Structure and function of spliceosomal DEAH-box ATPases.

Splicing of precursor mRNAs is a hallmark of eukaryotic cells, performed by a huge macromolecular machine, the spliceosome. Four DEAH-box ATPases are essential components of the spliceosome, which play an important role in the spliceosome activation, the splicing reaction, the release of the spliced mRNA and intron lariat, and the disassembly of the spliceosome. An integrative approach comprising X-ray crystallography, single particle cryo electron microscopy, single molecule FRET, and molecular dynamics simulations provided deep insights into the structure, dynamics and function of the spliceosomal DEAH-box ATPases.

Conformational dynamics of the RNA binding channel regulates loading and translocation of the DEAH-box helicase Prp43.

The DEAH-box helicase Prp43 has essential functions in pre-mRNA splicing and ribosome biogenesis, remodeling structured RNAs. To initiate unwinding, Prp43 must first accommodate a single-stranded RNA segment into its RNA binding channel. This allows translocation of the helicase on the RNA. G-patch (gp) factors activate Prp43 in its cellular context enhancing the intrinsically low ATPase and RNA unwinding activity. It is unclear how the RNA loading process is accomplished by Prp43 and how it is regulated by its substrates, ATP and RNA, and the G-patch partners. We developed single-molecule (sm) FRET reporters on Prp43 from Chaetomium thermophilum to monitor the conformational dynamics of the RNA binding channel in Prp43 in real-time. We show that the channel can alternate between open and closed conformations. Binding of Pfa1(gp) and ATP shifts the distribution of states towards channel opening, facilitating the accommodation of RNA. After completion of the loading process, the channel remains firmly closed during successive cycles of ATP hydrolysis, ensuring stable interaction with the RNA and processive translocation. Without Pfa1(gp), it remains predominantly closed preventing efficient RNA loading. Our data reveal how the ligands of Prp43 regulate the structural dynamics of the RNA binding channel controlling the initial binding of RNA.

Regulation of the DEAH/RHA helicase Prp43 by the G-patch factor Pfa1.

The DEAH/RHA helicase Prp43 remodels protein-RNA complexes during pre-messenger RNA (mRNA) splicing and ribosome biogenesis. The helicase activity and ATP turnover are intrinsically low and become activated by G-patch (gp) factors in the specific cellular context. The gp motif connects the helicase core to the flexible C-terminal domains, but it is unclear how this affects RecA domain movement during catalysis and the unwinding of RNA substrates. We developed single-molecule Förster Resonance Energy Transfer (smFRET) reporters to study RecA domain movements within Prp43 in real time. Without Pfa1(gp), the domains approach each other adopting predominantly a closed conformation. The addition of Pfa1(gp) induces an open state, which becomes even more prevalent during interaction with RNA. In the open state, Prp43 has reduced contacts with bound nucleotide and shows rapid adenosine diphosphate (ADP) release accelerating the transition from the weak (ADP) to the strong (apo) RNA binding state. Using smFRET labels on the RNA to probe substrate binding and unwinding, we demonstrate that Pfa1(gp) enables Prp43(ADP) to switch between RNA-bound and RNA-unbound states instead of dissociating from the RNA. ATP binding to the apo-enzyme induces the translocation along the RNA, generating the unwinding force required to melt proximal RNA structures. During ATP turnover, Pfa1(gp) stimulates alternating of the RecA domains between open and closed states. Consequently, the translocation becomes faster than dissociation from the substrate in the ADP state, allowing processive movement along the RNA. We provide a mechanistic model of DEAH/RHA helicase motility and reveal the principles of Prp43 regulation by G-patch proteins.

Structural basis for RNA translocation by DEAH-box ATPases.

DEAH-box adenosine triphosphatases (ATPases) play a crucial role in the spliceosome-mediated excision of pre-mRNA introns. Recent spliceosomal cryo-EM structures suggest that these proteins utilize translocation to apply forces on ssRNAs rather than direct RNA duplex unwinding to ensure global rearrangements. By solving the crystal structure of Prp22 in different adenosine nucleotide-free states, we identified two missing conformational snapshots of genuine DEAH-box ATPases that help to unravel the molecular mechanism of translocation for this protein family. The intrinsic mobility of the RecA2 domain in the absence of adenosine di- or triphosphate (ADP/ATP) and RNA enables DEAH-box ATPases to adopt different open conformations of the helicase core. The presence of RNA suppresses this mobility and stabilizes one defined open conformation when no adenosine nucleotide is bound. A comparison of this novel conformation with the ATP-bound state of Prp43 reveals that these ATPases cycle between closed and open conformations of the helicase core, which accommodate either a four- or five-nucleotide stack in the RNA-binding tunnel, respectively. The continuous repetition of these states enables these proteins to translocate in 3'-5' direction along an ssRNA with a step-size of one RNA nucleotide per hydrolyzed ATP. This ATP-driven motor function is maintained by a serine in the conserved motif V that senses the catalytic state and accordingly positions the RecA2 domain.