Molecular basis for the activation of human spliceosome

Abstract

The spliceosome executes pre-mRNA splicing through four sequential stages: assembly, activation, catalysis, and disassembly. Activation of the spliceosome, namely remodeling of the pre-catalytic spliceosome (B complex) into the activated spliceosome (B^act complex) and the catalytically activated spliceosome (B^* complex), involves major flux of protein components and structural rearrangements. Relying on a splicing inhibitor, we have captured six intermediate states between the B and B^* complexes: pre-B^act, B^act-I, B^act-II, B^act-III, B^act-IV, and post-B^act. Their cryo-EM structures, together with an improved structure of the catalytic step I spliceosome (C complex), reveal how the catalytic center matures around the internal stem loop of U6 snRNA, how the branch site approaches 5'-splice site, how the RNA helicase PRP2 rearranges to bind pre-mRNA, and how U2 snRNP undergoes remarkable movement to facilitate activation. We identify a previously unrecognized key role of PRP2 in spliceosome activation. Our study recapitulates a molecular choreography of the human spliceosome during its catalytic activation.

Fig. 1. Cryo-EM structures of six distinct conformational states of the human B^act complex.

a Schematic representation of the activation process of the human spliceosome. The B complex, containing U5.U4/U6 tri-snRNP, is remodeled by the RNA helicase BRR2, resulting in the release of U4 snRNP and recruitment of the NTC and NTR complexes. In the B^act-to-B^* transition catalyzed by PRP2, the BS is brought into the close proximity of 5′SS for the branching reaction. b Cryo-EM structures of six distinct intermediate states between the B and B^* complexes. These complexes, named pre-B^act, B^act-I, B^act-II, B^act-III, B^act-IV, and post-B^act, are resolved at average resolutions of 3.4, 3.0, 4.2, 3.0, 3.3, and 3.0 Å, respectively. All complexes, with components displayed in color-coded surface representation, are shown in the same orientation. c Cryo-EM structure of the human C complex at an average resolution of 3.4 Å. All structural images in this manuscript were prepared using ChimeraX and PyMol.

Fig. 2. Conformational changes of the RNA elements.

a Overall structure of the RNA elements in the pre-B^act complex. The color-coding scheme is preserved throughout this manuscript. In pre-B^act, U6 ISL and helices Ib and II of U2/U6 duplex are initially formed; but helix Ia is yet to form. The BS is ~60 Å away from 5′SS. The 5′-exon remains anchored to loop I of U5 snRNA. b Structure comparison of the RNA elements between the pre-B^act and B^act-I complexes. The U2/BS duplex and helix II remain unchanged. Helix Ia appears in B^act-I. The 5′SS moves toward the BS by ~10 Å, with notable movements for U5 and U6 snRNAs. All structure alignments reported in this manuscript are based on PRP8, unless stated otherwise. c Close-up views on the maturation of U6 ISL. In the pre-B^act to B^act-I transition, helix Ia is formed, and the key nucleotide U74 of U6 snRNA is bulged out. d Pairwise comparison of the RNA elements among the B^act-I, -II, -III, and -IV complexes. The distance between 5′SS and the BS remains unchanged at about 50 Å. In the transition of B^act-I to B^act-II, U2 snRNA undergoes a marked translocation and helix II is shifted slightly. In the transition of B^act-III to B^act-IV, helix II moves to the position where it is found in the C complex. e Structure comparison of the RNA elements between B^act-IV and post-B^act. U2 snRNA undergoes notable movements with the BS moving towards 5′SS by about 20 Å. f Structure comparison of the RNA elements between the post-B^act and C complexes. After the branching reaction, the BS is linked to 5′SS in the C complex, and U2 snRNA undergoes a pronounced translocation.

Fig. 3. Structure and function of PRP2 in the human B^act complex.

a PRP2 in the B^act-I complex. PRP2 is attached to the peripheral region of the spliceosome and loosely contacts the N-terminal region of SF3B1. N the N-terminus of SF3B1, C the C-terminus of SF3B1. b PRP2 in the B^act-II complex. In the B^act-I to B^act-II transition, PRP2 undergoes a pronounced translocation, moving to the cavity vacated by the RES complex. The PPT sequences of pre-mRNA are loaded into PRP2. c A close-up view on PRP2 in B^act-II. The loop between the N-terminal HEAT repeats of SF3B1 interacts with the RecA2 domain of PRP2, and the BS is anchored on the C-terminal HEAT repeats of SF3B1. The PPT sequences of pre-mRNA are bound to PRP2. d The N-terminal helices of PRP2 directly associate with the C-terminal helices of SKIP, and the linker helix and RRM domain of PPIL4. e A close-up view on the interface between the PPT of pre-mRNA and PRP2. Residues Arg503, Leu709, and Arg998 directly contact the PPT sequences. f Depletion of endogenous PRP2 from the nuclear extract. Compared to the untreated sample, the amount of PRP2 in the nuclear extract is sharply reduced as confirmed by the western blot. g Deletion of the N-terminal extension or mutation of key residues in PRP2 impairs splicing. Shown here are the reverse transcription-PCR results of in vitro splicing reaction. These results have been repeated for three times to confirm the analysis. Source data are provided as a Source Data file.

Fig. 4. Composition fluxes during activation of human spliceosome.

a The structures of six human B^act complexes, in surface representation with select components color-coded, reveal a series of compositional changes during activation of the human spliceosome. In the transition of pre-B^act to B^act-I, the splicing factor KIN17 is dissociated; CWC22, SRRM2, CWC27, RNF113A, and PRP2 are recruited. During the B^act-I to B^act-II transition, CWC27, SRRM1, SF3B6, and RES complex are released; PPIL1 and PPIL4 are recruited. In the transition of B^act-II to B^act-III, PRP17 and the NTC core are recruited. In the transition of B^act-III to B^act-IV, PPIL2 and the SF3a components SF3A1/SF3A2 are released; SYF2 is recruited. In post-B^act, RNF113A and SF3A3 are released. b A close-up view on the interface between the ZnF and WH domain of KIN17 and U6/5′SS duplex.

Fig. 5. Rearrangements of U2 snRNP during human spliceosome activation.

a, b U2 snRNP, comprising U2 snRNA, SF3a, SF3b and the core, undergoes remarkable positional changes in a stepwise manner during spliceosome activation. In the B-to-pre-B^act transition, U2 snRNP undergoes a large translocation, moving toward the main body of spliceosome. U2 snRNP has a series of minor positional and conformational changes from pre-B^act to B^act-IV. Notably, the SF3b component SF3B6 and the SF3a components SF3A1/SF3A2 are dissociated in the transitions of B^act-I to B^act-II and B^act-III to B^act-IV, respectively. The last component SF3A3 of the SF3a complex is released in the B^act-IV to post-B^act transition. In the B^*/C complexes, the SF3b complex is dissociated; U2 snRNP core undergoes prominent movements.