Review 1: "Multiscale Effects of Perturbed Translation Dynamics Inform Antimalarial Design"

RR:C19 Evidence Scale rating by reviewer:

• Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review:

Malaria parasites depend on efficient protein synthesis for infection and replication within the human red blood cell stage, making mRNA translation a promising target for drug development. Anton et al. used in situ cryoEM to reveal previously unknown details of mRNA translation, including a unique bifurcation in the decoding stage, opening an avenue for new antimalarial therapies. 

The authors' work here is an important step forward in understanding the fundamentals of translational control in Plasmodium falciparum. However, we have uncertainty about the accuracy of their interpretations of the reference material. Here is one minor correction: RSP16 used vs RPS16. Further, there is no evidence by Blumqvist et al. (ref 18) to suggest that PfRACK1 is important for translational regulation. There is no discussion about monosome vs polysome RACK1 binding in P. falciparum. This paper importantly shows that PfRACK1 is necessary for parasite survival during the IDC and its localization to the parasite cytoplasm, and that is all. The corresponding author of this paper was integral in one of the previous Plasmodium falciparum cryoEM structures that were missing PfRACK1.  

Additionally, references 19 and 20 do not suggest that PfRACK1 is not involved in translational regulation. However, given the previous structures, it may perform other duties in conjunction with its role in translational regulation. Citation 19 (a review article) references previous literature in support of RACK1’s important role in translational regulation. Citation 20 uses biochemical evidence to support the role of PfRACK1 binding to the ribosome, but that its binding affinity is different than that of mammalian RACK1 and again uses previous literature to support the necessity of RACK1-ribosome interaction in mRNA translation and translational regulation.

Anton et al. uncovered the consensus structure of Pf80S ribosome in a native state with structure details that support RACK1 binding, which had been missing in previous CryoEM structures. They bridged the missing information between the structure and binding assays. 

• Does the manuscript confirm previous work or refute the current understanding? Yes 

• How well does the manuscript position the work within the current literature/understanding? We addressed the concerns from our side, but in general, the manuscript acknowledged the current state of literature and understanding in the field. 

• Is there clarity regarding the recommended actions that result from the findings? Yes

• Do authors pay attention to ethics, diversity, and inclusion? I have not seen any ethical concerns in this manuscript.