Structural basis of catalytic activation in human splicing

Pre-mRNA splicing follows a pathway driven by ATP-dependent RNA helicases. A crucial event of the splicing pathway is the catalytic activation, which takes place at the transition between the activated Bact and the branching-competent B* spliceosomes. Catalytic activation occurs through an ATP-dependent remodelling mediated by the helicase PRP2 (also known as DHX16)1–3. However, because PRP2 is observed only at the periphery of spliceosomes3–5, its function has remained elusive. Here we show that catalytic activation occurs in two ATP-dependent stages driven by two helicases: PRP2 and Aquarius. The role of Aquarius in splicing has been enigmatic6,7. Here the inactivation of Aquarius leads to the stalling of a spliceosome intermediate—the BAQR complex—found halfway through the catalytic activation process. The cryogenic electron microscopy structure of BAQR reveals how PRP2 and Aquarius remodel Bact and BAQR, respectively. Notably, PRP2 translocates along the intron while it strips away the RES complex, opens the SF3B1 clamp and unfastens the branch helix. Translocation terminates six nucleotides downstream of the branch site through an assembly of PPIL4, SKIP and the amino-terminal domain of PRP2. Finally, Aquarius enables the dissociation of PRP2, plus the SF3A and SF3B complexes, which promotes the relocation of the branch duplex for catalysis. This work elucidates catalytic activation in human splicing, reveals how a DEAH helicase operates and provides a paradigm for how helicases can coordinate their activities.

2. Representations of pink, green, and broken lines should be indicated in Fig. 3a.
3. P.2, line 39: It is not clear to me how the authors determined that PRP2 translocates 18 nucleotides from the periphery towards the spliceosome core.Is the region of the pre-mRNA that PRP2 binds to on the Bact structure clearly visualized so that the distance of the translocation can be measured?4. P.3, line 53: The description of "The first reaction………within the C complex spliceosome" is imprecise.The C complex is the product of the first reaction, and is a stable form of the complex that can be isolated and analyzed.

P.3, line 68:
The authors appear unaware of and have failed to cite previous studies on yeast related to how PRP2 may drive catalytic activation (Liu, 2012).Two other DEAH RNA helicases, PRP16 and PRP22, have also been characterized in yeast, and a similar mechanism for remodeling the spliceosome in the second catalytic step and for the release of mature mRNA has been proposed (Chung, 2019;Schwer, 2008).The authors should discuss if this mechanism is general for DEAH RNA helicases.6. P.5, line 120: How can one explain the observation that PRP2 on Sc Bact exhibits the conformation corresponding to a post-ATP hydrolytic state prior to PRP2 activity?7. P.10, line 238: PRP2….translocated to "an upstream" position.8. P.12: "SPP2" instead of "Spp2" for consistency.9. P.12: This result is consistent with a previous report that the NTD of Sc PRP2 is dispensable (Edwalds-Gilbert, 2004), which should be included in the Discussion.
10.The model in Fig. 5b should be explained in more detail, in particular, how the hypothetical intermediate is generated.It would also be more appropriate to place the summary of the respective results in the legend of Fig. 5b than in the Discussion.

Referee #2:
This manuscript provides critical new concepts to our understanding of spliceosomal activation, i.e., the process by which, just prior to catalytic step one, the first-step nucleophile -the branch site adenosine complexed with U2 snRNA -is released from SF3b1 and moves 50A into the active site.
The authors present a ~2.9A cryo-EM structure of a human spliceosomal complex stalled by a mutation in the ATPase Aquarius.This represents a previously unknown intermediate in human spliceosome assembly/activation, which is not present in the yeast system (which does not have an Aquarius ortholog).The manuscript provides key new insights into the function of both PRP2 and Aquarius ATPases in spliceosome activation.Lines 130-131, "Whether DEAH-box helicases, including PRP2, pull their substrates from a distance or by stepwise translocation along the RNA strand remained unclear.":This is a big point, and I want to reiterate it and say that I entirely agree with the authors' assertion here.I don't think that we have known what any spliceosomal ATPase really does, and this is a big step forward in understanding PRP2 function.By stalling the complex at the AQR step, the authors can, for the first time, infer exactly what PRP2 translocates on: "PRP2 advanced 18 nts [on the pre-mRNA] towards the spliceosome core during the Bact-to-BAQR transition".I think that this point and its concomitant effects on the structure are incredibly important and an appropriate topic of a paper in Nature.Overall, this is one of the few spliceosome structural papers in which we actually learn new, unanticipated features of the mechanism.Specific Comments: 1. Line 83, "Following up on our previous work 6": This needs a bit more explanation.
2. The mechanism of translocation termination of PRP2 by "molecular brake elements" proposed by the authors is very interesting.It would be useful to (briefly) compare this to what is known about the termination of translocation of other helicases.For example, is it similar, or not, to the mechanism by which MAGOH-Y14 inhibits eIF4AIII and locks it onto RNA within the exon junction complex (Ballot et al., NSMB, 2005; PMID 16170325)?
3. The action of the ATPases as molecular clamps seems a lot like what was proposed by Melissa Moore for DExH/D proteins (Shibuya et al., NSMB, 2004;PMID 15034551).
4. Line 377, Fig. 1, "Key subunits are color-coded": This could be clarified.I understand that the colors in the molecule structure schematic are a rough match with the colors for the components list, shown in a, lower.However, there are some inconsistencies.For example, PRP2 is shown in brown, but it is not color-matched in Fig. 1b, where PRP2 is inconsistently written in red.Why is the RES complex indicated in light blue, when the only protein component included, BUD13, is dark blue?Overall, there are too many colors and shades of colors here to be able to follow.

Lines 268-271:
There is a high richness of new detail provided throughout.However, one statement, "The two helicases must coordinate to liberate the duplex from SF3B-RES constraints and hand it over to the catalytic center, while keeping it unaltered -a task that is best accomplished sequentially, by two molecular motors", leaves me wanting more info.Why is this "best accomplished" by two molecular motors?This is a critical difference between mammals and yeast, and I think it deserves to be explained further.Here, this is stated as a fact; as such, it needs further explanation.Alternatively, this could be stated as a conjecture that is, so far, without adequate explanation.

Referee #3:
The manuscript by Schmitzova et al. presents the structure of a new spliceosome assembly intermediate, Baqr, captured via a dominant-negative mutation in the helicase Aquarius.Aquarius is the only SF1-family helicase that is involved in splicing and its function is not well understood, in particular, that it is absent in some model organisms (e.g., S. cerevisiae).The complex presented by the authors was stalled between Bact and B*/C states and provides important mechanistic insights into this transition.Some of the key findings include: 1. Dissection of the Bact to B* transition into two steps, implying that PRP2 and Aquarius act in a sequential manner.
2. PRP2 was captured in a position suggesting that it translocates along the pre-mRNA in order to displace a specific subset of splicing, including the RES complex.It was previously proposed that PRP2 would remain attached to SF3B1 and act on the branch helix by pulling from a distance, but the exact mechanism of this remodeling remained elusive.The Baqr structure provides a conceptually new mechanism for this rearrangement.
3. PRP2 action alone is sufficient to unfasten the branch helix from the SF3b complex by reopening SF3B1 and therefore priming it for further remodeling.
4. SKIP and PPIL4 associate with intron RNA exiting PRP2 and could act as a molecular brake to achieve precise control over the extent of PRP2 translocation.
Overall it is a beautiful work providing new and exciting insights into the mechanism of one of the key steps in spliceosome assembly.Technically, the biochemical and structural aspects of the work are of high quality.The manuscript is clearly written and easy to follow, with all findings accompanied by appropriate figures.
I would recommend publication of this work in Nature upon addressing some of my comments below.
Major points: 1.While I understand the authors' logic that PRP2 is required for the Bact to Baqr transition and that Aquarius is needed further down the line, I am not entirely convinced if one can simplify the whole process by stating that the two helicases act in a sequential manner.One could imagine a situation where the action of Aquarius could only be needed to enable PRP2 to continue its translocation until the branch helix is completely liberated.This could happen, for example, by translocating the remote parts of the intron upstream from the branch site to release some potential tensions in the system to allow PRP2 to continue its unwinding.In other words, it is possible that IBC-K829A mutation would keep IBC locked on one position of intron, preventing PRP2 from further translocation.What would speak in favor of such a hypothesis is the fact that Prp2 alone is capable of performing catalytic activation in some species (i.e., S. cerevisiae) without the need for Aquarius.The sequential action of the two helicases is an important point of the paper, and alternative scenarios should be at least thoroughly considered in the Discussion.Ideally, the authors should try to come up with a feasible experiment showing that PRP2 is not needed to complete the catalytic activation after initial translocation or prove that Aquarius is really needed to complete it.
2. The authors propose that SKIP and PPIL4 act as a molecular brake that stops PRP2 from unwinding branch duplex and suggest that both proteins act as negative regulators of PRP2 in a context-dependent manner.This is potentially a very important point as such a regulation mechanism is conceptually new and might also be used by other spliceosomal helicases.However, based on the structure alone, the causality remains ambiguous here (i.e., it is not clear to me if SKIP/PPIL4 could bind to PRPF2 and actively induce its opening/inhibition, or do they just associate with the repositioned PRP2, which is inhibited from translocating further by some other means).It would significantly strengthen the manuscript if the authors provided some experimental evidence that SKIP and PPIL4 can indeed influence the intrinsic activity of PRP2 (for example, by an in vitro helicase assay).

The key question that remains open is what is the role of Aquarius in the catalytic activation
process?Although Aquarius was used by the authors to stall spliceosomes, the manuscript does not discuss its role in this process as all the analyses are focussed on PRP2.What is particularly puzzling is the fact that Aquarius binds upstream of the branch site, and given that it has 3'-5' RNA unwinding activity (De et al., NSMB, 2015), it is very difficult to imagine how it could exert any force on the branch helix, which still needs to be remodeled.By translocating with this polarity, it should create slack rather than tension between itself and the branch helix (see also point 1).I believe that the authors should comment on this aspect of their work.In particular, since the title "Structural basis of catalytic activation in human splicing" implies insights into the whole process of catalytic activation, but here only part of this process was analyzed.
Minor comments: 1. Line 68, "by a mechanism that remains largely hypothetical": I would suggest rephrasing to "by a mechanism that remains largely unknown".2. Line 167, "Surprisingly, after releasing the RES complex from RNA and former protein contacts, the RES complex binds, via BUD13531-534, to a composite structure formed by the hook of PRP2NTD, the slightly relocated C-terminal MA3 domain of CWC22 and PRP8 (Fig. 3a and Extended Data Fig. 13b,c).":This is a bit misleading in suggesting that the entire RES complex is relocated, while it is only a small fragment of Bud13 that rebinds.This should be rephrased to avoid any confusion.
3. It would be helpful to see some examples of high-resolution fitting, as current cryo-EM density snapshots (Ext.Data Fig. 8) show mostly secondary structure elements and do not support highresolution data claimed by the FSC.This is relevant given the highly anisotropic angular distribution of the 3D reconstruction, which could cause a mismatch between the resolution estimate and observed features.
4. There is a numbering problem with Ext.Data Figs.(12,14,14); 13 is missing.5.In Table S2 some entries are color-coded, but the meaning of that is not explained.

Author Rebuttals to Initial Comments:
The referee's comments are in black and the point-by-point response in blue.Although the release of U1 and U4 leads to activation of the spliceosome to form the RNA catalytic core, the branch helix is still 50 Å away from the catalytic center hindered by SF3B.Biochemical and proteomic studies have revealed that PRP2 plays a crucial role in removing SF3 complexes to allow the positioning of the branch site at the catalytic center.How PRP2 functions in remodeling of the spliceosome has been studied mostly in yeast, which does not have an Aquarius homolog.The use by the authors of the Aquarius mutant to arrest the spliceosome intermediate at catalytic activation represents an excellent approach for identifying the intermediate form in the human system and delineating detailed mechanisms for this process.The results demonstrate that PRP2 translocates from the periphery of the spliceosome toward the spliceosome core to displace components binding on the branch site downstream region.These results also argue against the pulling model for the action of DEAH RNA helicases.They also echo findings from previous studies in yeast, showing that PRP2 binds to the branch site downstream region, and then translocates in a 3' to 5' direction toward the branch site to displace SF3 complexes (Liu, 2012).The structure presented in this work uncovers details of the interactions among components on BAQR and, by comparing the structure of BAQR with those of Bact and B*, reveals how PRP2 and Aquarius may function in remodeling of the human spliceosome.Moreover, it reveals an interesting two-step process for catalytic activation of the human spliceosome.Overall, this paper represents a significant advance in our understanding of the mechanism of catalytic activation, and should be of great interest to people in the splicing field.
However, the paper falls short in that the Discussion is too superficial, simply representing a summary of the results.The authors should compare their findings with previous work on yeast to explore mechanistic similarities and differences.Yeast does not have an Aquarius homolog, so PRP2 is responsible for the entire process.PRP2 has been extensively studied in yeast.A model for how PRP2 may function in catalytic activation has also been proposed.These works should be cited and discussed.
We are very thankful for these suggestions.In the revised manuscript, we dedicate an entirely new section, with a subheading, to the catalytic activation in budding yeast and the parallels with human splicing (P13-P14).We have also included a model of catalytic activation in yeast, where we integrate our findings and the previously published structural, biochemical and genetics data (Fig. 5c).
We tried to cite all essential references, especially from the labs of Soo Chen Cheng, Reinhard Lührmann, Ren Jang Lin, Yigong Shi and Aaron Hosking.
It would also be interesting to discuss if the spliceosome stalls the same way in the absence of Aquarius as when a dominant-negative mutant of Aquarius is present, and how Aquarius might work in the second phase of the process based on the structural information for B AQR .
Besides acting as a helicase, Aquarius seems to possess a structural role in maintaining the stability of the pentameric IBC, securing the recruitment of the five components to the spliceosome (De et al., NSMB, 2015).The absence of Aquarius may therefore prevent proper incorporation of the other IBC components, including SYF1, at the B-to-B act transition.Misincorporation of SYF1 (due to Aquarius's absence) might affect the microenvironment of the catalytic center, possibly preventing the formation of a functional B act complex.Two main reasons suggest this scenario.
First, SYF1 interacts extensively with SYF3, whose N-terminal helical repeats (tetratricopeptides, TPRs) bind U6 within B act , very close to the catalytic center, suggesting an essential role of the two proteins in assisting the formation of the RNA-based catalytic center.Second, the yeast homologs of SYF1 and SYF3 are essential for cell viability and belong to the Nineteen Complex (NTC).NTC in yeast is essential for the catalytic center formation (in particular for the association of U5 and U6 with the spliceosome after the dissociation of U4; Chan et al., Science 2003).
To conclude, we estimate that the absence of Aquarius might affect the B-to-B act transition due to the scaffolding function of this helicase.In contrast, Aquarius K829A stalls the spliceosome later, at the B AQR state, as it affects the helicase function.
It would also be interesting to discuss .., how Aquarius might work in the second phase of the process based on the structural information for B AQR .
In the revised manuscript, we discuss how Aquarius might work in the second phase of the catalytic activation (P12-P13); we amend the model from Fig. 5b with an additional intermediate and have added the related Extended Data Fig. 9.

Comments:
1.The diagram in Fig. 1b is misleading.It gives the impression that RES is removed from the spliceosome during the transition from Bact to BAQR, yet RES is only removed away from the pre-mRNA without being released from the spliceosome.
The RES complex loses contact with the RNA, and nearly all interfaces with the spliceosome (PRP8, and the SF3B complex), while remaining flexibly anchored via a short region of BUD13.We would refer to this event as a destabilization rather than dissociation.It is common in human spliceosomes that proteins dissociate gradually, remaining attached flexibly after fulfilling the specific function.
For example, the PRP2 helicase and, to a reasonable extent, SF3A/B proteins are still present in the composition of C complexes (presumably by flexible anchoring), although they have been structurally "undocked" from the spliceosome (see, for example, the proteomic analyses from Agafonov et al., Mol Cell Biol, 2011).Therefore, we suggest referring to destabilization events rather than dissociation, whose meaning is more stringent.To avoid confusion, we explained in the legend of Fig. 1b that we indicate the recruited or destabilized subunits and that the destabilization does not equate to complete dissociation.
2. Representations of pink, green, and broken lines should be indicated in Fig. 3a.
Pink, green, and broken lines represent the PPT, PPT region bound by SF3B1 within B act , and PPT equivalent region visible within yeast B act structure.We have added this description in the legend of Figure 3a.
3. P.2, line 39: It is not clear to me how the authors determined that PRP2 translocates 18 nucleotides from the periphery towards the spliceosome core.Is the region of the pre-mRNA that PRP2 binds to on the Bact structure clearly visualized so that the distance of the translocation can be measured?
In the revised manuscript, we explain with more clarity our analysis.We included the Extended Data Fig. 5a-e, with a detailed description of the modeling.We also corrected an error -PRP2 has translocated 19 nucleotides, not 18, as we stated before.We apologize for the error.
In short, the pre-mRNA region that Prp2p binds is visible in the yeast B act spliceosomes from Shi lab (nts 28 to 34 , PDB 7DCO, Bai et al., 2021) and consistent with the biochemistry from Soo Chen Cheng lab (Liu et al., 2012).The local resolution around PRP2 is low in Hs B act , yet good enough to enable unambiguous localization of PRP2 in a virtually equivalent position as in Sc B act (Zhang et al., 2018).The intron is visible until the nucleotide 19 in Hs B act .Superposition between the human and yeast B act complexes allows counting the missing nucleotides between 19 and PRP2's binding sites.In this way, we estimate that human PRP2 binds the nucleotides 26 -32, versus 28 -34 in yeast (there is a difference of two nucleotides between human and yeast, as the latter has a two-base insertion).Of note, the translocation of 19 nucleotides by PRP2 is consistent with the stringent requirement for at least 32-34 nucleotides downstream of the BS-A for efficient branching (Bessonov et al., RNA, 2010).

P.3, line 53:
The description of "The first reaction………within the C complex spliceosome" is imprecise.The C complex is the product of the first reaction, and is a stable form of the complex that can be isolated and analyzed.
We agree and have corrected the error by stating "The branching reaction occurs..... during the C complex formation" (P3, lines 50-52)".

P.3, line 68:
The authors appear unaware of and have failed to cite previous studies on yeast related to how PRP2 may drive catalytic activation (Liu, 2012).Two other DEAH RNA helicases, PRP16 and PRP22, have also been characterized in yeast, and a similar mechanism for remodeling of the spliceosome in the second catalytic step and for the release of mature mRNA has been proposed (Chung, 2019;Schwer, 2008).The authors should discuss if this mechanism is general for DEAH RNA helicases.
We apologize and have amended the manuscript accordingly.We acknowledge the importance of this work (Liu 2012) and other papers dedicated to the Prp2p investigation in budding yeast, and we have included a thorough analysis of the catalytic activation in budding yeast (see the new section from P13).We have also depicted a diagram of the catalytic activation in yeast (Fig. 5c), by integrating previously published data and the current work.
In principle, the DEAH helicases PRP16 and PRP22 might follow a similar mechanism as PRP2, rather than acting as winches (which would agree with the earlier models derived from yeast splicing).
In particular, cryo-EM structures of C complexes show that PRP16 interacts with its targets CCDC49 and CCDC94 (Cwc25p and Yju2p in yeast), at the 3' end of the branched intron, before their release.
Reminiscent of how PRP2 strips away the RES complex, PRP16 might translocate in the 3'-to-5' direction to displace the two-step I factors, promoting substrate repositioning for exons ligation.Furthermore, a comparison between variants of C* complexes shows that PRP22, slightly but significantly, changes its position from the periphery of the spliceosome (Zhan, Mol Cell, 2022).
Although the RNA strand bound by PRP22 is not visible due to the local low resolution, the relocation of PRP22 might suggest translocation along the intron.We discuss these aspects in the revised manuscript (P11, lines 260-272).6. P.5, line 120: How can one explain the observation that PRP2 on Sc B act exhibits the conformation corresponding to a post-ATP hydrolytic state prior to PRP2 activity?To stall a B act complex, the authors (Bai et al., Science 2020) have used recombinant Prp2p carrying the ATPase-defective mutant K252A.Because this mutation in the Walker motif prevents ATP binding, the two RecA domains are relaxed in the opened conformation, equivalent to the post-ATP hydrolytic state.We refer to the above on P5, lines 109-111.7. P.10, line 238: PRP2….translocated to "an upstream" position.
In the revised manuscript, we capitalize the names of human proteins (by following the nomenclature suggested in Kastner et al., CSHP in Biology, 2019).For instance, we refer to the human PRP2 and yeast Prp2p; or human GPKOW and yeast Spp2p.Besides, we introduce the names of the proteins by explicitly stating which is human and which is yeast, to avoid confusion.9. P.12: This result is consistent with a previous report that the NTD of Sc PRP2 is dispensable (Edwalds-Gilbert, 2004), which should be included in the Discussion.
Interestingly, although PRP2 NTD is dispensable in budding yeast, Prp2p contains the "hook" element of the NTD, whose human homolog interacts with CWC22 HEAT10 and the RES complex in B AQR .As the yeast Cwc22p HEAT10 is required for the productive role of Prp2p (Yeh et al., Mol Cell Biol, 2011), Prp2p's hook element might play a conserved role in linking the function of Cwc22p and Prp2p in catalytic activation in yeast.The dispensable quality of NTD in yeast (Edwalds-Gilbert, 2004) may suggest that other subunits might compensate for its function without altering the function of Prp2p.
We found these correlations of potential interest and have included them in the manuscript (P14, lines 333-338).This manuscript provides critical new concepts to our understanding of spliceosomal activation, i.e., the process by which, just prior to catalytic step one, the first-step nucleophile -the branch site adenosine complexed with U2 snRNA -is released from SF3b1 and moves 50A into the active site.
The authors present a ~2.9A cryo-EM structure of a human spliceosomal complex stalled by a mutation in the ATPase Aquarius.This represents a previously unknown intermediate in human spliceosome assembly/activation, which is not present in the yeast system (which does not have an Aquarius ortholog).The manuscript provides key new insights into the function of both PRP2 and Aquarius ATPases in spliceosome activation.Lines 130-131, "Whether DEAH-box helicases, including PRP2, pull their substrates from a distance or by stepwise translocation along the RNA strand remained unclear.":This is a big point, and I want to reiterate it and say that I entirely agree with the authors' assertion here.I don't think that we have known what any spliceosomal ATPase really does, and this is a big step forward in understanding PRP2 function.By stalling the complex at the AQR step, the authors can, for the first time, infer exactly what PRP2 translocates on: "PRP2 advanced 18 nts [on the pre-mRNA] towards the spliceosome core during the Bact-to-BAQR transition".I think that this point and its concomitant effects on the structure are incredibly important and an appropriate topic for a paper in Nature.
Overall, this is one of the few spliceosome structural papers in which we actually learn new, unanticipated features of the mechanism.Specific Comments: 1. Line 83, "Following up on our previous work 6": This needs a bit more explanation.
We are more specific in the revised manuscript by stating, "After identifying the IBC as a complex that delivers Aquarius to the splicesome (De et al., 2015).." (P4, lines 75-77).
2. The mechanism of translocation termination of PRP2 by "molecular brake elements" proposed by the authors is very interesting.It would be useful to (briefly) compare this to what is known about the termination of translocation of other helicases.For example, is it similar, or not, to the mechanism by which MAGOH-Y14 inhibits eIF4AIII and locks it onto RNA within the exon junction complex (Ballot et al., NSMB, 2005; PMID 16170325)?
There are notable differences between the inhibition of eIF4AIII and PRP2.We mention this aspect in the revised manuscript (P10, lines 250-252).
MAGOH-Y14 partially embraces the helicase and stabilizes the closed conformation.In the case of PRP2, SKIP intercalates between the RecA domains, stabilizing the open conformation and preventing translocation and ATP hydrolysis.Besides, PPIL4 forms a brake shoe that prevents translocation by PRP2.Most likely, the two manners of inhibition respond to different needs -in one case, a DEAD box that acts primarily as an immobile clamp; in the second case, a translocase that actively walks along the RNA and should stop only after fulfilling its role.

The action of the ATPases as molecular clamps seems a lot like what was proposed by Melissa
Moore for DExH/D proteins (Shibuya et al., NSMB, 2004;PMID 15034551).
Indeed, eIFAIII appears to act as an RNA clamp and 'place holder' for the attachment of additional proteins to RNA in a sequence-independent manner.PRP2 is arrested to prevent the continuation of translocation that might dissociate or alter the branch duplex.4. Line 377, Fig. 1, "Key subunits are color-coded": This could be clarified.I understand that the colors in the molecule structure schematic are a rough match with the colors for the components list, shown in a, lower.However, there are some inconsistencies.For example, PRP2 is shown in brown, but it is not color-matched in Fig. 1b, where PRP2 is inconsistently written in red.Why is the RES complex indicated in light blue, when the only protein component included, BUD13, is dark blue?
Overall, there are too many colors and shades of colors here to be able to follow.
We fully agree and have amended Fig. 1 by removing inconsistencies in color coding.We believe that the revised Fig. 1a is much easier to follow.

Lines 268-271:
There is a high richness of new detail provided throughout.However, one statement, "The two helicases must coordinate to liberate the duplex from SF3B-RES constraints and hand it over to the catalytic center, while keeping it unaltered -a task that is best accomplished sequentially, by two molecular motors", leaves me wanting more info.Why is this "best accomplished" by two molecular motors?This is a critical difference between mammals and yeast, and I think it deserves to be explained further.Here, this is stated as a fact; as such, it needs further explanation.Alternatively, this could be stated as a conjecture that is, so far, without adequate explanation.
We fully agree and have updated the manuscript, discussing thoroughly why human catalytic activation requires two helicases, and the putative mechanism of Aquarius (P12-P13, lines 273-315).
Furthermore, we dedicated a section comparing the yeast and human catalytic activation (P13-P14, lines 317 -345).We have also added a diagram about the yeast catalytic activation (Fig. 5c) that can be readily compared with the human system (Fig. 5b) Line 378: "spliceosomes" should be "spliceosome".

Referee # 1 :
In this manuscript, the authors present the cryo-EM structure of a human spliceosome intermediate stalled at the catalytic activation step.They used a dominant-negative Aquarius mutant to block the splicing reaction, and arrested the spliceosome halfway through catalytic activation.The structure, which they call BAQR, reveals several interesting features distinct from that of Bact, including the localization of PRP2 near the spliceosome core and the open conformation of SF3B1.The N-terminal disordered domain of PRP2, which is visualized only in BAQR, interacts with spliceosomal components PPIL4 and SKIP, and the authors propose that it plays a regulatory role for structural changes.BAQR represents a spliceosome intermediate in the transition from Bact to B*.A model is proposed for how PRP2 may mediate structural changes of the spliceosome from Bact to BAQR.
10.The model in Fig. 5b should be explained in more detail, in particular, how the hypothetical intermediate is generated.It would also be more appropriate to place the summary of the respective results in the legend of Fig. 5b than in the Discussion.For clarity's sake, we expanded the schematic with an additional intermediate and have explained in the legend of Fig. 5b how the intermediates were generated.The hypothetical intermediate between B act and B AQR considers (i) that the molecular brake can form only after PPIL4's binding to the RNA (which in turn occurs only after dissociation of the RES complex by the translocation of PRP2) and (ii) the advance of PRP2 should progressively strip the intron from SF3B1 and promote the opening of SF3B1 towards the "loose" conformation.We generated the hypothetical intermediate between B AQR and B* by considering that Aquarius should induce complete displacement of the branch duplex from SF3B, which assumes the release of BS-A from the binding pocket on SF3B1 and SF3B1's transition from the loose to the open conformation.This transition would involve the interface between SF3B1 and PRP8, resulting in the dissociation of SF3B (together with SF3A, PPIL4, and PRP2) from PRP8.All these explanations are in the legend of Fig. 5b.Referee #2: