Global Release of Translational Repression Across Plasmodium’s Host-to-Vector Transmission Event
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Abstract
Malaria parasites must be able to respond quickly to changes in their environment, including during their transmission between mammalian hosts and mosquito vectors. Therefore, before transmission, female gametocytes proactively produce and translationally repress mRNAs that encode essential proteins that the zygote requires to establish a new infection. This essential regulatory control requires the orthologues of DDX6 (DOZI), LSM14a (CITH), and ALBA proteins to form a translationally repressive complex in female gametocytes that associates with many of the affected mRNAs. However, while the release of translational repression of individual mRNAs has been documented, the details of the global release of translational repression have not. Moreover, the changes in spatial arrangement and composition of the DOZI/CITH/ALBA complex that contribute to translational control are also not known.
Therefore, we have conducted the first quantitative, comparative transcriptomics and DIA-MS proteomics of Plasmodium parasites across the host-to-vector transmission event to document the global release of translational repression. Using female gametocytes and zygotes of P. yoelii , we found that nearly 200 transcripts are released for translation soon after fertilization, including those with essential functions for the zygote. However, we also observed that some transcripts remain repressed beyond this point. In addition, we have used TurboID-based proximity proteomics to interrogate the spatial and compositional changes in the DOZI/CITH/ALBA complex across this transmission event. Consistent with recent models of translational control, proteins that associate with either the 5’ or 3’ end of mRNAs are in close proximity to one another during translational repression in female gametocytes and then dissociate upon release of repression in zygotes. This observation is cross-validated for several protein colocalizations in female gametocytes via ultrastructure expansion microscopy and structured illumination microscopy. Moreover, DOZI exchanges its interaction from NOT1-G in female gametocytes to the canonical NOT1 in zygotes, providing a model for a trigger for the release of mRNAs from DOZI. Finally, unenriched phosphoproteomics revealed the modification of key translational control proteins in the zygote. Together, these data provide a model for the essential translational control mechanisms used by malaria parasites to promote their efficient transmission from their mammalian host to their mosquito vector.
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Reviewer #1 (Evidence, reproducibility and clarity (Required)):
This manuscript provides a detailed analysis of RNA and protein dynamics during transmission of the rodent malaria model P. yoelii from the mouse host to an in vitro ookinete culture setting (mimicking the mosquito midgut environment). This group and others have shown experimentally that a substantial number of mRNAs is stored in the female Plasmodium gametocyte, ready to be translated following initiation of ookinete development. The process is akin to maternal deposition of mRNA in oocytes of metazoans. With this manuscript the authors provide a significant contribution to the field of …
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Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
This manuscript provides a detailed analysis of RNA and protein dynamics during transmission of the rodent malaria model P. yoelii from the mouse host to an in vitro ookinete culture setting (mimicking the mosquito midgut environment). This group and others have shown experimentally that a substantial number of mRNAs is stored in the female Plasmodium gametocyte, ready to be translated following initiation of ookinete development. The process is akin to maternal deposition of mRNA in oocytes of metazoans. With this manuscript the authors provide a significant contribution to the field of translational control in Plasmodium parasites as they explore the translational activation during the early hours of zygote-to-ookinete development. The paper presents RNAseq and mass-spec analyses of female gametocytes and for the first time for 6-hour zygotes (ie a fertilized female gamete); the zygote datasets are much improved and more comprehensive than the only other performed in 2008 in P. gallinaceum. Using comparative analyses of transcriptome and proteome data (including published datasets) the authors arrive at a list of 198 transcripts that are translationally repressed in the gametocyte and translated within 6 hours of fertilization in the zygote. Many of these mRNAs are known to be involved in zygote to ookinete transformation. BioID is finally used to explore changes in mRNP protein composition between the female gametocyte and the zygote.
The paper is generally well written. The authors present a lot of data (also in comparison with published data). Sometimes perhaps the main message could be simplified / streamlined in section titles (Quantitative Proteomics by DIA-MS is not very informative. The outcome of the analysis would be more telling).
Response: We have revised section headers to clarify the content.
A considerable proportion of the DIA mass-spec proteomics results section is very technical. The paper describes a biological phenomenon rather than a technical mass-spec advance. Can these technical details be moved to the methods section?
Response: As this is one of the first published instances of using DIA-MS to Plasmodium, we want to keep this information in the main text to help our community adopt these approaches. While these details are highly technical, they are also some of the major advances of this project.
On the other hand, a bit more detail could be provided in the main text. For example, the age of the zygotes is never mentioned. This is important, please add this. The main manuscript text has 16 mentions of the word "many". As the authors are in possession of the data, please provide, if missing, (in parenthesis) the absolute numbers, maybe in an "x out y" format. Please clearly state the number of biological and/or technical replicates used for transcriptome and proteome analyses in the main text, figures and/or figure legends. How many protein coding genes are encoded in the P. yoelii genome?
Response: Several of these requested details are noted in the materials and methods. We have added this information to the main manuscript now as well. We have also revised the manuscript to replace some instances of “many” with specific numbers unless it adversely impacted the flow of the sentence to do so.
The authors claim that only zygotes (fertilized females) have surface-exposed Pys25 (a surface protein they use to affinity-purify zygotes) but not gametocytes. I could not find the experimental data for this in the paper. The cited reference #22 also does not appear to show this. In Figure 2C Pys25 is shown to be translated in gametocytes. In this context it may be important to note that in the related P. berghei the related protein P28 is expressed even in the absence of fertilization (Billker 2004; DOI: 10.1016/s0092-8674(04)00449-0). It may not be relevant whether translation requires fertilization, but the authors claim it affects trafficking of the Pys25 protein to the surface, so it needs to be shown. A reference to an infertile P. yoelii line would be great.
Response: We have corrected the reference supporting the surface exposure of p25 on zygotes. The observation by Billker and colleagues about Pbs28 is also of interest, but outside of the scope of this study as we did not investigate the fertilization event itself here.
It is highly commendable that all data is provided throughout the manuscript. For readability, may I suggest that the authors add labels to individual sheets within an excel file from A to Z, and do so also within the manuscript. That would really help; the most relevant data sets could then be identified quickly. For example, line 184 refers to 276 zygote proteins in which sheet of which table?
Response: While this labeling system would also be effective, we have provided a README tab for our files that quickly directs the reader to the relevant tab (as we do for our previous publications).
Section 176 onwards: here the authors combine P. falciparum and P. yoelii proteomics data. Please explain why you excluded any of the available P. berghei proteome data such as the male and female gametocyte proteome? The same question applies to 294 onwards.
Response*: We compared our datasets with those of Lasonder et al. NAR 2016 because that study was also focused on translational repression of mRNAs and provided both RNA-seq and proteomic datasets of female gametocytes (although not of zygotes). *
The comparative transcriptome-proteome analysis arrives at 198 translationally repressed mRNAs. Could the authors provide one or two alternatives using less stringent parameters? The list in P. falciparum and P. berghei is considerably larger (500+ and 700+).
Response: We could have reduced the stringency of our thresholds to arrive at a far larger number, but prefer to retain higher confidence in those we are scoring as translationally repressed and then released for translation. We provide all of the pertinent data in the supplemental files if readers would like to adjust these thresholds to see which additional mRNAs may also be regulated.
The turboID data is informative but somewhat speculative in regard to spatial rearrangements within these mRNPs. Figure 6 presents the RNA helicase to bind the 5' end of mRNAs that are associated with polyribosomes and I assume being translated. Is this association realistic? The RNA helicase DOZI homolog of yeast (Dhh1) is also involved in decapping. Response: We provide Figure 6 as our working model of how the reorganization of the DOZI/CITH/ALBA complex could occur based on available data from this study and others. Future studies are warranted to determine if DOZI remains associated with monosomes vs. polysomes, but current data indicate that DOZI can bind to eIF4E when translational repression is not imposed.
Specific comments:
title Is global the appropriate word? Some transcripts appear to be translated later.
Response: We believe it does apply appropriately to these data.
Line 30/32 Please re-phrase the sentence. There is: Cell Host Microbe 2012 Jul 19;12(1):9-19. doi: 10.1016/j.chom.2012.05.014.
Response: We conclude that the sentence is correct as written, even in considering Sebastian et al. Cell Host & Microbe 2012.
30 Perhaps add ookinete that establishes infection rather than the zygote. For a general readership, a brief description of the sexual life cycle might be useful
Response: It is not possible to get into these nuances in the Abstract. This information is covered in the main text and the works that are cited.
32 DOZI/CITH/ALBA complex would require some explanation for a more general reader
Response: It is not possible to get into these nuances in the Abstract. This information is covered in the main text and the works that are cited.
36-37 I believe zygotes were collected 6 hours after fertilization. Does that qualify as soon after fertilization? Motile ookinetes are generated within 20 hours and motility can be seen before that.
Response: Yes, we think this qualifies as the process is not synchronous, but relies on when male gametes encounter and fuse with female gametes.
37 Essential functions for what?
Response: It is not possible to get into these nuances in the Abstract. This information is covered in the main text and the works that are cited.
39 Is the spatial arrangement of this mRNP known?
Response*: Some interactions of members of this complex were known (DOZI with eIF4E, ALBA4 with PABP1), but not the overall spatial arrangement. These findings are novel to this study. *
40 Can you briefly allude to the "recent, paradigm-shifting models of translational control"
Response: It is not possible to get into these nuances in the Abstract. This information is covered in the main text and the works that are cited.
44 Products = mRNA
Response: We have stated it as products because the maternal cell provides more than just mRNAs that are essential to further development post-fertilization.
45 Oocyte in metazoans ?
Response: Yes, this is the correct term. The context here is in higher eukaryotes.
60/62 Please re-phrase the sentence. There is: Cell Host Microbe 2012 Jul 19;12(1):9-19. doi: 10.1016/j.chom.2012.05.014.
Response: We conclude that the sentence is correct as written, even in considering Sebastian et al. Cell Host & Microbe 2012.
81 PbDozi Plasmodium berghei DOZI
Response: We have added this clarifying text here as suggested.
84/85 Please rephrase and cite Nucleic Acids Res. 2008 Mar;36(4):1176-86. doi: 10.1093/nar/gkm1142. Epub 2007 Dec 23. and Cell Host Microbe 2012 Jul 19;12(1):9-19. doi: 10.1016/j.chom.2012.05.014.
Response: As noted above for other comments, we hold that the current phrasing is accurate even when considering these important publications.
88 Please define the timepoints throughout this manuscript. What age are the zygotes? How many hours post-induction? Please define the time for ookinete development somewhere in the introduction
Response: The timepoint used for zygote collection is now included in the main text in addition to its previous inclusion in the Materials and Methods section. As we have not studied the ookinete stage here, we have opted to keep the introduction focused on the key details for this study.
104 Please add the age (in hours) of these zygotes from the time of starting the in vitro cultures. From the methods section it looks like 6 hours.
Response: The timepoint used for zygote collection is now included in the main text in addition to its previous inclusion in the Materials and Methods section.
103/105 I can find no evidence for P25 (Pys25) expression relying on fertilization in the cited paper (22). The SOM has no reference to Pys25 either. Please show data or reference published data that there is no translation and trafficking of Pys25 in unfertilized female gametes, ie those that are placed in ookinete medium. In this respect it may be important to note that unfertilized Plasmodium berghei females placed in ookinete medium translate P28, the P25 paralog (https://www.sciencedirect.com/science/article/pii/S0092867404004490?via%3Dihub)
Response: We have corrected the reference supporting the surface exposure of p25 on zygotes. The observation by Billker and colleagues about Pbs28 is also of interest, but outside of the scope of this study as we did not investigate the fertilization event itself here.
104 What cell line was used for the zygotes?
Response*: The PyApiAP2-O::GFP transgenic parasite line was used here. These details are included in the manuscript and supporting information. *
114 The number of transcripts detected in gametocytes is quite small compared to the twice as large proteomics dataset. See for example also Lasonder 2016 for P. falciparum detected transcripts: 4477 different sense transcripts were identified, 98% of which were shared between MG and FG.
Response: Yes, the number of mRNAs or proteins scored as detected differs based on thresholds applied. We prefer to err on the side of higher stringency as noted above.
117 Does the 194 up-in-gametocytes dataset include the 81 not found in zygotes?
Response: No, these 194 are detected in both datasets, but are more abundant in gametocytes than zygotes.
117 Could you indicate some of the genes in the plot?
Response: Several hits of special note are described in the text. We have opted to keep the figure clear and streamlined.
Fig1 How were the upregulated transcripts identified? 1647 are shown to be specific to zygotes in 1B, yet only 685 are shown in 1C to be upregulated. Do the transcripts found exclusively in zygotes not count? Are these transcripts likely the result of de novo transcription? How old are these zygotes when the libraries are made?
Response: The details of the RNA-seq processing are provided in the MakeFile, the supplementary tables, and the manuscript. The README tab provides descriptions of what processing occurred between sequential tabs. As noted above, zygotes were collected at 6 hours.
132 Many? How many? Please provide a precise number.
Response: These details are now in the revised manuscript.
134 Please explain why p28 would be differentially abundant in the zygote rather than the female gametocyte. That would require de novo transcription of this gene. If there is experimental evidence for the de novo transcription of p28 and other translationally repressed transcripts in the zygote please cite the references. Can you name a few more examples here? P25 for example, ap2-o, or anything published and experimentally validated. What about AP2-o and AP2-Z? Both are known to be translationally repressed.
Response: We state in the original manuscript that there is not a significantly different mRNA abundance of pys28.
139 Please define how many members of the IMC?
Response*: We have now replaced “many” with the number of IMC members we have detected, which is also shown in supporting tables. *
156 Can you provide a number of how many parasites were used in total or per run. And how many biological and technical replicates were analysed?
Response: These details are provided in the Materials and Methods.
169 The number of proteins detected in the gametocyte sample is twice the size of transcripts. IS this to be expected?
Response*: This reflects the sensitivity of the assays run for transcriptomics and proteomics. *
170 How many samples were analyzed? One gametocyte and one zygote sample?
Response: Yes, for the creation of the DIA-MS spectral library, a single biological replicate was used in addition to in silico library approaches. This information is provided in the next sentence.
176 Why did you not include P. berghei in the meta-analysis?
Response: We compared these results to all of the published Plasmodium proteomes in PlasmoDB.
184 Please refer to an excel table here.
Response: We have pointed to the relevant supporting files in this section.
184 145 proteins: do you mean orthologs in general or orthologs with a gene/protein annotation other than unknown function?
Response: We use the standard form of ortholog throughout the manuscript.
190 142 proteins: do they all have orthologs in P. falciparum?
Response: No, not all proteins in our dataset have unambiguous orthologues in P. falciparum, and this is accounted for in our data processing approaches.
Figure 2C P25 is not exclusive to zygotes here and also found in the gametocyte sample.
Response: That is correct. It is known that p25 is expressed in female gametocytes, but that the localization changes in the zygote.
190 shortlist
Response: The spelling of “short list” as two words is an appropriate American spelling of this term.
219 onwards Does the list of 198 transcripts exclusively arise from your RNAseq and proteomics comparison? Or does it include falciparum data as outline in section 176 onwards, ie the list of 276 proteins that only are detected in zygotes?
Response: Yes, this list of 198 mRNAs is derived from our datasets only using our defined thresholds. The details of this are provided in the manuscript.
224 Early zygote? At 6 hours do the parasites not start to transform, elongate?
Response: This process is not synchronous, as it is affected by the timing of gamete fusion.
225 >5-fold. Is this an arbitrary decision?
Response: This threshold has been used by our group and others in prior studies, and was partially informed by the behavior of previously characterized transcripts.
227 1417 mRNAs: they are from which dataset?
Response: These are from our datasets with P. yoelii, as described in the manuscript.
228/229 Please explain why DOZI and CITH are in the list of 198 repressed transcripts? They are present in the gametocyte. Are they upregulated>5 fold?
Response: Yes, they meet our criteria for this regulation, and in the manuscript we note that we believe that they are self-regulated and likely have continuing roles in early mosquito stage development.
259 ... as they are already translated in the gametocyte?
Response: Yes. Translational repression allows for the existence of some of the protein in the initial timepoint. This differs from translational silencing which does not.
295 Is this from the 198 TR list S4?
Response: No. Transcripts that remain repressed would not be in the list of 198, as the protein was not detected in zygotes.
294 onwards How many putatively falciparum transcripts are there? How many were identified in P. berghei? How many are common to all? A Venn diagram perhaps to compare the different studies
Response: There is substantial overlap between the species with respect to the presence of syntenic orthologues in this dataset. However, because we did not conduct experiments with P. falciparum or P. berghei here, we do not want to make claims that they are similarly regulated or potentially have a reader misinterpret a figure to that effect.
301 How many transcripts were found associated with Plasmodium berghei DOZI and/or CITH in female gametocytes? How many of those were abundantly detected as protein in zygotes, or had no difference in protein abundance between gametocytes and zygotes, or even greater abundance in female gametocytes?
Response: These details are now provided in the revised manuscript.
303/305 Please indicate the numbers of translationally repressed transcripts identified for P. falciparum and berghei.
Response: These data are provided in Supporting Information Table 4.
317/319 Please add the promoter used for tid-GFP
Response: We have now added this information to the Materials and Methods.
320 Please elaborate on the spatial organization of the DCA complex.
Response: This has not been previously characterized, and this entire section is dedicated to the experimental data and interpretations of how the DOZI/CITH/ALBA complex may be organized.
321/322 Have precise binding sites of DOZI and ALBA4 really been shown experimentally in the cited papers? In relation to 5' and 3' ends of the mRNA? Please cite Braks et al. paper.
Response: Yes. The association of DOZI with eIF4E and ALBA4 with PABP1 are established in the literature, in some cases by multiple independent laboratories. The Braks publication does not address the binding of these proteins, and thus is not cited.
323 What is the first generation BioID enzyme? BirA*
Response: Yes. The first generation enzyme is called BirA*
323 Please cite relevant Kyle Roux and Alice Ting for the original enzymes
Response: We have now added these citations to this sentence.
327 Could you show images of ALBA4::TurboID::GFP, DOZI::TurboID::GFP and cytosolic (free) TurboID? Perhaps stained with fluorescently labelled streptavidin and / or against GFP? In the gametocyte and zygote samples?
Response: We attempted to stain with monoclonal antibodies that are reactive against biotin and there was insufficient specificity, hence why such data is not included. We conclude that all of the other data that supports this approach suffices to demonstrate its rigor.
331 What is the age of these zygotes? Where they affinity purified?
Response: As throughout the manuscript, zygotes were collected at 6 hours. Details of experimental purifications are provided in the materials and methods.
Fig S4 Please indicate whether ALBA4 and DOZI were tagged endogenously
Response: Yes. The endogenous loci for both ALBA4 and DOZI were modified to include the C-terminal TurboID and GFP tags.
421/430 Please add a few references here
Response: We do not believe that specific references are warranted for these general statements.
429 translational repression?
Response: Yes. These statements set the stage for the use of translational repression.
445 966 proteins in gallinaceum? The zygote cultures in that study were 2-3 hours. How old were the cultures in your study?
Response: As throughout the manuscript, zygotes were collected at 6 hours.
481 Please explain / cite why repression is energetically costly.
Response: These details are provided in both the introduction and discussion sections. The energetic cost of translational repression is both the cost to produce the transcripts without immediately/fully utilizing it for translation, in addition to the energetic cost to impose the regulation.
501 Please add the time-point of RNA and protein sampling. How many hours into ookinete development? What is the time from cardiac puncture through FACS sampling of gametocytes.
Response: We have provided all of these details in the materials and methods for female gametocytes and zygotes. We did not look at ookinetes in this study.
711/713 Do you have any images that show the successful purification of zygotes away from gametocytes? Secondly, please provide a reference for the statement that unfertilized female gametocyte do not express surface exposed Pys25.
Response*: We do not have captured images of these zygotes, but confirmed them during collection using microscopy. The reference for surface exposure of Pbs25 is now provided earlier in the manuscript as well. *
711/716 Were parasites lysed and mechanically homogenised?
Response: We have provided all of these details in the materials and methods for female gametocytes and zygotes.
Figure 6 What is the evidence that DOZI stays associated with mRNA that is being translated? Rather than mRNA that is being decapped. Please add the references that unequivocally show that DOZI and ALBA4 bind to opposite ends of repressed mRNAs.
Response: This is our working model of these data. It is feasible that these complexes could form off of mRNA as well. Publications describing the interactions of DOZI with eIF4E and ALBA4 with PABP1 are provided in the manuscript. It is well established that eIF4E binds to the m7G cap of the 5’ end of mRNAs, and PABP1 binds to the poly(A) tail at the 3’ end of mRNAs.
Reviewer #1 (Significance (Required)):
The experiments in the manuscript are carefully conducted. Apart from a P. gallinaceum study from 2009 this is the first comprehensive analysis of the transcriptome and proteome of a Plasmodium zygote (developing ookinete) at 6 hours post-fertilization. The data are used to explore the temporal aspect of activation of translation during the first quarter of the 20-24 hour ookinete developmental period. The study will be of interest to the field, specifically those scientists working to understand translational control, ookinete development, and those developing intervention strategies to prevent mosquito infection and thus malaria transmission.
Response: We appreciate Reviewer 1’s extensive feedback and positive remarks about the significance of our study. We have revised our manuscript to reflect this constructive feedback.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
Main findings
Taking a multi-omic approach, the authors provide quantitative evidence for translation repression of ~200 mRNAs in Plasmodium yoelii female gametocytes. These mRNAs are then translated, and proteins detected by 6 hours after activating gametocytes. They accomplish this by performing a comparative global analysis of the transcriptome and proteome between female gametocytes and early zygotes that provides an intresting resource. The authors also use proximity labelling of the DOZI/CITH/ALBA4 repression complex, and these data suggest the complex may disassemble in the zygote or change its composition.
Major points
Line 181-184: The authors state that there is no evidence of how the DCA complex selects specific mRNAs for translation repression. While the exact mechanisms have not been fully elucidated, Braks et al (2008, doi:10.1093/nar/gkm1142) suggested a role of the untranslated regions (UTRs) in translation repression of transcripts in Plasmodium berghei female gametocytes. They identified a uridine-rich 47-base element in the 5'UTR and or 3'UTR that was associated with translationally repressed transcripts and validated it experimentally. Considering this finding, I would recommend an amendment of the statement and to include the earlier work. I would also like to see additional analysis to check if this U-rich motif or other motifs are associated with the translationally repressed transcripts identified in the current study. The current study should be better powered to conduct such an analysis.
Response: We have now added a comment and citation in the revised text about this study in Lines 86-88. Understanding the full importance of this element is challenging, as the Plasmodium transcriptome is highly enriched in A’s and U’s due to the highly skewed A/T content of its genome. Perhaps for this reason, we did not see an association of this motif with the identified mRNAs.
The authors used zygotes that expressed GFP tagged AP2-O, however, there is no explanation of the significance of using this line.
Response: This line is described in the Materials and Methods and supporting information. It was used to provide further validation of the production of zygotes.
Minor points
In line 106-107, the authors refer to figure SI, this figure is about genomic locus and genotyping PCR for the PyApiAP2-O::GFP parasites but there is no intext description of why this specific line was used.
Response: We have provided this information in the revised manuscript.
Statement in line 122-124 "It is likely that....." should go into the discussion not results.
Response: We have placed this single sentence immediately after presenting these data here to aid reader comprehension.
Statement in line 171-175: "In addition to providing confirmatory...." Should be in the discussion not on the results.
Response: We view this sentence as a concluding remark of this section of data that also places this information in context for the reader.
In Fig. 4 A and B, could the colour scheme be changed so that the proteins that are not in both samples (and probably contain many unspecifically detected proteins) appear less prominent?
Response: We appreciate this suggestion and have adjusted these plots accordingly in the revised manuscript.
Reviewer #3 (Significance (Required)):
Why is the paper interesting. Translation repression of mRNA at a global level in the female gametocytes has been studied previously in rodent malaria parasites investigated, but prior to the current study, the release of mRNA from translation repression in the mosquito stages has only been demonstrated for specific transcripts. By characterizing and quantitating changes in protein abundance between macrogamete and zygote, coupled with transcriptomic analysis, the current work broadens our understanding of zygotic translation activation that is key to successful malaria parasite transmission to the mosquito.
This dataset provides a useful resource for the Plasmodium research community as it provides a more comprehensive view of how transcripts behave during the transitions from the mammalian host to the vector. It is one step in a broader endeavour towards finding genes crucial for parasite transmission that could be targeted for interventions.
How translational repression and derepression is regulated remains unknown, although some of the molecular players have been identified. This paper shows proximity labelling and expansion microscopy data of the ribonuclear protein complex thought to mediate repression. Although the specific mechanistic insights provided by the experiments shown here remain relatively limited, the work demonstrates interesting new avenues for how translational derepression in Plasmodium can be studied.
Response: We also appreciate Reviewer 3’s excellent feedback and positive remarks about the significance of our study. The revised manuscript addresses these comments, and we believe it is further strengthened because of it.
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Referee #2
Evidence, reproducibility and clarity
Main findings
Taking a multi-omic approach, the authors provide quantitative evidence for translation repression of ~200 mRNAs in Plasmodium yoelii female gametocytes. These mRNAs are then translated, and proteins detected by 6 hours after activating gametocytes. They accomplish this by performing a comparative global analysis of the transcriptome and proteome between female gametocytes and early zygotes that provides an intresting resource. The authors also use proximity labelling of the DOZI/CITH/ALBA4 repression complex, and these data suggest the complex may disassemble in the zygote or change its composition.
Major points
- Line …
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Referee #2
Evidence, reproducibility and clarity
Main findings
Taking a multi-omic approach, the authors provide quantitative evidence for translation repression of ~200 mRNAs in Plasmodium yoelii female gametocytes. These mRNAs are then translated, and proteins detected by 6 hours after activating gametocytes. They accomplish this by performing a comparative global analysis of the transcriptome and proteome between female gametocytes and early zygotes that provides an intresting resource. The authors also use proximity labelling of the DOZI/CITH/ALBA4 repression complex, and these data suggest the complex may disassemble in the zygote or change its composition.
Major points
- Line 181-184: The authors state that there is no evidence of how the DCA complex selects specific mRNAs for translation repression. While the exact mechanisms have not been fully elucidated, Braks et al (2008, doi:10.1093/nar/gkm1142) suggested a role of the untranslated regions (UTRs) in translation repression of transcripts in Plasmodium berghei female gametocytes. They identified a uridine-rich 47-base element in the 5'UTR and or 3'UTR that was associated with translationally repressed transcripts and validated it experimentally. Considering this finding, I would recommend an amendment of the statement and to include the earlier work. I would also like to see additional analysis to check if this U-rich motif or other motifs are associated with the translationally repressed transcripts identified in the current study. The current study should be better powered to conduct such an analysis.
- The authors used zygotes that expressed GFP tagged AP2-O, however, there is no explanation of the significance of using this line.
Minor points
In line 106-107, the authors refer to figure SI, this figure is about genomic locus and genotyping PCR for the PyApiAP2-O::GFP parasites but there is no intext description of why this specific line was used.
Statement in line 122-124 "It is likely that....." should go into the discussion not results. Statement in line 171-175: "In addition to providing confirmatory...." Should be in the discussion not on the results. In Fig. 4 A and B, could the colour scheme be changed so that the proteins that are not in both samples (and probably contain many unspecifically detected proteins) appear less prominent?Significance
Why is the paper interesting.
Translation repression of mRNA at a global level in the female gametocytes has been studied previously in rodent malaria parasites investigated, but prior to the current study, the release of mRNA from translation repression in the mosquito stages has only been demonstrated for specific transcripts. By characterizing and quantitating changes in protein abundance between macrogamete and zygote, coupled with transcriptomic analysis, the current work broadens our understanding of zygotic translation activation that is key to successful malaria parasite transmission to the mosquito.
This dataset provides a useful resource for the Plasmodium research community as it provides a more comprehensive view of how transcripts behave during the transitions from the mammalian host to the vector. It is one step in a broader endeavour towards finding genes crucial for parasite transmission that could be targeted for interventions.
How translational repression and derepression is regulated remains unknown, although some of the molecular players have been identified. This paper shows proximity labelling and expansion microscopy data of the ribonuclear protein complex thought to mediate repression. Although the specific mechanistic insights provided by the experiments shown here remain relatively limited, the work demonstrates interesting new avenues for how translational derepression in Plasmodium can be studied.
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Referee #1
Evidence, reproducibility and clarity
This manuscript provides a detailed analysis of RNA and protein dynamics during transmission of the rodent malaria model P. yoelii from the mouse host to an in vitro ookinete culture setting (mimicking the mosquito midgut environment). This group and others have shown experimentally that a substantial number of mRNAs is stored in the female Plasmodium gametocyte, ready to be translated following initiation of ookinete development. The process is akin to maternal deposition of mRNA in oocytes of metazoans. With this manuscript the authors provide a significant contribution to the field of translational control in Plasmodium parasites …
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Referee #1
Evidence, reproducibility and clarity
This manuscript provides a detailed analysis of RNA and protein dynamics during transmission of the rodent malaria model P. yoelii from the mouse host to an in vitro ookinete culture setting (mimicking the mosquito midgut environment). This group and others have shown experimentally that a substantial number of mRNAs is stored in the female Plasmodium gametocyte, ready to be translated following initiation of ookinete development. The process is akin to maternal deposition of mRNA in oocytes of metazoans. With this manuscript the authors provide a significant contribution to the field of translational control in Plasmodium parasites as they explore the translational activation during the early hours of zygote-to-ookinete development. The paper presents RNAseq and mass-spec analyses of female gametocytes and for the first time for 6-hour zygotes (ie a fertilized female gamete); the zygote datasets are much improved and more comprehensive than the only other performed in 2008 in P. gallinaceum. Using comparative analyses of transcriptome and proteome data (including published datasets) the authors arrive at a list of 198 transcripts that are translationally repressed in the gametocyte and translated within 6 hours of fertilization in the zygote. Many of these mRNAs are known to be involved in zygote to ookinete transformation. BioID is finally used to explore changes in mRNP protein composition between the female gametocyte and the zygote.
The paper is generally well written. The authors present a lot of data (also in comparison with published data). Sometimes perhaps the main message could be simplified / streamlined in section titles (Quantitative Proteomics by DIA-MS is not very informative. The outcome of the analysis would be more telling).
A considerable proportion of the DIA mass-spec proteomics results section is very technical. The paper describes a biological phenomenon rather than a technical mass-spec advance. Can these technical details be moved to the methods section?
On the other hand, a bit more detail could be provided in the main text. For example, the age of the zygotes is never mentioned. This is important, please add this. The main manuscript text has 16 mentions of the word "many". As the authors are in possession of the data, please provide, if missing, (in parenthesis) the absolute numbers, maybe in an "x out y" format. Please clearly state the number of biological and/or technical replicates used for transcriptome and proteome analyses in the main text, figures and/or figure legends. How many protein coding genes are encoded in the P. yoelii genome?
The authors claim that only zygotes (fertilized females) have surface-exposed Pys25 (a surface protein they sue to affinity-purify zygotes) but not gametocytes. I could not find the experimental data for this in the paper. The cited reference #22 also does not appear to show this. In Figure 2C Pys25 is shown to be translated in gametocytes. In this context it may be important to note that in the related P. berghei the related protein P28 is expressed even in the absence of fertilization (Billker 2004; DOI: 10.1016/s0092-8674(04)00449-0). It may not be relevant whether translation requires fertilization, but the authors claim it affects trafficking of the Pys25 protein to the surface, so it needs to be shown. A reference to an infertile P. yoelii line would be great.
It is highly commendable that all data is provided throughout the manuscript. For readability, may I suggest that the authors add labels to individual sheets within an excel file from A to Z, and do so also within the manuscript. That would really help; the most relevant data sets could then be identified quickly. For example, line 184 refers to 276 zygote proteins in which sheet of which table?
Section 176 onwards: here the authors combine P. falciparum and P. yoelii proteomics data. Please explain why you excluded any of the available P. berghei proteome data such as the male and female gametocyte proteome? The same question applies to 294 onwards.
The comparative transcriptome-proteome analysis arrives at 198 translationally repressed mRNAs. Could the authors provide one or two alternatives using less stringent parameters? The list in P. falciparum and P. berghei is considerably larger (500+ and 700+).
The turboID data is informative but somewhat speculative in regard to spatial rearrangements within these mRNPs. Figure 6 presents the RNA helicase to bind the 5' end of mRNAs that are associated with polyribosomes and I assume being translated. Is this association realistic? The RNA helicase DOZI homolog of yeast (Dhh1) is also involved in decapping.
Specific comments:
title Is global the appropriate word? Some transcripts appear to be translated later.
Line 30/32 Please re-phrase the sentence. There is: Cell Host Microbe 2012 Jul 19;12(1):9-19. doi: 10.1016/j.chom.2012.05.014.
30 Perhaps add ookinete that establishes infection rather than the zygote. For a general readership, a brief description of the sexual life cycle might be useful
32 DOZI/CITH/ALBA complex would require some explanation for a more general reader
36-37 I believe zygotes were collected 6 hours after fertilization. Does that qualify as soon after fertilization? Motile ookinetes are generated within 20 hours and motility can be seen before that.
37 Essential functions for what?
39 Is the spatial arrangement of this mRNP known?
40 Can you briefly allude to the "recent, paradigm-shifting models of translational control"
44 Products = mRNA
45 Oocyte in metazoans ?
60/62 Please re-phrase the sentence. There is: Cell Host Microbe 2012 Jul 19;12(1):9-19. doi: 10.1016/j.chom.2012.05.014.
81 PbDozi Plasmodium berghei DOZI
84/85 Please rephrase and cite Nucleic Acids Res. 2008 Mar;36(4):1176-86. doi: 10.1093/nar/gkm1142. Epub 2007 Dec 23. and Cell Host Microbe 2012 Jul 19;12(1):9-19. doi: 10.1016/j.chom.2012.05.014.
88 Please define the timepoints throughout this manuscript. What age are the zygotes? How many hours post-induction? Please define the time for ookinete development somewhere in the introduction
104 Please add the age (in hours) of these zygotes from the time of starting the in vitro cultures. From the methods section it looks like 6 hours.
103/105 I can find no evidence for P25 (Pys25) expression relying on fertilization in the cited paper (22). The SOM has no reference to Pys25 either. Please show data or reference published data that there is no translation and trafficking of Pys25 in unfertilized female gametes, ie those that are placed in ookinete medium. In this respect it may be important to note that unfertilized Plasmodium berghei females placed in ookinete medium translate P28, the P25 paralog (https://www.sciencedirect.com/science/article/pii/S0092867404004490?via%3Dihub)
104 What cell line was used for the zygotes?
114 The number of transcripts detected in gametocytes is quite small compared to the twice as large proteomics dataset. See for example also Lasonder 2016 for P. falciparum detected transcripts: 4477 different sense transcripts were identified, 98% of which were shared between MG and FG.
117 Does the 194 up-in-gametocytes dataset include the 81 not found in zygotes?
117 Could you indicate some of the genes in the plot?
Fig1 How were the upregulated transcripts identified? 1647 are shown to be specific to zygotes in 1B, yet only 685 are shown in 1C to be upregulated. Do the transcripts found exclusively in zygotes not count? Are these transcripts likely the result of de novo transcription? How old are these zygotes when the libraries are made?
132 Many? How many? Please provide a precise number.
134 Please explain why p28 would be differentially abundant in the zygote rather than the female gametocyte. That would require de novo transcription of this gene. If there is experimental evidence for the de novo transcription of p28 and other translationally repressed transcripts in the zygote please cite the references. Can you name a few more examples here? P25 for example, ap2-o, or anything published and experimentally validated. What about AP2-o and AP2-Z? Both are known to be translationally repressed.
139 Please define how many members of the IMC?
156 Can you provide a number of how many parasites were used in total or per run. And how many biological and technical replicates were analysed?
169 The number of proteins detected in the gametocyte sample is twice the size of transcripts. IS this to be expected?
170 How many samples were analyzed? One gametocyte and one zygote sample?
176 Why did you not include P. berghei in the meta-analysis?
184 Please refer to an excel table here.
184 145 proteins: do you mean orthologs in general or orthologs with a gene/protein annotation other than unknown function?
190 142 proteins: do they all have orthologs in P. falciparum?
Figure 2C P25 is not exclusive to zygotes here and also found in the gametocyte sample.
190 shortlist
219 onwards Does the list of 198 transcripts exclusively arise from your RNAseq and proteomics comparison? Or does it include falciparum data as outline in section 176 onwards, ie the list of 276 proteins that only are detected in zygotes?
224 Early zygote? At 6 hours do the parasites not start to transform, elongate?
225 >5-fold. Is this an arbitrary decision?
227 1417 mRNAs: they are from which dataset?
228/229 Please explain why DOZI and CITH are in the list of 198 repressed transcripts? They are present in the gametocyte. Are they upregulated>5 fold?
259 ... as they are already translated in the gametocyte?
295 Is this from the 198 TR list S4?
294 onwards How many putatively falciparum transcripts are there? How many were identified in P. berghei? How many are common to all? A Venn diagram perhaps to compare the different studies
301 How many transcripts were found associated with Plasmodium berghei DOZI and/or CITH in female gametocytes? How many of those were abundantly detected as protein in zygotes, or had no difference in protein abundance between gametocytes and zygotes, or even greater abundance in female gametocytes?
303/305 Please indicate the numbers of translationally repressed transcripts identified for P. falciparum and berghei.
317/319 Please add the promoter used for tid-GFP
320 Please elaborate on the spatial organization of the DCA complex.
321/322 Have precise binding sites of DOZI and ALBA4 really been shown experimentally in the cited papers? In relation to 5' and 3' ends of the mRNA? Please cite Braks et al. paper.
323 What is the first generation BioID enzyme? BirA*
323 Please cite relevant Kyle Roux and Alice Ting for the original enzymes
327 Could you show images of ALBA4::TurboID::GFP, DOZI::TurboID::GFP and cytosolic (free) TurboID? Perhaps stained with fluorescently labelled streptavidin and / or against GFP? In the gametocyte and zygote samples?
331 What is the age of these zygotes? Where they affinity purified?
Fig S4 Please indicate whether ALBA4 and DOZI were tagged endogenously
421/430 Please add a few references here
429 translational repression?
445 966 proteins in gallinaceum? The zygote cultures in that study were 2-3 hours. How old were the cultures in your study?
481 Please explain / cite why repression is energetically costly.
501 Please add the time-point of RNA and protein sampling. How many hours into ookinete development? What is the time from cardiac puncture through FACS sampling of gametocytes.
711/713 Do you have any images that show the successful purification of zygotes away from gametocytes? Secondly, please provide a reference for the statement that unfertilized female gametocyte do not express surface exposed Pys25.
711/716 Were parasites lysed and mechanically homogenised?
Figure 6 What is the evidence that DOZI stays associated with mRNA that is being translated? Rather than mRNA that is being decapped. Please add the references that unequivocally show that DOZI and ALBA4 bind to opposite ends of repressed mRNAs.
Significance
The experiments in the manuscript are carefully conducted. Apart from a P. gallinaceum study from 2009 this is the first comprehensive analysis of the transcriptome and proteome of a Plasmodium zygote (developing ookinete) at 6 hours post-fertilization. The data are used to explore the temporal aspect of activation of translation during the first quarter of the 20-24 hour ookinete developmental period. The study will be of interest to the field, specifically those scientists working to understand translational control, ookinete development, and those developing intervention strategies to prevent mosquito infection and thus malaria transmission.
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