Length-Dependent Translation Efficiency of ER-Destined Proteins

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 

The study by Sahinbegovic et al. deals with the mechanisms governing the translation of signal sequence-bearing mRNAs for endoplasmic reticulum – targeted proteins. The authors conclude that the long mRNAs remain on the ER while being translated, whereas the short mRNAs face a translation pause. According to the authors, this should lead to a drop in the translation efficiency of small ER-targeted proteins.

 The manuscript presents potential interesting data. However, further controls for the experiments and a more detailed discussion of the results are required.

Points: 

1.) Please add a molecular weight marker for the proteins in figure 2.   

2.) The authors have performed a fractionation assay. However, the data in figure 4 do not show necessary controls for the quality of the separation. Therefore, the authors should present marker proteins as control for the separation steps.

3.) The authors analyse especially the relation of different mRNA versions to the ER membrane. Therefore, the authors should perform an additional experiment to analyse specifically the association with the ER membrane and not just with a general membrane fraction.

4.) The authors cite the analysis of the expression dataset (Cunningham et al. 2022), which reveals a clear trend of enhanced transcription towards shorter mRNAs. Please provide a discussion of the similarities and differences to this paper.

5.) Please explain the differences to the cited studies Guo et al. 2014 and Rogers et al. 2017.

6.) The style of the references in the text is not the style of the journal.

 

Comments on the Quality of English Language

A moderate editing of English language is required.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The study by Sahinbegovic et al. describes an interesting observation that the size/length of ER-destined mRNAs influences the amount of generated proteins. The study is interesting, and the proposed model is original and has great potential; however, some experimental data are not very convincing. Considering the journal's policy regarding fast publication, I am not asking completion of additional experiments. Still, I advise you to "soft" some of the conclusions and eliminate overstatements. My critiques are listed below:

 

1. The gap in knowledge is missing.
2. The model organism should be indicated in the Abstract and the Introduction.
3. "The apparent translation deficiency for shorter, ER-localized mRNAs is not affected components of the ribosome stalling pathway": please correct the grammar.
4. "As expected, RPS10 ubiquitination increased upon expression of the reporter with the poly-A sequence (K)20 but not a single IgL domain": I don't see the difference. Bands quantification is needed. Have you done this experiment using proteasome inhibitors? This should be indicated in the main text.
5. "Therefore, we deleted the ER destination signal peptide from IgL (ΔSP IgL) and fused the same signal sequence to the Ig-like domain of FLNA (SP FLNA)": where precisely the SP was placed on the reporter mRNA? Was it the real 5’-end of the mRNA reporter? Have you placed any 5'UTR? Why you used the 2A sequence? Have you inserted any linker sequences between IgL domain-encoding sequences? If yes, what is the length? Bottom line, it is critical to describe the reporter-based experimental system (construct) used here in better detail and provide a rationale.
6. No reference to Figure 2C.
7. Fig 4B&C: "Relative ratio of free (cytosolic) and membrane-bound (ER-associated) mRNA encoding 1 and 2 IgLλ domains. Significance was compared using the two-tailed Student's t test, * = P, 0.05, ns = non-significant. Error bars represent the mean ±SD.": not clear what the readout is and how this ratio was calculated. Are these qPCR-based signals? Please, provide these details in the Figure 4B&C legend and explain how you can distinguish between "long" and "short" mRNAs.
8. Ribo Mega-SEC: Please, provide an explanation of the methodology for this assay.
9. Related to Figure 4D: I see a colossal 60S:40S imbalance. Comment on this, please. Overall, this experiment is useless, as you analyze the total mRNA pool, and only a small % belongs to your plasmid-derived mRNA. "As expected, the cells with abundant mRNA encoding two IgL domains resemble significantly enriched polysomal pool compared to cells expressing single IgL ORF.": This is an overstatement; please, re-phrase the conclusion.
10. I believe any structural predictions of the used mRNA reporters will benefit this study. As such, if the authors can model 1xIgL vs. 2xIgL mRNA structure that likely, demonstrates no significant structural differences (except one extra IgL-encoding region), it will help the conclusion that secondary mRNA structure does not contribute to the observed effects.

Comments on the Quality of English Language

The article is written in good English; however, I found a few things that needed to be corrected (for example, I found a few typos: "Absorbancde" (Figure 4D legend); "polysomal pool compared." ). Please, proofread your manuscript. Please, proofread your manuscript thoroughly. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed all of my points.