Molecular Dynamics Simulations Elucidate the Molecular Basis of Pre-mRNA Translocation by the Prp2 Spliceosomal Helicase

Abstract

The spliceosome machinery catalyzes precursor-messenger RNA (pre-mRNA) splicing by undergoing at each splicing cycle assembly, activation, catalysis, and disassembly processes, thanks to the concerted action of specific RNA-dependent ATPases/helicases. Prp2, a member of the DExH-box ATPase/helicase family, harnesses the energy of ATP hydrolysis to translocate a single pre-mRNA strand in the 5′ to 3′ direction, thus promoting spliceosome remodeling to its catalytic-competent state. Here, we established the functional coupling between ATPase and helicase activities of Prp2. Namely, extensive multi-μs molecular dynamics simulations allowed us to unlock how, after pre-mRNA selection, ATP binding, hydrolysis, and dissociation induce a functional typewriter-like rotation of the Prp2 C-terminal domain. This movement, endorsed by an iterative swing of interactions established between specific Prp2 residues with the nucleobases at 5′-and 3′-ends of pre-mRNA, promotes pre-mRNA translocation. Notably, some of these Prp2 residues are conserved in the DExH-box family, suggesting that the translocation mechanism elucidated here may be applicable to all DExH-box helicases.

Tracing Allostery in the Spliceosome Ski2-like RNA Helicase Brr2

“…Instead, the PCA of NCBrr2 ATP reveals that PC1 corresponds to the NcTOC (Figure D) motion, while PC2 describes a type-writer-like rotatory motion between NSec63 and NRecA1–2 domains (Figure F). This movement was observed and suggested to promote RNA translocation in Prp2 ATPase/helicase. − Remarkably, in the complete Brr2 structure, the C-cassette hinders this movement, and it is only the coordinated effort of the N- and C-cassettes that drives RNA translocation (Figure E).…”

“…We remark that this study does not allow us to formulate a hypothesis on Brr2 translocation mechanism due to the lack of structural information on the snRNA binding to the C-cassette. 27 This lack of knowledge also prevents unraveling the molecular basis of RNA-driven allosteric regulation of Brr2 ATPase activity and comparing it with that observed in other DExH helicases. 16 RNA-dependent helicases play key roles in distinct aspects of RNA metabolism.…”

“…We also supply information for future mutagenesis studies to establish their relevance in allosteric signaling within Brr2. We remark that this study does not allow us to formulate a hypothesis on Brr2 translocation mechanism due to the lack of structural information on the snRNA binding to the C-cassette . This lack of knowledge also prevents unraveling the molecular basis of RNA-driven allosteric regulation of Brr2 ATPase activity and comparing it with that observed in other DExH helicases .…”

Frontiers and Challenges of Computing ncRNAs Biogenesis, Function and Modulation

“…All these functional movements, underlying spliceosome remodeling and its progression along the splicing cycle, are driven by the concerted action of specific RNA-dependent ATPases/helicases . The molecular mechanism of this important class of proteins has only recently started to be elucidated via molecular simulations. , Yet, their relevance as a potential drug target to modulate pre-mRNA splicing has already been proven …”

“…339 The molecular mechanism of this important class of proteins has only recently started to be elucidated via molecular simulations. 340,341 Yet, their relevance as a potential drug target to modulate pre-mRNA splicing has already been proven. 342 Diseases-Associated Mutations Trigger Aberrant RNA Metabolism.…”