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The manuscript Cytoplasmic protein-free mRNA induces stress granules by two G3BP1/2-dependent mechanisms assesses the effects of microinjected mRNA in the formation of stress granules. They found that injecting low amounts of RNA triggers stress granule formation by activating PKR, and high concentrations of untranslating mRNAs induces stress granules through a mechanism independent of PKR, and partially in cells lacking G3BP proteins. This work provides strong support for previously established models of stress granule assembly. They use a novel method of microinjecting RNA which allowed them to directly study the effects of changes of concentration of protein free mRNAs in the cell, which has previously not been done to this extent. It demonstrates clearly that the formation of stress granules is dependent on the concentration of its components and can be triggered directly by increasing the cytoplasmic concentration of non-translating mRNAs.

Major comments –

The manuscript tells a fairly complete story, but I’d like to bring up a couple of alternative interpretations and ideas for the author’s consideration that would make the conclusions more robust:

1.      The authors interpret the results as acute increase of free mRNA saturates the capacity of RNA-binding proteins to prevent RNA-RNA interactions. There are other (or concurrent) interpretations of how the cell might be reacting to the acute increase of mRNAs. For example, does this increase of mRNA affect translation? The new mRNA may saturate the translation machinery leading to shutdown of translation without eIF2-alpha phosphorylation. The authors could explore this idea with a puromycin translation assay.

2.      The authors’ explanation of puromycin on page 19 is not completely correct. They state “puromycin treatment releases most mRNA from ribosomes…” Puromycin induces premature termination of translation, leading to the disassembly of the polysome but does not prevent continued loading of the initiation complex.

Also in this paragraph, they hypothesize that “naked mRNAs will be bound quickly by RNA-binding proteins, reducing the effective concentrations of naked mRNAs…” How would this hypothesis explain how drugs that release mRNA from ribosomes cause stress granules without additional stressors and without phosphorylating eIF2-alpha (See for example, pateamine A in Dang et al, 2006, JCB)? As mentioned above, puromycin doesn’t prevent loading of the initiation complex, while pateamine A does. It follows that the continued loading of initiation machinery and transiently elongating ribosomes in puromycin treatment keeps mRNAs from being incorporated into a stress granule. However, if that is what the authors mean they should make it clear that “RNA-binding proteins” includes the translation initiation complex. This makes exploring if additional mRNA is saturating the translation machinery even more interesting.

Minor points –

1.      Injecting large amounts of RNA, which contain dsRNA regions, might also trigger RNase L activation which can lead to the formation of G3BP positive puncta called RNase L bodies (described in Burke et al., 2020 JCB). The authors might want to rule out puncta formed in PKR deletion cells are RNase L bodies by performing a RNase L knockout.

2.      The amount of mRNA in the cell line used here (page 8, line 14) has been published in Khong et al 2017 (Mol. Cell). Using this more recent data, the amount of mRNA post-injection would be higher. 

3.      Some experiments were quantified using SG+ cells. I’m curious to see the quantification of stress granule area or ratio of stress granule area to cytoplasm area. I think this would be an excellent addition to the binary SG+ cells quantification method.

4.      I find it curious that the total amount of RNA in the cell does not change that much according to oligo-d(T) staining even with 300 ng/uL (Figure 1b). Is this analysis within the linear range of poly-d(T) staining? I think the highest concentrations used in this manuscript (600 ng/uL) would cause a significant change and could be used as a positive control for the assay.

5.      How many cells were micro-injected? I recommend adding that information to the methodology.

Competing interests

The authors declare that they have no competing interests.