PREreview of Cytoplasmic protein-free mRNA induces stress granules by two G3BP1/2-dependent mechanisms

Cytoplasmic protein-free mRNA induce stress granules by two G3BP1/2-dependent mechanisms

            In this manuscript, the authors demonstrate that microinjected mRNAs can promote stress granule formation in cells via two different mechanisms. When the mRNA concentration is low, the in vitro transcribed dsRNA can trigger a PKR-dependent mechanism that phosphorylates eIF2α, leading to translation repression and stress granule formation. However, at high mRNA concentration and when PKR is inactivated, the cells can activate a PKR-independent mechanism to assemble stress granules. Furthermore, they show that both the PKR-dependent and PKR-independent mechanisms are influenced by G3BP1/2. Overall, this manuscript is interesting and uses a new method of microinjection to provide substantial support for existing models of stress granule assembly.

The manuscript made several conclusions, and some of them would require further work to make a robust conclusion.

Major point:

1.     In cells injected with high concentrations of mRNA, it is possible stress granules are forming due to mRNA translation being repressed by other eIF2α-independent mechanisms. This analysis would be stronger if the authors perform a SUnSET assay to measure changes in mRNA translation upon injecting a high concentration of mRNA.

2.     In Fig. 1B, the authors used poly(A) staining to show that microinjection of mRNA did not affect the total mRNA amount in cells. One potential concern is that due to abundance of poly(A) signals at baseline level, the signal detection might be at saturation and no longer at the dynamic range to detect changes, if any. The authors could complement this by performing a Northern blot against poly(A) tails at the linear range of detection.

3.     In their final conclusion, the authors propose that endogenous ribosome-free mRNAs released during ribosome run-off is insufficient to induce stress granule formation. They performed experiment by microinjecting 300ng/uL ftz mRNA and treating with puromycin in cells that cannot phosphorylate eIF2α. However, they have previously shown in Supple Fig. 2E that stress granules formation is restored without the need for puromycin in these cell lines. Thus, the experiment would have been more convincing if the authors performed this experiment with 75ng/uL ftz mRNA.

4.     The authors used puromycin and harringtonine to induce translation shut-off and ribosome run-off. However, there are some caveats regarding the effects of these drugs. While puromycin is known to disassemble polysomes from mRNAs, it does not usually lead to stress granule formation (Kedersha et al., 2000, JCB). Similarly for harringtonine, while the drug traps a vacant 80S ribosome at the start of the transcript and leads to ribosome run-off for elongating ribosomes, it does not promote stress granule assembly (Fedorovskiy et al., 2023, Biochemistry (Moscow)). Therefore, it would strengthen the conclusion if the authors revise their interpretation of the data presented in Fig. 6 based on these caveats.

5.     The authors could consider incorporating more parameters when quantifying stress granule assemblies. Apart from counting the number of stress granule positive cells, it would be informative to see if treatments alter stress granule size and number. For example, in the images shown in Fig. 5A and 6E, stress granules size looks smaller in mutant cells compared in WT cells.

Minor point:

1.     For one of the yellow arrows in Fig. 1F, the cell contains TIA-1+ assemblies too

2.     Specify what is defined as docking of PB to SG? What’s the range of distance that is defined as docking?

3.     Typo in Fig. 2C, y-axis

4.     Lacks p-eIF2α staining in Fig. 2D to show that shPKR is working, and ftz mRNA FISH in Fig. 2E to show its localization

5.     Would prefer if the quantification of SG area in between shCtrl and shPKR in Fig. 2E is shown

6.     Rescue the “no SG” phenotype in Fig. 2 by putting WT-PKR or OE WT p-eIF2α to show that it is a direct effect of the ISR.

7.     Lacks ftz mRNA staining in Fig. 3D to show that ftz mRNA localizes to SG in absence of p-eIF2a activation

8.     Wrong citation: (see quantification in Fig. 5A) à should be Fig. 5B

9.     No SD for the WT injected with Puromycin column in Fig. 6C

10. Missing total number of cells that is injected with RNA and quantified for each experiment

Competing interests

The authors declare that they have no competing interests.