PREreview of Autophagy activity is inhibited by hnRNP R

This present review was prepared as a practical work from the graduate-level course "How to Read and Evaluate Scientific Papers and Preprints" from the University of São Paulo, in which the primary goal was to afford students the chance to review scientific articles, develop critical and constructive discussions on the endless frontiers of knowledge, and gain insight into the procedure of peer review process.

Evidence, Reproducibility, and Clarity

In this study, Changhe Ji presents a potential pathway of autophagy regulation, which has not been extensively explored, and involves an RNA-binding protein (hnRNP R) with a role in the regulation of autophagy. To demonstrate this hypothesis, the author employs appropriately designed experiments with necessary controls. The study provides evidence that hnRNP R negatively regulates autophagy, although some apparent contradictions need further analysis or discussion. In addition, the manuscript contains several spelling errors and sentence structure issues, which difficult the readability and interpretation of the content.

Major Comments:

·         The introduction provides reasonable information about autophagy, however, it is a bit repetitive. So, I suggest to eliminate repeated statements and to incorporate the available information about RNA binding proteins (hnRNP R) (not given in the introduction) in this section. A brief overview on the role of the hnRNP R in the regulation of autophagy could be helpful for non-specialized readers. It would be interesting as well to reorganize the structure of the paragraphs and to correct spelling and grammatical mistakes all along the manuscript.

·         According to the title, the abstract and the introduction, the main objective of the investigation is to demonstrate that the hnRNP R has a role in the regulation of autophagy. In fact, most of the experiments carried out by the author point to demonstrate this. In addition, in the results several experiments are presented to verify the relationship between autophagy and stress granules in a hnRNP R dependent manner. Thus, I suggest the author to mention in advance that another important objective of the study was to investigate the relationship between stress granules and autophagy.

·         To investigate how the hnRNP R affects autophagy, the author uses hnRNP R knockdown (KD) cells and measures the levels of autophagy marker proteins by western blot: ATG9, LAMP1, p62, ATG3, LC3I and LC3II. Figures 1A and 1B show that the levels of ATG9, LC3 II, LAMP 1 increase in hnRNP R KD cells treated with bafilomycin, which corroborates that the loss of hnRNP R activates autophagy, as suggested by the author. It is surprising that p62 levels have decreased since bafilomycin prevents the fusion of lysosomes and autophagosomes and therefore would impair the degradation of p62 present in autophagosomes. The author should discuss better this finding.

·         In the topic: ''Loss of hnRNP R activates autophagy activity'' (second paragraph of the results section): much explanation is provided on the interpretation of the monitoring of the autophagy flow by measuring the levels of LC3 II. I suggest the author to move this to the introduction or discussion sections.

·         In addition to measuring LC3 II levels by western blot, the author complements this result using the Tandem-fluorescent LC3 assay, which is an elegant way to confirm the same result. However, the result of this experiment (Figure 2) was not well explained. I suggest the author to explain in more detail the results shown in Figure 2 and how they reached the conclusion of this experiment.

·         The author mentions in the abstract that they found that autophagy has no connection with stress granules, but the immunoprecipitation experiments show that the stress granule protein G3BP1 interacts with ATG9. In the discussion, the author states: ''Consistent with previous finding by other research [55], we also found that G3BP1 can also bind with ATG9, this indicates ATG9 can also tether the stress granule protein to the autophagosome, we also found that hnRNP R link the binding of G3BP1 with ATG9''. This in fact, confirms that autophagy has a connection to stress granules. Thus, the information given in the abstract was contradictory.

·         The confocal microscopy is an effective way to confirm co-localization of two objects (i.e. proteins). The immunofluorescence images are very clear and beautiful. However, I suggest to better describe the criteria used to define co-localization. The author mentions that in Figure 5, there are more stress granules that co-localize with LAMP1 after hnRNP R depletion and that in Figure EV4 some p62 dots co-localize with stress granules in hnRNP R -/- cells. However, looking at the respective figures these proteins appear not to be co-localized.

·         In the discussion, the author concludes that hnRNP R plays an important role in the regulation of autophagy. This conclusion is well supported by the increase of LC3 II in hnRNP R knockdown and knockout cells. However, the author soon after mentions that the increase of LC3 II in hnRNP R knockdown and knockout cells after Bafilomycin A1 treatment is due to bafilomycin toxicity and that autophagy is activated consequently to keep the cells alive. According to the literature, bafilomycin causes accumulation of LC3 II and p62, by inhibiting lysosomal degradation, and the increase in LC3 II reflects the amount of LC3 II that would have been degraded by autophagy during the treatment period and not due to bafilomycin toxicity as suggested by the author. Please clarify this point.

Minor Comments:

·         "Another interesting finding is LAMP1 protein was increased in hnRNP R knockdown cells (Figure 1), we also use confocal image to confirm the downregulation of LAMP1 in hnRNP R knockdown cells." In the previous sentence the word "downregulation" appears to have been used instead of "upregulation".

·         I suggest the author to define the abbreviations in full the first time they are used, e.g., "Ubiquitin proteasome system (UPS)" and then start using the abbreviation directly.

Competing interests

The author declares that they have no competing interests.