Research Article

Mechanism of spliceosome remodeling by the ATPase/helicase Prp2 and its coactivator Spp2

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Science  08 Jan 2021:
Vol. 371, Issue 6525, eabe8863
DOI: 10.1126/science.abe8863

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Remodeling an RNA processing machine

Splicing of precursor messenger RNA (pre-mRNA) is carried out by the spliceosome, a highly dynamic, supramolecular complex that undergoes assembly, activation, catalysis, and disassembly. These essential spliceosome remodeling events are driven by a conserved family of adenosine triphosphatase (ATPase)/helicases. In the presence of its coactivator Spp2, the ATPase/helicase Prp2 associates with the activated spliceosome and translocates the single-stranded pre-mRNA toward its 3′ end. Bai et al. now report the cryo–electron microscopy structures of Prp2 both before and after recruitment into the activated spliceosome. These structures and the associated biochemical analysis reveal how Prp2 remodels the activated spliceosome and how Spp2 safeguards the function of Prp2.

Science, this issue p. eabe8863

Structured Abstract

INTRODUCTION

Splicing of precursor messenger RNA (pre-mRNA) is carried out by the spliceosome. During each splicing cycle, the spliceosome undergoes assembly, activation, catalysis, and disassembly. The assembled spliceosome exists in eight major functional states. Spliceosome remodeling between neighboring states is driven by conserved adenosine triphosphatase (ATPase)/helicases. Together with its coactivator Spp2, the ATPase/helicase Prp2 harnesses the energy of adenosine 5′-triphosphate (ATP) binding and hydrolysis to translocate 3′-to-5′ on the single-stranded pre-mRNA, thus remodeling the activated spliceosome (known as the Bact complex) into the catalytically activated spliceosome (the B* complex). The B* complex catalyzes the branching reaction.

Since 2015, cryo–electron microscopy (cryo-EM) structures of all major states of the assembled spliceosome have been determined at near-atomic resolutions, generating crucial information on the active site and overall organization of the spliceosome. How spliceosome remodeling occurs, however, remains poorly understood because none of the ATPase/helicases in the presence of the spliceosome has been visualized in atomic details owing to limited resolution. In particular, it remains largely unknown how Prp2 remodels the Bact complex and why Prp2 requires its coactivator Spp2.

RATIONALE

To address these questions, we need to examine the detailed structural features of Prp2 and Spp2 in the Bact complex and compare these features with those of Prp2 and Spp2 in isolation. Atomic resolution is required, which may need improvement in sample preparation. Structure-guided biochemical analyses may be needed to corroborate the conclusions.

RESULTS

We used galactose-inducible expression of an ATPase-defective Prp2 mutant to stall remodeling of the endogenous Bact complex. This strategy resulted in marked enrichment of the Bact complex in the final cryo-EM sample. We determined the cryo-EM structure of the S. cerevisiae Bact complex at 2.5 Å—the highest resolution achieved for an intact spliceosome to date—which allows atomic identification of 12 new proteins, including Prp2 and Spp2 (see the figure). Prp2 weakly associates with the spliceosome and fails to function in the absence of Spp2. Spp2 uses its C-terminal sequences to stably associate with Prp2 and its N-terminal sequences to anchor on the spliceosome, thus tethering Prp2 to the Bact complex and allowing Prp2 to function. In the spliceosome, pre-mRNA is loaded into a featured channel between the N and C halves of Prp2, where Leu536 from the N half and Arg844 from the C half serve as barbed wires to prevent backward sliding of pre-mRNA toward its 5′ end. Conserved residues in the channel make hydrogen bonds mainly to the backbone phosphates, but not the nucleobases, of the pre-mRNA, explaining its sequence-independent recognition by Prp2.

We then determined the cryo-EM structures of Prp2 in three related functional states: free Prp2, Spp2-bound Prp2, and Spp2-bound ADP-loaded Prp2. These structures, together with that of Prp2 in the Bact complex and other published information, yield a working mechanism for Prp2. Relative movement between the RecA1 and RecA2 domains of Prp2, driven by ATP binding and hydrolysis, results in pre-mRNA translocation. In step 1, ATP binding to RecA1 is predicted to trigger the movement of RecA2 toward RecA1, allowing Leu536 to push the nucleobase of pre-mRNA to translocate by one nucleotide toward its 3′ end. Because of the translocation, Arg844 loses its association with the departing nucleobase and associates with the newly arrived upstream nucleobase. In step 2, ATP hydrolysis leads to the relaxation of RecA2 back to its original position, allowing Leu536 to shift its interactions to an upstream nucleobase. The movement of RecA2 is safeguarded by Arg844, which may prevent backward sliding of pre-mRNA. Last, ADP is released, and Prp2 is reset for the next cycle. In our model, Leu536 and Arg844 serve as two hands to alternately bind and push pre-mRNA toward its 3′ end.

CONCLUSION

As a coactivator, Spp2 stably associates with Prp2 and tethers it to Bact, enabling Prp2 function. ATP binding and hydrolysis trigger interdomain movement in Prp2, which allows Leu536 and Arg844 to alternately bind and push pre-mRNA unidirectionally toward its 3′ end.

Mechanism of action for the ATPase/helicase Prp2 in the activated spliceosome.

(A) Structure of the Bact complex. (B) Spp2 uses its N-terminal sequences to anchor on the spliceosome and its C-terminal sequences to associate with Prp2, tethering Prp2 to the spliceosome and enabling its function. (C) Leu536 (L536) and Arg844 (R844) of Prp2 prevent backward sliding of pre-mRNA by only allowing 5′-to-3′ movement of the RNA bases.

Abstract

Spliceosome remodeling, executed by conserved adenosine triphosphatase (ATPase)/helicases including Prp2, enables precursor messenger RNA (pre-mRNA) splicing. However, the structural basis for the function of the ATPase/helicases remains poorly understood. Here, we report atomic structures of Prp2 in isolation, Prp2 complexed with its coactivator Spp2, and Prp2-loaded activated spliceosome and the results of structure-guided biochemical analysis. Prp2 weakly associates with the spliceosome and cannot function without Spp2, which stably associates with Prp2 and anchors on the spliceosome, thus tethering Prp2 to the activated spliceosome and allowing Prp2 to function. Pre-mRNA is loaded into a featured channel between the N and C halves of Prp2, where Leu536 from the N half and Arg844 from the C half prevent backward sliding of pre-mRNA toward its 5′-end. Adenosine 5′-triphosphate binding and hydrolysis trigger interdomain movement in Prp2, which drives unidirectional stepwise translocation of pre-mRNA toward its 3′-end. These conserved mechanisms explain the coupling of spliceosome remodeling to pre-mRNA splicing.

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