Helicases are omnipresent enzymes, spread through the whole phylogenetic tree of life as well as viruses, since their unwinding capabilities are of striking importance for numerous cellular pathways of RNA and DNA metabolism. Based on sequence alignments, helicases were subdivided into six superfamilies (SF) which come in two different flavors (Singleton et al., 2007; Fairman-Williams et al., 2010; Jankowsky and Fairman, 2007; Pyle, 2011). Most members of the SF1 or SF2 are monomeric, in contrast to the members of SF3 to SF6 which assemble as hexamers (Singleton et al., 2007). The latter are composed of either RecA- or AAA⁺-like domains, thus being distinct from the SF1 and SF2 helicases which are exclusively composed of two RecA-like domains in juxtaposition with each other.

The SF2 is by far the largest superfamily, comprising a vast number of RNA helicases with implications in numerous RNP metabolism pathways, such as translation initiation and termination, pre-mRNA editing and splicing, mRNA export as well as ribosome biogenesis (Ozgur et al., 2015). The centerpiece of these helicases is formed by the two RecA-like domains, harboring conserved motifs which are crucial for these NTPase-dependent RNA helicases to accomplish their function. The motifs I, II, V and VI are required for nucleoside triphosphate binding and hydrolysis, motifs Ia, Ib and IV are involved in RNA binding and the motif III participates in coupling the NTPase and the unwinding activity (Caruthers and McKay, 2002; Cordin and Beggs, 2013; Cordin et al., 2012). Most SF2 helicases exhibit extensions at the N- and C-termini of the helicase core, which is also true for the DEAH-box proteins, a subfamily of the DEAH/RHA family. The C-terminal extension is highly conserved in DEAH-box helicases and is composed of three domains: a winged helix (WH), a ratchet and an oligosaccharide binding (OB) fold (Walbott et al., 2010; He et al., 2010). The ratchet domain was originally named according to the homologous domain of the DNA helicase Hel308 (Büttner et al., 2007), but recent studies on MLE suggested that this domain has no ratcheting function and should be renamed (Prabu et al., 2015). Therefore it is hereinafter referred to as ratchet-like domain. In DEAH-box proteins the degrees of freedom of the helicase core are reduced by the interactions between the C-terminal domains and the two RecA-like domains in comparison to those DEAD-box proteins lacking such C-terminal domains. The conservation of the N-terminal extension is distinctly lower and they dramatically differ in length between the members of this subfamily. Reported functions for this N-terminal extension are, among others, the involvement in the recruitment as shown for Prp16 (pre-mRNA processing factor 16) as well as for Prp22 or for the subcellular targeting to the nucleus as elucidated for Prp43 (Wang and Guthrie, 1998; Schneider and Schwer, 2001; Fouraux et al., 2002).

The latter mentioned helicase, Prp43, is an outstanding member of the DEAH-box subfamily since it has implications in different substantive cellular processes. The first reported function of Prp43 is its involvement in pre-mRNA splicing. In the course of this process, Prp43 acts at the latest stage of the splicing cycle and it is required to dismantle the intron-lariat spliceosome into the excised lariat and the U2•U5•U6 snRNPs (Arenas and Abelson, 1997; Fourmann et al., 2013, 2016a). Recently, the target substrate of Prp43 during this process was revealed which is the RNA network between the U2 snRNP and the branch site of the intron (Fourmann et al., 2016b). Indeed, in the spliceosome, Prp43 was crosslinked exclusively to the pre-mRNA and not to any snRNAs. Furthermore, Prp43 is also a key player for the disassembly of stalled spliceosomes (Pandit et al., 2006; Semlow and Staley, 2012; Koodathingal et al., 2010). During these spliceosomal processes, Prp43 binds RNA in a sequence-independent manner. Prp43 action is also essential for ribosome biogenesis. Here, it is involved in the maturation of 18S and 25S rRNAs with several reported binding sites on intermediates which vary in sequence and thus make a sequenced-based binding mechanism unlikely for Prp43 (Lebaron et al., 2005; Bohnsack et al., 2009). Lately, the involvement of Prp43 in a third distinct cellular function was illuminated and it was unraveled that Prp43 is also involved in the promotion of apoptosis (Heininger et al., 2016).

The distribution of Prp43 between these three fundamentally different processes is regulated by a fine-tuned interplay of the helicase itself and cofactors. These cofactors belong to the family of G-patch proteins, which share an intrinsically disordered glycine-rich region, the G-patch motif (Aravind et al., 1999). Apart from being involved in recruitment, these G-patch proteins also increase the NTPase and stimulate the helicase activity of Prp43. To date, in S. cerevisiae five G-patch proteins were identified out of which four interact with Prp43. In the context of splicing, Prp43 function is modulated by Ntr1 (Nineteen complex-related proteins 1) (Tanaka et al., 2007; Tsai et al., 2005, 2007; Boon et al., 2006). During rRNA maturation, Prp43 is associated with Pfa1 (Prp forty-three associated 1) or Gno1 (G-patch nucleolar protein 1) (Lebaron et al., 2005, 2009; Bohnsack et al., 2009). Moreover, its apoptotic function is implemented by the interaction with Cmg1 (Cytoplasmic and mitochondrial G-patch protein 1) (Heininger et al., 2016). The remaining G-patch protein from yeast is Spp2 (Suppressor of PRP protein 2) which interacts with another spliceosomal DEAH-box helicase, Prp2 (Warkocki et al., 2015).

Despite intensive studies, the exact modus operandi of the DEAH-box helicases and thus of Prp43 is still elusive to date. This is primarily attributable to a lack of crystal structures of members of the DEAH-box subfamily in an active state and with bound interaction partners. Recently, the crystal structure of the related DExH-box helicase MLE (Maleless), which belongs to another DEAH/RHA subfamily, provided a first insight into how this helicase might function. The complex of MLE with a U₁₀-RNA and ADP•AlF₄^- led to a proposed mechanism of how RNA is translocated through its interior tunnel driven by conformational changes of a Hook-Loop in the RecA2 domain (Prabu et al., 2015).

So far, three crystal structures of Prp43 in the post-catalytic ADP-bound state have been available from S. cerevisiae or C. thermophilum (Walbott et al., 2010; He et al., 2010; Tauchert et al., 2016). The latter mentioned organism might be a valuable alternative for structural studies on DEAH-box proteins, since several crystal structures of spliceosomal RNA helicases from this thermophilic fungus were solved only recently (Tauchert et al., 2016; Tauchert and Ficner, 2016; Absmeier et al., 2015). Owing to this, intensive crystallization trails with Prp43 from C. thermophilum were performed leading to three new crystal structures. All three structures show Prp43 in an active state, since it was crystallized with a non-hydrolysable ATP-analog (ADP•BeF₃^-) as well as one of these structures show Prp43 with an additional U₇-RNA. The Prp43•U₇-RNA•ADP•BeF₃^-complex unveils the structural basis of why Prp43 can bind RNA in a sequence-independent manner. The Prp43•ADP•BeF₃^- structures illustrate how Prp43 binds and releases complex RNA substrates due to massive rearrangements of the C-terminal domains which affect an opening of the interior RNA-binding tunnel which is formed by the two RecA-like and the C-terminal domains. The hydrolysis of an ATP molecule induces conformational rearrangements of the RecA-like domains which, in turn, allow the translocation of the RNA through the interior tunnel via the so far unrecognized RF motif present in a β-turn of all DEAH-box helicases. According to our results, the translocation mechanism of MLE is very unlikely to be compatible for Prp43. All proposed functional mechanisms of Prp43 are strengthened by functional analyses of structure-based mutants in this study.

Prp43 was known to bind RNA in a sequence-independent manner which is explained by the ctPrp43ΔN•U₇•ADP•BeF₃^- complex structure revealing the almost entire absence of interactions between the bases of the RNA and Prp43. The observed K_D value in the μM range suggests that the RNA binding of Prp43 is transient, which is in line with the low number of hydrogen bonds primarily formed by four out of the seven nucleotides, and the high B-factors for U1-U3 and their alternative conformations. However, this unselective and transient binding mode of RNA by Prp43 is perfectly in line with its molecular function. Prp43 helicase activity is required in several distinct cellular processes. Target sites in rRNA maturation and pre-mRNA splicing are very different and thus selective RNA binding would not be compatible with Prp43’s mode of function. Furthermore, a tight binding of substrate RNA would be undesirable since it would decrease the efficiency of Prp43 and prolong the required time for the release of substrate RNA.

The comparison of the RNA-binding mode of Prp43 with that of MLE, a DExH-box protein from D. melanogaster that was previously crystallized with a U₁₀-RNA and the transition state analog ADP•AlF₄^- (Prabu et al., 2015), reveals several differences but also striking similarities. Despite the fact that the architecture of the conserved domains is almost identical and the relative position of the bound RNA in the helicase is similar, MLE binds poly-uridine RNA in a sequence-dependent manner. Numerous bases are specifically recognized by MLE and a tighter and much more elaborated interaction network between MLE and the RNA is present. Nonetheless, there are also noticeable similarities concerning the RNA interactions. In Prp43, four main-chain amide interaction interactions between residues of the RecA1 or RecA2 domains and the RNA are present, one with each of the tightly bound nucleotides (U4-U7). Glu 316 interacts with U4, Gly 349 with U5, Arg 153 with U6 and Arg 180 with U7 (Figure 1e). There are also two threonine residues of conserved motifs that interact with the RNA. Thr 195 from motif Ib and Thr 381 from motif V are involved in hydrogen bonding via their sides chains with the phosphates of the nucleotides U7 and U5, respectively. Interestingly, these main-chain amide interactions are completely conserved in MLE. Despite the fact that the residues themselves are not conserved in MLE, the position at which the interaction takes place is virtually identical (Figure 1—figure supplement 4). The corresponding interacting residues and nucleotides in MLE are Trp 663-U6, Ser 692-U7, Arg 443-U8 and Arg 470-U9. Furthermore, the interactions with the conserved threonine residues are also present in MLE (Thr 486-U9 and Thr 717-U7). Moreover, there are three additional side chain interactions which are conserved between Prp43 and MLE. The interaction of Arg 201 (motif Ib) with the ribose of the U7, the hydrogen bonding of Asn 382 with the ribose of U4 and the interaction of Lys 403 with the phosphate moiety of U4 in Prp43 have identical counterparts in MLE. These counterparts are Arg 492-U9, Asn 718-U6 and Arg 739-U6.

Having unraveled the conserved interactions with RNA between the two members of the DEAH/RHA subfamilies, the question arose whether these interactions are present also in the related family of viral NS3/NPH-II helicases. Members of the NS3/NPH-II family also contain a helicase core composed of two RecA-like domains with the protruding β-hairpin in the RecA2 domain. In the NS3/NPHII helicase family there is only one C-terminal domain present, which is not homologous to any C-terminal domain of the DEAH/RHA helicases, and no RNA-binding tunnel is formed. Owing to the fact that numerous NS3 helicases are encoded by human pathogenic viruses, such as hepatitis C, Zika or yellow fever, these helicases have been subject of intensive studies and various crystal structures are available. The comparison with Prp43 was restricted to the NS3 hepatitis C virus (HCV) helicase, as NS3 HCV binds RNA also in a sequence-independent manner and a crystal structure with a bound ATP-analog (ADP•BeF₃^-) and U₈-RNA is available making a direct comparison with Prp43 very feasible (PDBid: 3o8r) (Appleby et al., 2011). The comparison between Prp43 and NS3 HCV revealed that the unspecific interactions between the RNA backbone and the main-chain amides as well as the two interactions with the conserved threonines from motif Ib and V are also present in NS3 HCV (Figure 1—figure supplement 4). In NS3 HCV, the main-chain amides of the residues Lys 371, Arg 393, Val 232 and Gly 255 interact with the nucleotides U4-U7 and Thr 269 as well as Thr 411 are involved in hydrogen bonding via their site chains with U7 and U5, respectively. Hence, this RNA-binding mode appears to be conserved for the sequence-unspecific interaction of the RecA-like domains of several members of the DEAH/RHA and NS3 families. However, there are also differences in the RNA binding between the DEAH/RHA and NS3 families, e.g. a functionally important tryptophan of NS3 members (Trp 501 in NS3 HCV), which stacks on the nucleotide at the 3’ end, is not conserved in DEAH-box helicases.

With the set of new Prp43 structures novel insights into the mechanism of RNA loading are provided. Considering the fact that the RNA-binding site of Prp43 is in its interior tunnel and the circumstance that the substrates of Prp43 are highly folded RNA networks, RNA loading to Prp43 is not trivial. The Prp43 target sites in the spliceosome and in the pre-ribosomal subunits are surrounded by complex tertiary structures without single-stranded overhangs. Here, we could demonstrate that in the ATP-bound state Prp43 is capable of adopting an open conformation in which numerous interactions between the RecA-like domains and the C-terminal domains are disrupted converting the RNA-binding tunnel into a groove and thus enabling Prp43 to bind to single-stranded RNA regions within folded RNA stretches (Figure 3). This rearrangement of the C-terminal domains of Prp43 with bound ADP•BeF₃^- has not been observed in any other crystal structure of a DEAH- or DExH-box helicase, which demonstrates that Prp43 exhibits a higher degree of flexibility as initially expected from the previously determined crystal structures of Prp43•ADP complexes.

Since structures of both the ATP- and ADP-bound state of Prp43 are now available, an in-depth analysis of conformational rearrangements which occur at the active site is possible. One can assume that these rearrangements almost exclusively occur after the release of the γ-phosphate owing to the fact that the helicase cores of Prp43 with the ATP-analog ADP•BeF₃^- and of MLE (chain A) with the transition state analog ADP-AlF₄^- superpose very well with an r.m.s.d. value of 0.70 Å for 235 C_α (Figure 6—figure supplement 1). ATP hydrolysis causes the movement of one β-turn and one loop, located at the RNA-binding site of the RecA1 and RecA2 domain, respectively (Figure 7a). The β-turn in the RecA1 domain, the Hook-Turn, was shown to be of crucial importance for Prp43’s helicase activity, its capability to disassemble ILSs and also for its RNA-induced ATPase activity. In contrast to this, the Hook-Loop, located in the RecA2 domain, does neither affect the unwinding activity nor the ATPase activity of Prp43. Remarkably, the integrity of this Hook-Loop was known to be essential for the helicase activity of MLE and it was therefore proposed that the Hook-Loop is presumably required in all DExH- but also all DEAH-box helicases for the translocation of RNA (Prabu et al., 2015). Unexpectedly, but according to our results, the difference in the mechanism of how DEAH-box and DExH-box helicases do translocate RNA through their interior tunnel seems to be tremendous. For Prp43, exclusively the interaction between the Hook-Turn and the RNA is required for successful translocation of the RNA. The mutation of the Hook-Loop, which is essential in MLE, is effectless in Prp43. Furthermore, the Hook-Turn is not required for RNA binding but it is involved in the RNA-induced stimulation of the ATPase activity. This is supported by the finding that the ctPrp43-IDSB mutant still exhibits RNA-stimulated ATPase activity. For this mutant, RNA binding in the tunnel is unlikely but the stimulation might be caused by the binding of the single stranded RNA only to the tunnel exit as this is formed, among others, by the Hook-Turn.

To further elaborate the difference in the RNA-translocation mechanism between the DEAH- and DExH-box subfamilies the conservation of the Hook-Turn and the Hook-Loop residues in family members from S. cerevisiae, ctPrp43 and MLE was analyzed (Figure 7—figure supplement 1). The RF motif the Hook-Turn is conserved in all DEAH-box helicases, while in the DExH-box helicases, this motif is only present in MLE. Between DEAH-box and DExH-box helicases, the Hook-Loop is not conserved (Yxx vs. Hxx), but conservation among the individual members is noticeable. These findings are perfectly in line with our interpretation of the activity of the Hook-Turn and Hook-Loop mutants, which clearly hint at the fact that the RNA-translocation mechanism between DEAH- and DExH-box helicases is strikingly different. The Hook-Turn is presumably the key feature of genuine DEAH-box helicases for RNA translocation through the interior binding tunnel and the Hook-Turn RF motif should be added to the list of conserved DEAH-box helicase motifs. Like for MLE, the structure of the Prp43-RNA complex does not provide any indication for a ratcheting function of the C-terminal helical-bundle domain, which supports the idea of renaming this domain.

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Author details

1. Marcel J Tauchert

   Department of Molecular Structural Biology, Institute for Microbiology and Genetics, GZMB, Georg-August-University Göttingen, Göttingen, Germany

   Contribution

   MJT, Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing—original draft, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

2. Jean-Baptiste Fourmann

   Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

   Contribution

   J-BF, Formal analysis, Investigation, Visualization, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

3. Reinhard Lührmann

   Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

   Contribution

   RL, Supervision, Funding acquisition, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-6403-4432

4. Ralf Ficner

   Department of Molecular Structural Biology, Institute for Microbiology and Genetics, GZMB, Georg-August-University Göttingen, Göttingen, Germany

   Contribution

   RF, Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Project administration, Writing—review and editing

   For correspondence

   rficner@uni-goettingen.de

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-1739-6086

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