The CHARGE syndrome-associated protein FAM172A controls AGO2 nuclear import

AGO2 has long been known to shuttle between the cytosol and the nucleus of mammalian cells and this study helps to understand how this occurs.

that has been the subjected of conflicting and unreliable previous studies. On re-reading the paper, I believe the fundamental problem is that much of the data is either indirect or descriptive. The authors over-interpret the data -injecting too much certainty into their conclusions. In both the Response and the manuscript, the language often lacks scholarly restraint and goes beyond what the data justify. Many of the experiments are also complex and have ambiguous interpretations. I acknowledge that these are difficult experiments and that authors should be encouraged to publish the data they have, not the data we might wish them to have if they were to expend months of effort responding to reviewers. A manuscript that better describes the limitations of the experimental approaches and has more modest and thoughtful conclusions might be a better addition to the confused literature on this topic.

Reviewer #1 Review
Comments to the Authors (Required): CHARGE syndrome is a neural crest disorder mainly caused by mutation of the chromatin remodeler-coding gene CHD7. Alternative causes include mutation of other chromatin and/or splicing factors. One of these additional players is the poorly characterized FAM172A, which the authors previously found in a complex with CHD7 and the small RNA-binding protein AGO2 (Argonaute-2) at the chromatin-spliceosome interface. AGO2 is a small RNA-binding protein best known as a major component of the RNA induced silencing complex (RISC), which orchestrates post-transcriptional gene silencing in the cytoplasm. However, there are increasing lines of evidence supporting non-canonical roles of AGO2 in the nucleus. Most of the work linking AGO proteins and their nuclear functions has been performed in plants and yeast but little is known about the potential nuclear AGO protein functions in mammals. In agreement with these canonical and non-canonical functions, AGO2 has been reported to shuttle between the cytosol and the nucleus of mammalian cells. However, the molecular mechanism behind AGO2 nuclear import remains unknown. In this work, the authors showed by combining biochemistry and microscopy approaches that the NLS-containing FAM172A regulates AGO2 nuclear import via a direct protein-protein interaction, which is influenced by the status of CK2-mediated phosphorylation of FAM172A. In addition, perturbation of this process is functionally relevant, contributing to some of the cellular and molecular defects previously identified in the Fam172aTp/Tp 102 mouse model of CHARGE syndrome. Overall, the experiments are well designed and the conclusions shed light about the mechanism that imports AGO2 into the nucleus. Moreover, this study strengthens the notion that non-canonical nuclear functions of AGO2 and associated regulatory mechanisms are clinically relevant in human disease. However, there are critical points that require further clarification to support the proposed molecular mechanism: 1) In figure 2, the authors showed by coimmunoprecipitation experiments that the PAZ domain-containing N-terminal half of AGO2 as being essential for mediating the interaction with FAM172A. However, when the authors evaluated the functional relevance of this direct interaction for AGO2 nuclear localization they did not test the localizacion of this construct either by biochemical fractionation or microscopy analysis. The prediction from the proposed model is that the deletion of the PAZ domain should impair the import of AGO2 into the nucleus.
2) In figure 4, the authors concluded by mutational studies of FAM172A that CK2-mediated phosphorylation of FAM172A is important for FAM172A-dependent nuclear import of AGO2. If the proposed working model is correct CK2 knockdown should reduce AGO2 nuclear import 3) In figure 5, the authors showed the functional relevance of FAM172A-regulated nuclear import of AGO2 via rescue experiments in Fam172aTp/Tp MEFS, by combining cell proliferation assays and splicing defects. To this end, the authors tested FAM172A and its mutant versions or AGO2. The authors concluded that overexpression of WT AGO2 (which increases overall levels in both cytoplasm and nucleus) partially rescued while another version engineered to contain a strong NLS (3 copies of SV40 monopartite NLS) fully rescued the proliferation rate of Fam172aTp/Tp MEFS. However, to reinforce the working model the authors should test the PAZ domain deletion evaluated in figure 2 to demonstrate that AGO2 nuclear import reduction is unable to rescue cell proliferation and splicing defects.

Reviewer #2 Review
Comments to the Authors (Required): In their manuscript "The CHARGE syndrome-associated protein FAM172A controls AGO2 nuclear import" Sallis, Pilon and coworkers investigate nuclear import of the RISC-component Argonaute-2 (AGO2), which, despite its very prominent function(s) has not been elucidated so far. Of note, AGO-2 does not contain a (recognizable) NLS or NES. The authors make the point that the protein FAM172A functions as a mediator of nuclear import, most likely allowing a "piggyback" mechanism for import of AGO2. Furthermore, the authors investigate the effects of phosphorylation of FAM172A on nuclear import of the protein itself and on that of AGO2. The findings are well described and technically of high quality. The major concern here is the nature of the postulated complex of FAM172A and AGO2. As the authors mention, BiFC can lead to stabilization of very transient/low-affinity complexes (lane 183). Likewise, the biochemical characterization of the complex (Fig. 1) is not very convincing. The authors use low-salt buffers for all their binding/IP-experiments. Are those interactions stable in buffers containing physiological salt concentrations? Would AGO1, which seems to be unaffected by FAM172A, interact in low-salt buffers? Also, the mutant E229Q seems to interact with AGO2 to some extent ( Fig. 2A). The authors should characterize the postulated complex of FAM172A and AGO2 in more detail. This could also involve bona fide nuclear import complexes containing, for example, importin alpha (which was already identified as a potential binding partner of FAM172A (lane 276) and importin beta, leading to a real insight into the molecular mechanisms of AGO2-import. Without this, we are left with very interesting observations, pointing to a role of FAM172A in the subcellular localization of AGO2.
Minor points: Fig. 1D: The method used for fractionation should be briefly described. Fig. 1I: The log2-ratio after rescue with Myc-F172A is around "0", whereas it is around 1.5 in WT cells in Fig. 1B. The authors should comment on this partial rescue. It seems that the "predominant nuclear localization of AGO2" is not "efficiently reestablished" (as stated in the manuscript (lane 137-139).

Reviewer #3 Review
Comments to the Authors (Required): Argonaute is an important protein in the RNAi machinery. While Ago is often thought to function in the cytoplasm, it may also act in cell nuclei. Exactly how AGO shuttles between cytoplasm and nuclei is unknown in spite of several papers claiming to often insights. The strength of this manuscript is that it addresses an understudied research problem. The weaknesses is that the data are not sufficiently persuasive.
1) This is not a "CHARGE" syndrome paper. The introduction needs to be rewritten to focus on the main point of the paper. 2) In Figure 1, the microscopy results are qualitative. They do contribute to the paper, but are offset by the small change in Ago localization shown in the western (1C). Clearly, AGO can still enter the nucleus. The quantitative measures of Ago (total Ago and Ago in cytoplasm versus nucleus) are not well explained.
3) No western data is provided for the second part of Figure 1. 4) The question arises, if Ago localization is changing (not clear enough to this reviewer) is it a direct or indirect affect of mutating FAM172A. 5) The experiments in Figure 2 use recombinant proteins, which might overload the system and yield misleading results. Also, at best, the experiments suggest that FAM172A and Ago might interact inside cells. 6) I'm troubled that Figure 4 does not have western analysis of AGO localization 7) The premise for the Figure 5 is not clear. The differences are small. use of Cd44 in these cells for such a pivotal experiment is problematic. Cell proliferation may have little to do with any Ago function. This figure is not really needed anyway, the point of the paper is Ago import, not its function once it is in the nucleus.
In summary, the lack of clear, decisive western analysis of protein localization reduces my enthusiasm for this paper because that deficiency prevents it from rising about previous papers that often conflicting results. Even if the correlation between FAM expression and Ago important was stronger, I would remain concerned about whether the effect was direct or indirect. Thank you for submitting your manuscript entitled "The CHARGE syndrome-associated protein FAM172A controls AGO2 nuclear import" to Life Science Alliance. We invite you to submit a revised manuscript to acknowledge the limitations of the approach and temper the conclusions made, along the lines outlined by Reviewers 2 and 3.
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