Abstract
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.
1 Introduction
The spliceosome is a huge and highly dynamic macromolecular machine in the nucleus of eukaryotic cells that is responsible for the removal of most non-coding introns from pre-mRNAs (Wahl et al. 2009). It is composed of five large protein-RNA complexes, the uracil-rich small nuclear ribonucleoprotein particles (UsnRNPs), and numerous additional proteins (Figure 1). For each intron to be excised, the spliceosome is newly assembled in a step-wise manner and needs to undergo further conformational changes to reach a state that is capable of catalyzing the splicing reaction. Acting as a single-turnover enzyme, the spliceosome is disassembled after each exon ligation and the components are recycled in the next cycle.
Schematic representation of the splicing cycle. Cryo-EM structures of characterized spliceosomal complexes from S. cerevisiae during sequential assembly, activation, the two transesterification steps and final disassembly are shown in cartoon representation. The U-snRNAs are highlighted in different colors, whereas the protein components are displayed as gray semi-transparent cartoons. The eight spliceosomal ATPases are indicated at the corresponding step, crystal structures of the four DEAH-box proteins are shown as cartoon representations. The following PDB depositions were used for the depicted complexes: U1: 5uz5; E: 6n7p; A: 6g90; tri-snRNP: 5gan; pre-B: 5zwn, 5zwm; B: 5nrl; complex Bact: 7dco; B*: 6j6g; C: 7b9v; C*: 5mq0; P: 6bk8; ILS: 5y88.
Major steps in the pre-mRNA splicing cycle are the binding of the U1 snRNP to the 5′ splice site, and the recognition of the branch point site (BPS) and the adjacent polypyrimidine tract by the SF1/BBP and U2AF proteins yielding the spliceosomal E complex. Subsequently the U2 snRNP joins and binds to the BPS, leading to the displacement of SSF1/BBP and the formation of the A complex. This allows the pre-assembled U4/U6.U5 tri-snRNP to join the complex forming the pre-B complex that contains almost all components known to be necessary for the splicing reactions. Several conformational and compositional rearrangements lead to the release of the U1 snRNP, allowing the U6 snRNP to occupy its former position and thereby generate the B complex. Subsequently, the U4 snRNP is released and massive reorganizations of the snRNA interaction network lead to the formation of the Bact complex, which is then further remodeled to form the catalytically active B* complex. This B* spliceosome catalyzes the first step of the splicing reaction, called branching, where the 2′ hydroxyl group of the branch site adenosine nucleophilically attacks the 5′ splice site, leading to the cleavage of its phosphodiester bond. The resulting complex, containing the free 5′-exon and the lariat-intron 3′ exon, is denoted as C complex. Additional rearrangements of the spliceosome lead to the C* complex, which is competent to catalyze the second step of the splice reaction, where the 3′ splice site is attacked by the 3′ hydroxyl group of the 5′ exon, leading to exon joining. The post-splicing P complex contains the excised intron in lariat form and mature mRNA, which is released in a first disassembly step, yielding the ILS complex. In a second disassembly step, the remaining components dissociate. Most of the involved proteins and snRNPs are recycled to be used in subsequent cycles of splicing. The conformational rearrangements of the splicing components that drive the transitions from state to state are mainly promoted by a set of ATPases which dynamically remodel RNA secondary structures and facilitate binding as well as dissociation of RNPs.
Major insights into the architecture and function of the spliceosome have been gained by single particle cryogenic electron microscopy (cryo-EM). Initial cryo-EM studies of UsnRNPs and spliceosomal complexes gave first views of the overall shape, allowed to locate some of the proteins and showed the heterogeneity and dynamics of these complexes (Will and Lührmann 2011). However, due to limited resolution, they did not provide structural details such as the identification of secondary structure elements or even atomic models of proteins and RNAs.
During the past decade the resolution of cryo-EM three-dimensional (3D) reconstructions improved tremendously, leading to atomic models of almost all spliceosomal complexes. These structural models provided deep insights into the structure and function of the spliceosome, which have recently been summarized in several review articles (Borisek et al. 2021; Fica et al. 2017; Kastner et al. 2019; Plaschka et al. 2019; Tholen and Galej 2022; Wan et al. 2019; Wilkinson et al. 2020; Yan et al. 2019). In this review, we will focus on the structure and function of those DEAH-box ATPases which are required to drive the conformational changes of the spliceosome during its activation, the splicing reaction, the release of the reaction products and the subsequent disassembly of the spliceosome.
2 Functions of SF2 ATPases in the spliceosome
At least eight steps of the pre-mRNA splicing process require the action of ATPases belonging to the helicase superfamily 2 (SF2) (Figure 1). The SF2 ATPases involved in splicing belong to the DEAD-box, DEAH-box and Ski2-like subfamilies, which all are monomeric proteins. These proteins are RNA-dependent ATPases, as their activity is stimulated upon RNA-binding. They share a conserved helicase core formed by two RecA-like domains and exhibit additional variable N- and C-terminal extensions or domains (Figure 2A). In Saccharomyces cerevisiae and H. sapiens, respectively, the spliceosomal proteins Prp5/DDX46, Sub2/DDX39B and Prp28/DDX23 belong to the DEAD-box family, while Brr2/SNRNP200 is a Ski2-like helicase, and Prp2/DHX16, Prp16/DHX38, Prp22/DHX8 and Prp43/DHX15 are DEAH-box proteins. Prp5 and Sub2 are required for the formation of the pre-spliceosome, and Prp28 mediates the exchange of the U1 snRNP with the U6 snRNP at the 5′ splice site during the transition from the pre-B to the B complex (Chen et al. 2001; Kistler and Guthrie 2001; Xu and Query 2007). Brr2 unwinds the U4/U6 snRNA duplex, which leads to the release of the U4 snRNP and the formation of the Bact complex (Laggerbauer et al. 1998). The action of Prp2, which is strictly dependent on its cofactor Spp2, makes the BS accessible for the first step of the splicing reaction by displacing several splicing factors and making room for new ones (Kim and Lin 1996; King and Beggs 1990; Roy et al. 1995; Silverman et al. 2004). Thereby, the Bact complex is remodeled to the catalytically active B* complex. Prp16 is required for the second catalytic step of the splicing reaction, as it enables exon ligation by promoting the dissociation of several splicing factors (Burgess and Guthrie 1993; Tseng et al. 2011). Subsequent to the splicing reaction, Prp22 facilitates the release of the mature mRNA by disrupting its base-pairings with the U5 snRNA, obtaining the ILS spliceosome (Company et al. 1991; Schwer 2008). Finally, Prp43 is involved in the disassembly of the post-catalytic spliceosome by disrupting the interactions of the U6 snRNA with the U2 snRNA and the intron (Arenas and Abelson 1997; Fourmann et al. 2013). Besides their function as ATP-dependent motors that drive the transitions between spliceosomal complexes, most of these proteins also have a function in controlling splicing fidelity that is achieved through kinetic proofreading. To this end, the ATPase might limit the time window for a particular event in the splicing cycle. Splicing only proceeds normally, when the proofreading step is passed faster than the respective ATPase can act (Semlow and Staley 2012). An example for this mechanism is Prp22, which competes with the exon ligation reaction by pulling on the 3′ exon in the C* complex (Liu et al. 2017). Thereby, a suboptimal substrate could be removed from the spliceosome at this stage. Alternatively, the ATPase is capable of sensing suboptimal substrates and rejects them more quickly than canonical substrates. This is likely the case for Prp43 that together with its cofactors Ntr1 and Ntr2 senses and disassembles early spliceosomal complexes formed on suboptimal substrates (Fourmann et al. 2016, 2017).
Conserved sequence motifs and domains of spliceosomal SF2 ATPases. (A) ATPases belonging to the SF2 share eight to nine conserved sequence motifs spread over both RecA-like domains. The C-terminal domain architecture of DEAH-box and Ski2-like subfamilies is also conserved. (B) Domain organization and crystal structure of ctPrp43 in complex with U7-RNA and ADP-BeF3− (PDB ID: 5lta) in cartoon representation. Remaining residues of the truncated N-terminal domain (61–96) are shown in black, the RecA1 domain (97–273) in orange, the RecA2 domain (274–458) in blue, the winged-helix domain (WH, 459–526) in gray, the helix-bundle domain (HB, 527–640) in wheat and the OB-fold domain (OB, 641–764) in green.
3 Cryo-EM structures of spliceosomal complexes with DEAH-box ATPases
Several structures of spliceosomal complexes from S. cerevisiae (sc) and H. sapiens (hs) containing one of the DEAH-box proteins were obtained by means of single particle cryo-EM (Table 1). These are the Bact complex with Prp2, the C complex with Prp16, the C* complex with either Prp22 or Prp16, the P complex with Prp22, and the ILS complex with Prp43. A common feature of all DEAH-box ATPases in these complex structures is their localization at the periphery of the respective spliceosome (De Bortoli et al. 2021). In general, the local resolution of the spliceosome cryo-EM 3D reconstructions decreases from the rigid center to the rim due to increasing conformational flexibility and/or lower occupancy of transiently interacting components. Therefore, the assessed local resolutions of cryo-EM map areas corresponding to the DEAH-box ATPases in early cryo-EM 3D reconstructions have been much lower than the stated overall resolution, just in the range of 11.5–7.3 Å (Ficner et al. 2017). However, in very recent cryo-EM 3D reconstructions the improvement of the overall resolution resulted also in a substantial increase of the local resolutions of the peripheral DEAH-box ATPases (Table 1). One example is Prp2 that has been resolved at 3.7 Å in the yeast Bact complex (Bai et al. 2021). In some cases the improved quality of the cryo-EM maps also allowed tracing of the RNA to which the DEAH-box protein is bound. Thereby, the binding sites of Prp22 on the 3′ exon, of Prp16 on the intron and of Prp22 at the 3′ end of the pre-mRNA could be described in more detail (Bai et al. 2021; Liu et al. 2017; Wilkinson et al. 2021).
Spliceosomal DEAH-box ATPases in cryo-EM structures (hs: homo sapiens, sc: Saccharomyces cereviseae).
| Complex | ATPase | Source | PDB | EMBD | Res., Å | Local res.a (max), Å | Local res.a (avg ± SD), Å | Reference |
|---|---|---|---|---|---|---|---|---|
| ILS | Prp43 | sc | 5y88 | 6817 | 3.5 | 2.9 | 3.1 ± 0.3 | Wan et al. (2017) |
| DHX15 | hs | 6id1 | 9647 | 2.9 | 4.4 | 30.6 ± 20.0 | Zhang et al. (2019) | |
| P | Prp22 | sc | 5ylz | 6839 | 3.6 | 4.0 | 18.1 ± 24.9 | Bai et al. (2017) |
| Prp22 | sc | 6exn | 3979 | 3.7 | 4.1 | 19.6 ± 21.9 | Wilkinson et al. (2017) | |
| DHX8 | sc | 6bk8 | 7109 | 3.3 | 3.6 | 4.6 ± 1.8 | Liu et al. (2017) | |
| DHX8 | hs | 6icz | 9645 | 3 | 3.3 | 44.1 ± 30.8 | Zhang et al. (2019) | |
| DHX8 | hs | 6qdv | 4532 | 3.3 | 3.7 | 3.9 ± 0.3 | Fica et al. (2019) | |
| C* | Prp16 | sc | 5wsg | 6684 | 4 | 6.3 | 39.8 ± 27.9 | Yan et al. (2017) |
| Prp22 | sc | 5mq0 | 3541 | 4.2 | 4.6 | 7.0 ± 4.9 | Fica et al. (2017) | |
| DHX8 | hs | 5mqf | 3545 | 5.9 | 6.8 | 69.5 ± 33.5 | Bertram et al. (2017) | |
| DHX8 | hs | 5xjc | 6721 | 3.6 | 3.9 | 29.2 ± 24.9 | Zhang et al. (2017) | |
| DHX8 | hs | 7w5b | 32,321 | 4.3 | 4.7 | 26.4 ± 24.3 | Zhan et al. (2022) | |
| Pre-C∗-I | DHX8 | hs | 7w59 | 32,317 | 3.6 | 4.0 | 24.5 ± 21.7 | Zhan et al. (2022) |
| Pre-C∗-II | DHX8 | hs | 7w5a | 32,319 | 3.6 | 3.9 | 14.9 ± 14.6 | Zhan et al. (2022) |
| C | Prp16 | sc | 7b9v | 12,110 | 2.8 | 6.6 | 7.0 ± 0.5 | Wilkinson et al. (2021) |
| Prp16 | sc | 5lj5 | 4057 | 3.8 | 10.8 | 13.4 ± 3.8 | Galej et al. (2016) | |
| DHX38 | hs | 5yzg | 6864 | 4.1 | 7.2 | 7.2 ± 0.2 | Zhan et al. (2018) | |
| DHX38 | hs | 7a5p | 11,570 | 5 | 5.5 | 76.8 ± 34.0 | Bertram et al. (2020) | |
| Bact | Prp2 | sc | 5lqw | 4099 | 5.8 | 7.0 | 8.2 ± 1.1 | Rauhut et al. (2016) |
| Prp2 | sc | 7dco | 30,637 | 2.5 | 3.7 | 3.7 ± 0.3 | Bai et al. (2021) | |
| Prp2 | sc | 5gm6 | 9524 | 3.5 | 4.1 | 52.3 ± 34.5 | Yan et al. (2016) | |
| DHX16 | hs | 6ff7 | 4240 | 4.5 | 3.6 | 78.8 ± 16.0 | Haselbach et al. (2018) | |
| DHX16 | hs | 5z56 5z57 5z58 |
6889 6890 6891 |
5.1, 6.5, 4.9 |
4.5, 10.4, 7.1 |
35.2 ± 33.4 89.3 ± 18.6 72.3 ± 26.0 |
Zhang et al. (2018) | |
| DHX16 | hs | 7dvq | 30,878 | 2.9 | 3.8 | 27.5 ± 27.7 | Bai et al. (2021) | |
| BAQR | DHX16 | hs | 7qtt | 14,146 | 3.1 | 5.7 | 7.6 ± 8.2 | Schmitzova et al. (2023) |
-
aThe local map resolution of the helicase region was determined using the mapman utility of the Uppsala Software Factory (Kleywegt and Jones 1996) based on local map resolution calculations performed with the ResMap program (version 1.1.4). Maximum (max) and average (avg ± SD) resolutions of this region are shown. These have been calculated based on the deposited cryo-EM map fragment selected within a 3 Å radius around each atom constituting the helicase molecule.
4 Crystal structures of spliceosomal DEAH-box ATPases
Crystal structures of all spliceosomal DEAH-box ATPases have been determined at high resolutions and revealed important details required for understanding their function and mechanism (Table 2). The two RecA-like domains RecA1 and RecA2 form the helicase core and exhibit a fold highly conserved among the known structures of SF2 helicases (Pyle 2008). They harbor eight conserved sequence motifs known to play crucial roles in nucleotide and RNA binding as well as in coupling nucleotide hydrolysis to translocation and RNA unwinding. A characteristic feature of the RecA2 domain is an anti-parallel β-hairpin present in all DExH-box helicases, which protrudes out of the RecA2 domain thereby contacting the C-terminally located domains. These domains are conserved among the spliceosomal DEAH-box proteins, comprising a winged-helix (WH) domain, a helix bundle (HB) domain, and an OB-fold domain (Figure 2B). Up to now, only for Prp43 the structure of the short N-terminal extension could be resolved, as it is attached to the surface of the RecA1 and the C-terminal WH and HB domains, leading to a stable fold of the extension (He et al. 2010; Walbott et al. 2010). For all other spliceosomal DEAH-box proteins the N-terminal extensions have been predicted to be intrinsically unfolded and therefore were removed prior to crystallization.
Crystal structures of spliceosomal DEAH-box ATPases.
| ATPase | Complex | Organism | PDB | Resolution, Å | Reference |
|---|---|---|---|---|---|
| Prp43/DHX15 | ADP | ct | 5d0u | 2.9 | Tauchert et al. (2016) |
| sc | 2xau | 1.9 | Walbott et al. (2010) | ||
| sc | 3kx2 | 2.2 | He et al. (2010) | ||
| hs | 5xdr | 2.0 | Murakami et al. (2017) | ||
| CDP | sc | 5jpt | 2.9 | Robert-Paganin et al. (2017) | |
| ADP-BeF3− | ct | 5ltk, 5ltj | 1.8, 3.2 | Tauchert et al. (2017) | |
| ADP-BeF3−/RNA | ct | 5lta | 2.6 | Tauchert et al. (2017) | |
| ADPNP/RNA | sc | 5i8q | 4.2 | He et al. (2017) | |
| NKRF(gp) | hs | 6sh7 | 2.2 | Studer et al. (2020) | |
| NKRF(gp)/ADP | hs | 6sh6 | 1.9 | Studer et al. (2020) | |
| SUGP1(gp) | hs | 8ejm | 1.8 | Zhang et al. (2022) | |
| Prp22/DHX8 | apo | ct | 6i3o | 3.3 | Hamann et al. (2019) |
| ADP | hs | 6hyt, 6hys | 2.3, 2.6 | Felisberto-Rodrigues et al. (2019) | |
| RNA | ct | 6i3p | 2.8 | Hamann et al. (2019) | |
| RNA | hs | 6hyu | 3.2 | Felisberto-Rodrigues et al. (2019) | |
| Prp16/DHX38 | ADP | ct | 8cnt | 1.9 | Garbers et al. (2023) |
| Prp2/DHX16 | apo | ct | 6fa9 | 2.6 | Schmitt et al. (2018) |
| ADP | ct | 6fa5, 6faa, 6fac | 2.3, 2.0, 2.0 | Schmitt et al. (2018) | |
| ADP-BeF3−/RNA | ct | 6zm2 | 2.1 | Hamann et al. (2021) | |
| Spp2(gp)/ADP | ct | 6rma, 6rmb, 6rmc, 6rm8, 6rm9 | 2.1, 2.5, 2.6, 2.0, 1.9 | Hamann et al. (2020) |
Importantly, well resolved crystal structures of different functional complexes of Prp2, Prp22 and Prp43 have been obtained, e.g., of the apo state, the ADP complex, the ADB-BeF3− complex mimicking ATP, the RNA complex with bound ADP-BeF3−, as well as complexes with bound fragments of G-patch proteins for Prp43 and Prp2. All crystal structures of spliceosomal DEAH-box proteins deposited in the PDB are summarized in Table 2. Most interestingly, comparison of the atomic structural models of different functional states provided strong evidence for the mechanisms of RNA loading and ATP-dependent RNA translocation.
4.1 Nucleotide binding
The nucleotide binding pocket is located in the cleft between the RecA1 and RecA2 domains. The amino acids involved in the binding of ADP or the ATP analog ADP-BeF3− belong to the conserved sequence motifs I, II, V, or VI. The adenine base of ADP-BeF3− is bound via cation-π and in the case of ADP also by π–π interactions, but seems to be not specifically recognized as it does not form a unique set of hydrogen bonds (Hamann et al. 2021; He et al. 2010; Schmitt et al. 2018; Tauchert et al. 2016, 2017; Walbott et al. 2010). This distinction between ADP-bound and ATP-bound states is conserved between Prp43 and Prp2. The unspecific interactions with the base observed in DEAH-box ATPases significantly differ from the DEAD-box and Ski2-like helicases, which contain an additional binding motif, the Q-motif, that enforces adenine specificity. The glutamic acid residue of the eponymous DEAH-motif (motif II) is involved in the coordination of a water molecule and positions it in close spatial proximity to the γ-phosphate. During ATP hydrolysis, this water is thought to attack the γ-phosphate since it is in almost perfect orientation for a nucleophilic attack, hence supports a nucleophilic substitution mechanism. A detailed view of the catalytic mechanism at atomic level was recently obtained by MD simulations of Prp2, which also revealed the mechanism of allosteric activation upon binding of a single stranded RNA (Movilla et al. 2023).
Following hydrolysis, the reaction products ADP and Pi are released, however the order of these events remains unclear. Due to the narrow nucleotide entry site, ADP would have to dissociate first to clear the way for Pi release if both molecules leave the binding pocket on the same path. However, one of the Prp2 crystal structures revealed a putative separate exit tunnel for the phosphate anion which connects the γ-phosphate position of the active site to the surface of the protein (Figure 3A) (Hamann et al. 2021). In all other nucleotide-bound structures, the channel is occluded by residues of motif III (Figure 3B). Interestingly, random-accelerated molecular dynamics simulations with Prp2 and also Prp43 strongly supported Pi release through the tunnel, prior to dissociation of ADP, a mechanism which might also be conserved in other DEAH-box family members (Hamann et al. 2021).
Movements of the conserved sequence motif III allow the formation of a channel, connecting the γ-phosphate binding site to the protein surface. (A) Cross section of ctPrp2 with bound ADP (6fac; Schmitt et al. 2018) shown in surface representation with domains colored as in Figure 2B. ADP-BeF3− (from 6zm2; Hamann et al. 2021) was modeled into the active site of the ADP-bound structure in order to visualize the position of the γ-phosphate. The ADP moiety is depicted as balls and sticks and the BeF3− is shown as green spheres. The cross section reveals a direct connection of the γ-phosphate binding site with the protein surface that might serve as an exit channel. (B) The crystal structure of ctPrp2-ADP (6fac; Schmitt et al. 2018) is shown as cartoon model, ADP is depicted as sticks. The RecA1 and RecA2 domains are colored as in (A), the C-terminal domains are colored gray, motif III is highlighted in green and the putative phosphate exit channel is represented by black spheres. The inlet shows motif III exhibiting alternative conformations in other crystal structures of ctPrp2 (6faa, 6fa5, 6fa9: Schmitt et al. 2018; 6zm2: Hamann et al. 2021), blocking the putative exit channel. Adapted from Hamann et al. (2021).
4.2 RNA binding
Crystal structures of Prp43, Prp22 and Prp2 in complex with a single strand of RNA showed that the RNA is bound in a tunnel formed by both RecA domains and the C-terminal domains (Hamann et al. 2019, 2021; Tauchert et al. 2017). The spliceosomal DEAH-box proteins bind RNA in a sequence-independent manner, predominantly by polar interactions with the sugar-phosphate backbone (Figure 4). In the RNA-bound structures of Prp43, Prp22 and Prp2, the binding channel contacts seven to nine RNA nucleotides. The amino acids interacting with the 3′ end of the RNA strand are highly conserved between the three spliceosomal DEAH-box ATPases, leading to a stacked conformation of the RNA bases in this region. In contrast, the contacts to the 5′ end of the strand show a higher variability in terms of number of interactions and conformation. In Prp43, this region of the RNA only loosely associates with the protein, leading to two alternative conformations (Tauchert et al. 2017). In Prp22 on the other hand, this area is highly stabilized through stacking interactions with residues of the HB and OB-fold domains (Hamann et al. 2019).
RNA interaction of Prp43 from Chaetomium thermophilum. Crystal structure of ctPrp43 in complex with U7-RNA and the ATP-analog ADP-BeF3−, the model is displayed as cartoon representation. The domains are colored as in Figure 2B. The ssRNA binds in a channel formed the helicase core and the C-terminal domains. The inlet shows the protein RNA interactions in more detail. The RNA, exhibiting two alternative conformations for nucleotides U1–U3, as well as the amino acids involved in hydrogen-bonds or stacking interactions with the RNA nucleotides are shown as ball and stick models, hydrogen-bonds are depicted as dashed lines. The interaction pattern at the 3′ region involving the residues highlighted by oval shapes is also observed for ctPrp22 and ctPrp2 (ctPrp22-U12-RNA, PDB ID: 6i3p; ctPrp2-U12-RNA-ADP-BeF3−, PDB ID: 6zm2). The residues marked by a rectangular shape display unique interactions.
5 Structural insights into the mechanism of DEAH-box ATPases
5.1 ATP-dependent RNA-translocation
The comprehensive number of structures of DEAH-box ATPases in different functional states allowed to deduce a mechanism for ATP-driven RNA translocation (Hamann et al. 2019; He et al. 2017). A comparison between the RNA-bound structures in the nucleotide-free or ATP-bound state revealed that the proteins cycle between closed nucleotide-bound and open nucleotide-free conformations of the helicase core. In the closed conformation, a stack of four RNA nucleotides is present in the binding tunnel between a conserved loop in RecA1, and the β-hairpin in RecA2 (Figure 5A). When transitioning to the open state upon nucleotide release, the RecA2 domain shifts towards the 5′ end of the RNA, leading to the accommodation of one additional nucleotide in the channel, resulting in a five-nucleotide stack (Figure 5B). Domain closure shifts the RecA1 domain towards RecA2, omitting the 3′ most nucleotide from the channel. Due to the larger number of interactions of the RecA2 domain with the RNA backbone, the RNA stays stably attached to the RecA2 domain and does not slide back. Instead, the few RecA1 interactions are broken and subsequently replaced by the succeeding sugar-phosphate moiety pushed into that position. The continuous transition between these states enables the proteins to translocate in 3′–5′ direction along a ssRNA with a step-size of one RNA nucleotide per hydrolyzed ATP (Figure 5C). 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 (Hamann et al. 2019). This analysis of crystal structures, representing snapshots of defined states, was recently complemented by MD simulations of the RNA translocation process (Becker and Hub 2023a).
Translocation of DEAH-box ATPases. (A and B) The helicase cores of nucleotide-bound ctPrp43 (5lta; Tauchert et al. 2017) and nucleotide-free ctPrp22 (6i3p; Hamann et al. 2019) in complex with ssRNA are shown as cartoon models. The RecA1 and RecA2 domains are colored as in Figure 2B, the RNA is depicted in gray with stacked nucleotides highlighted in red. (A) Nucleotide-bound ctPrp43 adopts a closed conformation of the helicase core, incorporating a stack of four RNA nucleotides in the binding channel. (B) The helicase core of nucleotide-free ctPrp22 exhibits an open conformation, allowing the accommodation of an additional RNA nucleotide, leading to a five-stack. In both structures, the β-hairpin of the RecA2 domain interrupts the stack. (C) Schematic representation of translocation by DEAH-box ATPases. The helicase core is shown in gray, the RNA in blue and the stacked RNA nucleotides are depicted in green and yellow. Opening of the helicase core upon ADP release leads to the incorporation of an additional RNA nucleotide between the four-nucleotide stack and the β-hairpin. Subsequent domain closure induced by ATP binding pushes the RNA by one nucleotide through the binding channel. Continuous cycling between these conformations during ATP turnover enables translocation in 3′–5′ direction along ssRNA with a step-size of one RNA nucleotide per hydrolyzed ATP. Reproduced from Hamann et al. (2019).
5.2 Helicase activity
Due to similarities regarding the sequence and structure of other SF2 helicases, all spliceosomal DEAH-box proteins were thought to act as helicases that unwind double stranded RNA. Such an unwinding activity was actually reported for Prp43, Prp22, and Prp16 (Tanaka and Schwer 2006; Wagner et al. 1998; Wang et al. 1998), while for Prp2 no helicase activity could be observed (Bao et al. 2017; Hamann et al. 2021; Kim et al. 1992; Warkocki et al. 2015). Based on structural considerations, DEAH-box ATPases were proposed to act as processive helicases, as after duplex unwinding one of the resulting single strands remains trapped in the RNA binding tunnel (Walbott et al. 2010). However, the processivity of RNA unwinding was just recently demonstrated for Prp43 by means of single molecule FRET (Enders et al. 2022). While Prp43 on its own rapidly dissociates from ssRNA during ATP turnover, the interaction with its G-patch partner Pfa1 enables it to processively unwind RNA double-strands of up to 20 base-pairs.
The mechanism employed by DEAH-box helicases to unwind double-stranded RNA has been the subject of longstanding debates. While features that are implicated in unwinding have been identified, the mechanistic connection between translocation and duplex unwinding remains unclear. A conserved β-turn in the RecA1 domain, the hook-turn, has been shown to be crucial for Prp43 to unwind double stranded RNA and disassemble ILSs (Tauchert et al. 2017). The interaction of a loop, termed C-loop, protruding from the HB domain with the 5′ RNA region turned out to be required for the unwinding activity of Prp43. In Prp2, this C-loop interacts with a loop in the RecA2 domain instead (Figure 6). As the sequence of the C-loop differs between the four spliceosomal DEAH-box helicases but is conserved in each individual helicase among different organisms, it likely plays a role in regulating their unwinding activity (Hamann et al. 2021).
The conformation of the 5′ RNA region is determined by the C-terminal loop. (A) Superposition of the ctPrp2-ADP-BeF3−-RNA (gray, 6zm2) and ctPrp43-ADP-BeF3−-RNA structures (red, 5lta) via the helicase core shown as cartoon models. The 3′ region of the RNA is bound almost identically by ctPrp43 and ctPrp2, while the interactions with the 5′ region differ. (B) In ctPrp43, the base of U3 interacts with P557 and S555 of the C-terminal loop. (C) In ctPrp2, the C-terminal loop adopts an alternative conformation and does not interact with the RNA. This conformation is stabilized by hydrogen bonds with N548 in the RecA2 and R811 in the HB domain. Adapted from Hamann et al. (2021).
A direct interaction with double-stranded RNA regions might not be necessary for the spliceosomal DEAH-box ATPases to fulfill their functions. As they are located at the periphery of the spliceosome, they are rather distant to their folded targets which are often buried within large RNPs and thus inaccessible by direct translocation (De Bortoli et al. 2021). Therefore, a mechanism described as winching has been suggested, where the accessible single stranded region of the RNA is pulled through the DEAH-box ATPase. The resulting force is transmitted to the site of remodeling at the interior of the RNP, leading to rearrangements and conformational changes distant from the initial binding site (Semlow et al. 2016). However, a recent cryo-EM structural model showing the transformations from the human Bact to the B* spliceosome is not in line with such a winching mechanism for Prp2 (Schmitzova et al. 2023). Due to the inactivation of the SF1 helicase Aquarius the activation of the spliceosome stalls in a complex half of the way from Bact to B*. In this complex, denoted BAQR, Prp2/DHX8 has moved about 85 Å deep towards the spliceosome core by translocating about 19 nucleotides along the pre-mRNA and removing the proteins RBMX2 and SF3B1 from the intron. This cryo-EM structure also revealed atomic details of several fragments of the N-terminal domain (NTD) of Prp2, suggesting its role as a brake that might ensure termination of translocation (Schmitzova et al. 2023).
5.3 RNA-loading mechanism
Crystal structures of Prp43 in complex with the ATP analog ADP-BeF3− showed a conformation where the RNA binding tunnel opens up to form a groove that could facilitate RNA loading (Figure 7). In these structures, the center of mass of the C-terminal HB and OB domains is shifted by approximately 15 Å with respect to the helicase core while the WH domain acts as a hinge between the RecA2 and the HB domain (Tauchert et al. 2017). In contrast, the tunnel is fully closed in the ADP-bound state and encloses the ssRNA in an RNA-bound structure in presence of ADP-BeF3− (Tauchert et al. 2016, 2017). The open conformation would allow RNA binding in one single step, instead of inserting the strand through the narrow opening formed by the RecA2 and the HB and OB domains. Thereby, single stranded regions that are enclosed by folded RNA or RNA-bound proteins could be accessed. The functional relevance of the open tunnel conformation was demonstrated by introducing an artificial covalent link between the RecA and C-terminal domain preventing domain movement (Tauchert et al. 2017), and it was also observed by means of single molecule FRET (Enders et al. 2023).
Conformational dynamic of the RNA binding tunnel. Crystal structures of ctPrp43 + ADP (5d0u; Tauchert et al. 2016), ctPrp43 + U7-RNA + ADP-BeF3− (5lta; Tauchert et al. 2017) and ctPrp43 + ADP-BeF3− (5ltj, 5ltk; Tauchert et al. 2017) are shown as surface representation and colored as in Figure 2B. Structures are ordered according to the degree of C-terminal displacement. The RNA binding tunnel which is indicated in the ctPrp43 + ADP and ctPrp43 + U7-RNA + ADP-BeF3− structures opens into a cleft in the ctPrp43 + ADP-BeF3− structures. Adapted from Tauchert et al. (2017).
6 Regulation of substrate specificity and catalytic activity
The careful regulation of DEAH-box ATPases is essential for normal cellular function, as they play crucial roles in various aspects of RNA metabolism and their dysregulation is often connected to disease (Steimer and Klostermeier 2012). They show intrinsically low specificity for their RNA substrates because they primarily interact with the sugar-phosphate backbone (Ozgur et al. 2015; Sloan and Bohnsack 2018). Therefore, mechanisms that reduce indiscriminate interactions and ensure target specificity are needed. Additionally, some spliceosomal DEAH-box ATPases, such as Prp43 and Prp2, show inherently low enzymatic activity, the modulation of which provides an additional layer of regulative potential (Sloan and Bohnsack 2018). While regulation of RNA helicases can be achieved by various means, the most common mechanism is through the interaction with cofactor proteins that bind to the helicase to recruit them to their site of action and in many cases also modulate their activity (Bohnsack et al. 2021; Sloan and Bohnsack 2018). One potential interaction site for specific recruitment of DEAH-box proteins to the spliceosome is their N-terminal extension, as this region shows the least conservation between these otherwise highly similar proteins. The activity of two of the spliceosomal DEAH-box ATPases, Prp2 and Prp43, is regulated through the interaction with a class of specialized cofactor proteins, known as G-patch proteins (Roy et al. 1995; Tanaka et al. 2007).
The G-patch protein family comprises a variety of highly diverse proteins that only share the eponymous 40–50 amino acid-long glycine-rich consensus motif and a general implication in RNA metabolism (Aravind and Koonin 1999; Bohnsack et al. 2021). All G-patch proteins contain only a single consensus motif that is always located in an intrinsically disordered region. Besides seven highly conserved glycine residues, the G-patch motif contains three hydrophobic patches and an invariable aromatic amino acid following the second glycine (Figure 8). While five different G-patch factors have been identified in yeast, the increased regulatory complexity of higher eukaryotes is reflected by the repertoire of more than 20 human G-patch proteins (Bohnsack et al. 2021). The majority of these proteins has been shown to be involved in the regulation of Prp43 in yeast or its human homolog DHX15. The only other interactions characterized so far involve a single G-patch partner for Prp2 and its human homolog DHX16 (Hegele et al. 2012; Roy et al. 1995). For several human G-patch proteins, the target helicase remains to be identified (Bohnsack et al. 2021).
The G-patch motif. Sequence alignment of the G-patch domains of Spp2 from C. thermophilum, and SUPGP1 and NKRF from H. sapiens together with the G-patch domains of all G-patch proteins from S. cerevisiae and H. sapiens known to interact with Prp2 or Prp43/DHX15 in the spliceosomal context. Conserved hydrophobic residues are highlighted in yellow, glycine residues in red and the invariable aromatic residue in blue.
6.1 Helicase G-patch interactions in pre-mRNA splicing
In yeast, the remodeling action of Prp2 during the transition from the Bact to the B* complex strictly depends on the G-patch protein Spp2 (Kim and Lin 1996; King and Beggs 1990; Roy et al. 1995; Warkocki et al. 2015). While the G-patch protein is not required to recruit the ATPase to its binding site in the spliceosome, their interaction couples the ATPase activity of Prp2 to the remodeling of the Bact complex (Bai et al. 2021; Warkocki et al. 2015). In in vitro experiments, Spp2 was shown to stimulate Prp2’s ATPase activity in presence of ssRNA without affecting its affinity towards RNA (Warkocki et al. 2015). The human counterparts, DHX16 and GPKOW, directly interact involving the G-patch domain of GPKOW and have both been identified in the human Bact spliceosome (Hegele et al. 2012; Zang et al. 2014). Interestingly, mutations in the G-patch domain of GPKOW do not impair pre-mRNA splicing, indicating a different mechanism than in yeast spliceosomes (Zang et al. 2014).
Out of the four G-patch partners of Prp43 in yeast, only Ntr1 acts in the functional context of the spliceosome. It is part of the NTR complex and recruits Prp43 to the spliceosome, where the helicase mediates the disassembly of the ILS in the late states of splicing (Fourmann et al. 2016; Tanaka et al. 2007; Wan et al. 2017) and where it disrupts stalled spliceosome complexes associated with suboptimal or mutated pre-mRNA substrates (Fourmann et al. 2017; Mayas et al. 2010). In this context, the C-terminal domain of Ntr1 hinders a productive interaction of its G-patch motif with Prp43 in functional spliceosomes before the ILS stage, preventing their premature disassembly (Fourmann et al. 2017). In human cells, DHX15 interacts in a G-patch dependent manner with the Ntr1 homolog TFIP11 during ILS disassembly, implying a functional conservation (Yoshimoto et al. 2009). In early spliceosomal complexes, the G-patch proteins SUGP1, RBM5, RBM17 and ZGPAT have been identified as interaction partners of DHX15. While SUGP1 is involved in correct branch site recognition, RBM5 and RBM17 are regulators of alternative splicing and ZGPAT plays a role during tri-snRNP maturation (Bohnsack et al. 2021; Chen et al. 2017; Corsini et al. 2007; Niu et al. 2012; Zhang et al. 2022).
6.2 Binding mode of the G-patch motif
While the G-patch motif was easily identified as minimal interacting region of G-patch proteins, the corresponding interaction sites on the ATPase were only recently unambiguously characterized. Crystal structures of three ATPase-G-patch complexes show the almost identical interaction mode of Prp2-Spp2(gp) from Chaetomium thermophilum (ct), human DHX15-NKRF(gp) and DHX15-SUGP1(gp) (Figure 9A) (Hamann et al. 2020; Studer et al. 2020; Zhang et al. 2022). In all complexes the mostly unstructured G-patch motif reaches across the backside of the RNA binding channel, connecting the WH and RecA2 domains of the ATPase. Only the N-terminal part of the peptide forms a short α-helix that binds to a hydrophobic patch on the WH domain (Figure 9B). A loop at the C-terminus of the peptide constitutes the second major interaction surface and packs into a hydrophobic cleft on the RecA2 domain (Figure 9B). Here, the highest conformational variation in the ATPase-G-patch interaction can be found, as the arrangement of this loop differs slightly between the three G-patches and it even adopts two alternative conformations in the five available structures of the Prp2-Spp2(gp) complex (Hamann et al. 2020). The connecting linker region barely interacts with the ATPase, especially the conserved eponymous glycine residues make only few contacts. However, they are mainly responsible for the flexibility of this region, allowing conserved hydrophobic sidechains to contact hydrophobic cavities on the ATPase surface. The N-terminal helix contributes most to the overall binding affinity and is strictly required for complex formation while the C-terminal loop provides a weaker interaction site (Hamann et al. 2020; Studer et al. 2020). Both major interaction sites show strong sequence conservation between the two G-patch proteins and gp-interacting ATPases. Comparison to the cryo-EM map of the scPrp43-Ntr1 complex in the ILS shows a similar binding mode of the G-patch motif, suggesting a common interaction strategy (Hamann et al. 2020; Studer et al. 2020; Wan et al. 2017). Recent cryo-EM structures of isolated scPrp2 in complex with full-length Spp2 as well as scPrp2 in the spliceosomal context of the Bact complex also show two major interaction sites between the ATPase and its G-patch cofactor (Bai et al. 2021). The C-terminal interaction patch of Spp2 is virtually identical to the C-terminal loop characterized in the crystal structures. While the N-terminal site comprises the α-helix observed in the crystal structures, it extends beyond the conserved G-patch motif and includes a second α-helix. The residues of Prp2 that interact with this second helix are not conserved in Prp43 (Bai et al. 2021).
Binding mode of the G-patch motif. (A) The crystal structures of the ctPrp2-ADP-ctSpp2(gp) complex (left) and hsDHX15-ADP in complex with NKRF(gp) or SUGP1(gp) (right) are shown as cartoon models, with ctPrp2 and hsDHX15 displayed semi transparently and domains colored as in Figure 2B. Two alternative conformations of ctSpp2(gp) were observed in different crystal structures, representatives of both conformations are displayed in different shades of red (C1: 6rm8; C2: 6rm9; Hamann et al. 2020). NKRF(gp) (6sh6; Studer et al. 2020) and SUGP1(gp) (8ejm; Zhang et al. 2022) are colored in yellow and teal, respectively. (B) Overview of hydrophobic interactions between ctSpp2(gp) and ctPrp2. Hydrophobic residues of ctSpp2(gp) are colored yellow, while hydrophobic residues of ctPrp2 within 8 Å of the ctSpp2(gp) residues are highlighted in green. Glycine residues of ctSpp2(gp) are represented as red spheres. Adapted from Hamann et al. (2020).
The two remaining spliceosomal DEAH-box ATPases, Prp16 and Prp22, might be regulated by protein cofactors utilizing a similar mode of binding as observed for G-patch proteins. Although both proteins display disruptive substitutions at the N-terminal interaction site, excess portions of the cryo-EM map that occupy the space where the G-patch motif binds in the crystal structures is present in the cryo-EM 3D reconstructions of the respective spliceosomal complexes (Fica et al. 2017; Galej et al. 2016; Liu et al. 2017; Studer et al. 2020; Zhan et al. 2018).
6.3 Mechanism of regulation
Spp2 is thought to stimulate the ATPase activity of RNA-bound Prp2 by restricting the interdomain movements of the helicase core required for subsequent cycles of ATP hydrolysis (Bai et al. 2021; Warkocki et al. 2015). Comparison between high resolution cryo-EM derived atomic models in presence and absence of Spp2 revealed no apparent conformational changes in the ATPase. In consequence, the G-patch protein might mainly activate Prp2 by stabilizing its otherwise weak spliceosomal association through additional sites of contact (Bai et al. 2021).
All characterized G-patch partners of Prp43 stimulate the enzymatic activity of the helicase and enhance its affinity for ssRNA. Additionally, their isolated G-patch motifs can bind to the helicase and are sufficient for efficient stimulation (Chen et al. 2014; Christian et al. 2014; Fourmann et al. 2017; He et al. 2017; Heininger et al. 2016; Lebaron et al. 2009; Robert-Paganin et al. 2017; Tauchert et al. 2017). Interestingly, the identity of the G-patch motif is not connected to the cellular context as the G-patch motif of Pfa1 can replace Ntr1(gp) during ILS disassembly (Fourmann et al. 2017). Therefore, the G-patch motif seems to primarily stimulate Prp43 in its functional context, whereas guidance to the target sites involves the complete G-patch proteins. It was shown in smFRET experiments that interaction with a G-patch protein clearly modulates the domain motility of the helicase (Enders et al. 2022). The G-patch motif stimulates the ATPase activity by inducing an open conformation of the RecA domains that facilitates ADP release. As the interaction with ssRNA is the weakest in the ADP-bound state, the G-patch accelerates the transition to strong RNA binding during continuous ATP turnover, greatly reducing dissociation. Thereby, the G-patch interaction enables processive translocation and unwinding (Enders et al. 2022). The impact of the G-patch protein NKRF on the domain flexibility of DHX15, the human Prp43, was recently analyzed by MD simulations, revealing a more structured conformational ensemble with reduced flexibility of the relative domain arrangements in presence of the G-patch (Becker and Hub 2023b). Furthermore, the G-patch protein leads to a more stable open RNA tunnel, which is in line with recent smFRET data (Enders et al. 2023).
7 Conclusion and outlook
The four DEAH-box ATPases Prp2, Prp16, Prp22 and Prp43 are essential driving motors of the spliceosome. Our understanding of their function and regulation has tremendously improved within the past 10 years due to complementary structural, biochemical and biophysical studies. With exception of Prp2 all spliceosomal DEAH-box ATPases exhibit helicase activity, even though their function in the spliceosome appears to be winching a single stranded RNA and thereby inducing conformational changes and rearrangements inside the spliceosome. The activity of Prp2 and Prp43 is controlled by the G-patch proteins Spp2 and Ntr1, respectively, which also recruit these DEAH-box proteins to the corresponding target sites in the periphery of the spliceosome. However, Prp16 and Prp22 associate with the spliceosome via other protein-protein interactions, and it remains to be clarified whether these interactions also stimulate their ATPase and RNA-translocation activities. Prp16, Prp22 and Prp43 also contribute to the fidelity of the pre-mRNA splicing process, and a malfunction might play a role in various diseases, e.g., as shown for mutations in DHX38 (Prp16) and retinitis pigmentosa (Ajmal et al. 2014; Obuca et al. 2022).
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Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
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Research funding: Our work was funded by the Deutsche Forschungsgemeinschaft (DFG) SFB 860 (A02).
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Conflict of interest statement: The authors declare no conflicts of interest regarding this article.
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