supernatant; D215A, D215A mutant Prp43 protein.

FIG. 3.

Dynamic interactions of Prp43 with Ntr1-Ntr2. (A) Extracts prepared from Ntr1-HA- and Prp43-V5-tagged strains were precipitated with anti-HA antibody (lane 1). The precipitates were then incubated in buffer DK alone (lanes 2 and 3) or with 2 mM ATP (lanes 4 and 5) at 25°C for 20 min, and supernatant and pellet fractions were collected for Western blotting, probing with antibodies against V5, Ntr1, and Ntr2. T, total precipitate; P, pellet; S, supernatant. (B) Extracts prepared from Ntr1-HA-tagged strain were fractionated on an anti-HA antibody-conjugated protein A-Sepharose column. After being washed, the beads were divided into two aliquots and transferred to Eppendorf tubes. One tube was incubated with buffer DK containing 2 mM ATP at room temperature for 20 min, and this was repeated once. Both tubes were then eluted with the HA-tagged peptide, and elution was repeated four times. Individual fractions were then Western blotted with antibodies against Ntr1, Ntr2, and Prp43. T, total precipitate; FT, flowthrough. (C) Ntr1-depleted extracts (ΔNTR Ext), prepared from Ntr1-HA- and Prp43-V5-tagged strains, were supplemented with affinity-purified Ntr1-Ntr2 complex and immunoprecipitated with anti-Ntr1 antibody (α-Ntr1) following incubation in the presence or absence of ATP. The precipitate was analyzed by Western blotting, probing with anti-V5 antibody.

FIG. 4.

Association of NTR complex with U5 snRNP. (A) Extracts prepared from Ntr1-HA-, Ntr2-HA-, or Prp43-V5-tagged strains were precipitated with the anti-HA (α-HA) (lanes 5 and 6) or anti-V5 (α-V5) (lanes 7 and 8) antibody, and precipitates were analyzed by Northern blotting. Prp43-V5 extracts from which Ntr1 was depleted were also precipitated with the anti-V5 antibody (lane 8). Ntr1-HA extracts were precipitated with the anti-Smd1 (lane 3) or anti-Ntc20 (lane 4) antibody as controls. PAS, protein A-Sepharose. (B) Western and Northern blotting of affinity-purified NTR (lane 1) and Ntr1-Ntr2 complex (lane 2), probing with antibodies against Ntr1, V5, and Snu114 and with U5, respectively. (C) Western and Northern blotting of total extracts depleted with pre-Snu114 (lane 1), anti-Snu114 (lane 2), pre-Ntr1 (lane 3), or anti-Ntr1 (lane 4) serum, probing for Ntr1, Snu114, and U5. Pre, preimmune serum; Ab, antibody. (D) Splicing was carried out in Ntr1-depleted Snu114-3×HA extracts, and the reaction mixture was precipitated with the anti-Ntc20 antibody. The precipitate (lane 1) was then added to the affinity-purified NTR complex (lane 2), which contained untagged Snu114, and the supernatant (lane 4) was further precipitated with the pre-Ntr1 (lane 5) or anti-Ntr1 (lane 6) serum. (E) Snu114-3×HA mock-depleted extracts (M) (lane 1), Ntr1-depleted extracts (dNtr1) (lane 2), Snu114-depleted extracts (dSnu114) (lane 3), and the mixture of Ntr1- and Snu114-depleted extracts (dNtr1+dSnu114) were immunoprecipitated with the anti-Ntr1 antibody, followed by Western blotting, probing with anti-Ntr1 and anti-HA antibodies.

FIG. 5.

Antibody inhibition of splicing and the association of NTR with U5. (A) A 5-μl aliquot of extract was preincubated with 1.4 μg of anti-Ntr1 (lane 2) or 0.14 μg of anti-Ntr2 antibody (α-Ntr2) (lane 3) and used for splicing in a 10-μl reaction mixture. (B) Methods were as described for panel A, except splicing was carried out with five times the amounts of nonbiotinylated (lanes 1, 3, and 5) or biotinylated (lanes 2, 4, and 6) pre-mRNA, which was then precipitated with streptavidin-Sepharose followed by Western blotting. (C) Extracts were preincubated with (lanes 2 and 5) or without (lanes 1 and 4) anti-Ntr2 antibody and then precipitated with anti-Ntr1 antibody followed by Western (lanes 1 and 2) and Northern (lanes 3 to 5) blot analyses. M, mock-treated extracts; T, total RNA; IP, immunoprecipitation.

FIG. 6.

The association of NTR with U5 and with the spliceosomes formed in in vivo Ntr2-depleted extracts. (A) Splicing in wild-type (lanes 1 to 3 and 7) and in vivo Ntr2-depleted extracts alone (lanes 4 to 6 and 8) or with recombinant Ntr2 added (lane 9). (B) Western blotting of total extracts from wild-type (lane 1) and Ntr2-depleted (lane 2) strains. (C) Splicing in wild-type (lanes 1 and 3) or Ntr2-depleted (lanes 2 and 4) extracts by using nonbiotinylated (lanes 1 and 2) or biotinylated (lanes 3 and 4) pre-mRNA. Reaction mixtures were precipitated with streptavidin-Sepharose, followed by Western blot analysis of precipitates. (D) Wild-type (lane 1) or Ntr2-depleted (lane 2) extracts were immunoprecipitated with anti-Ntr1 antibody, and the precipitates were analyzed by Western blotting, probing for Ntr1, Ntr2, and Snu114, and by Northern blotting, probing for U5. WT, wild type; dNtr2 or d, in vivo Ntr2-depleted extract.

FIG. 7.

Interaction of Ntr2 with U5 and with the spliceosome. (A) Two-hybrid interactions of Ntr2 and Brr2. Ntr1 or Ntr2 was fused to the LexA DNA binding domain (LexA-BD) and Brr2 was fused to GAL4 activation domain (GAL4-AD) for two-hybrid assays using β-galactosidase as a reporter. (B) Immunoprecipitation (IP) of the spliceosome formed in Ntr1-depleted extracts without (lanes 2 to 6) or with the supplement of 0.1 μg of recombinant Ntr2 protein (lanes 7 to 11) with no antibody (lanes 3 and 8) or anti-Ntc20 (lanes 4 and 9), anti-Ntr1 (lanes 5 and 10), or anti-Ntr2 antibody (lanes 6 and 11). Lanes 1, 2, and 7 had 2 μl of splicing reaction mixtures; lanes 4 to 6 and 8 to 11 had immunoprecipitated materials from 20 μl of splicing reaction mixtures. PAS, protein A-Sepharose. (C) The Ntr1-depleted extract was incubated with recombinant Ntr2-HA prebound to the anti-HA antibody (α-HA) coupled to protein A-Sepharose (lane 4). Bound materials were analyzed by Northern blotting, probing with U1, U2, U4, U5, and U6. Lane 1, total RNA from 1 μl extract; lane 2, mock-depleted extract; lane 3, Ntr2 not added; lane 5, anti-HA antibody not included.

FIG. 8.

A diagram illustrating the interactions of Ntr1-Ntr2 with Prp43 and with U5 to mediate spliceosome disassembly. Double arrows indicate equilibrium between association and dissociation forms.