PREreview of Bacterial RNA promotes proteostasis through inter-tissue communication inC. elegans

The manuscript demonstrates that dietary RNAs from bacteria can have a systemic, beneficial impact on the healthspan of C. elegans by reducing protein aggregation through a mechanism involving systemic autophagy, ribonuclease-dependent bacterial-RNA species, RNAi machinery and the germline, and improved muscle function. This work highlights the potential of dietary components in promoting healthy aging. Overall, the textual content is well written, the figures are clear, and the results support the conclusion. However, there are several concerns that require further attention.

Major points:

1. Since the authors claim that worms fed on OP50 produce a significantly higher number of progenies with faster development, it’s better to show the daily brood size and the developmental time difference in the progenies between OP50 and HT115 conditions.

2. It is known that different diets affect worm’s mitochondrial morphology in germline. Even though the authors claimed muscle proteotoxicity is not due to VB12. However, a functional germline is indispensable to prevent muscle proteotoxicity.  Could the authors comment on the mitochondrial morphology in muscle cells when comparing different diets?

3. The authors should measure the pharyngeal pumping rate to evaluate if worms consume the same amount of food.

4. HSF-1 is essential for maintaining proteostasis and can suppress protein aggregation. Does activate HSF-1 reduce polyQ40 aggregation?

5. Since dsRNA/ssRNA in HT115 is important for healthspan, what levels of endogenous dsRNA/ssRNA do E. coli OP50 and HT115 generate? Is there any difference regarding aggregate formation in muscle cells between using OP50-derived RNA and HT115-derived RNA?

6. It is not clear which type(s) of RNA, other than bacteria-derived RNA, play an important role in reducing aggregate formation. It would be important to narrow down the identity of RNA types. Thus, the authors should examine whether the absence of different types of ribonucleases could reduce aggregate formation in muscle cells.

7. Clarification and discussion are needed for the mutants that have significant changes among groups in Fig 4C-H.

8. It is not clear when the bacterial RNA is important to promote proteostasis in worms. Does feeding HT115 E. coli at later ages after feeding OP50 E. coli reduce aggregate formation compared to feeding OP50 alone?

9. Please provide correction method used for proteomics data analysis for multiple testing and adjust the y axis of the volcano plots accordingly.

Minor:

1) Please define “fitness” used in this paper, and it’s not clear about the difference of fitness and healthspan.

2) Determining whether worms move or not by touching them is subjective, so it’s better to define the area of body wall muscle considered as paralyzed for evaluation in this paper.

3) There are some statements that do not clearly or fully conveyed the information from the figures. For example, line 78-80 for Figure 1A and 1B.

4) Line 54-56 may lack references.

5) Line 234: OP50(xu363), the name form is not consistent.

6) Figure 5: “Scale bars in all panels are 100 µm” should be deleted.

7) More details are needed for the conclusion of vitamin B12 is not involved.

8) How about fat metabolism difference contributing to proteostasis between OP50 and HT115? Please discuss.

9) Do the 194 significantly changed proteins from proteomic analysis include the key muscle genes that are studied in this paper?

10) There is no access to all the supplementary information, so it is not possible to evaluate the related observations or conclusions.

Competing interests

The authors declare that they have no competing interests.