FMRP Regulates Neuronal RNA Granules Containing Stalled Ribosomes, Not Where Ribosomes Stall

Reviewer #1 (Public review):

Summary:

The authors have investigated the role of FMRP in the formation and function of RNA granules in mouse brain/cultured hippocampal neurons. Most of their results indicate that FMRP does not have a role in the formation or function of RNA granules with specific mRNAs, but may have some role in distal RNA granules in neurons and their response to synaptic stimulation. This is an important work (though the results are mostly negative) in understanding the composition and function of neuronal RNA granules. The last part of the work in cultured neurons is disjointed from the rest of the manuscript, and the results are neither convincing nor provide any mechanistic insight.

Strengths:

(1) The study is quite thorough, the methods and analysis used are robust, and the conclusion and interpretation are diligent.

(2) The comparative study of Rat and Mouse RNA granules is very helpful for future studies.

(3) The conclusion that the absence of FMRP does not affect the RNA granule composition and many of its properties in the system the authors have chosen to study is well supported by the results.

(4) The difference in the response to DHPG stimulation concerning RNA granules described here is very interesting and could provide a basis for further studies, though it has some serious technical issues.

Weaknesses:

(1) The system used for the study (P5 mouse brain or DIV 8-10 cultured neuron) is surprising, as the majority of defects in the absence of FMRP are reported in later stages (P30+ brain and DIV 14+ neurons). It is important to test if the conclusions drawn here hold good at different developmental stages.

(2) The term 'distal granules' is very vague. Since there is no structural or biochemical characterization of these granules, it is difficult to understand how they are different from the proximal granules and why FMRP has an effect only on these granules.

(3) Since the manuscript does not find any effect of FMRP on neuronal RNA granules, it does not provide any new molecular insight with respect to the function of FMRP

Reviewer #2 (Public review):

In the present manuscript, Li et al. use biochemical fractionation of "RNA granules" from P5 wildtype and FMR1 knock-out mouse brains to analyze their protein/RNA content, determine a single particle cryo-EM structure of contained ribosomes, and perform ribo-seq analysis of ribosome-protected RNA fragments (RPFs). The authors conclude from these that neither the composition of the ribosome granules, nor the state of their contained ribosomes, nor the mRNA positions with high ribosome occupancy change significantly. Besides minor changes in mRNA occupancy, the one change the authors identified is a decrease in puromycylated punctae in distal neurites of cultured primary neurons of the same mice, and their enhanced resistance to different pharmacological treatments. These results directly build on their earlier work (Anadolu et al., 2023) using analogous preparations of rat brains; the authors now perform a very similar study using WT and FMR1-KO mouse brains. This is an important topic, aiming to identify the molecular underpinnings of the FMRP protein, which is the basis of a major neurological disease. Unfortunately, several limitations of this study prevent it from being more convincing in its present form.

In order to improve this study, our main suggestions are as follows:

(1) The authors equate their biochemically purified "RG" fraction with their imaging-based detection of puromycin-positive punctae. They claim essentially no differences in RGs, but detect differences in the latter (mostly their abundance and sensitivity to DHPG/HHT/Aniso). In the discussion the authors acknowledge the inconsistency between these two modalities: "An inconsistency in our findings is the loss of distal RPM puncta coupled with an increase in the immunoreactivity for S6 in the RG." and "Thus, it may be that the RG is not simply made up of ribosomes from the large liquid-liquid phase RNA granules."

How can the authors be sure that they are analysing the same entities in both modalities? A more parsimonious explanation of their results would be that, while there might be some overlap, two different entities are analyzed. Much of the main message rests on this equivalence, and I believe the authors should show its validity.

(2) The authors show that increased nuclease digestion (and magnesium concentration) led to a reduction of their RPF sizes down to levels also seen by other researchers. Analyzing these now properly digested RPFs, the authors state that the CDS coverage and periodicity drastically improved, and that spurious enrichments of secretory mRNAs, which made up one of the major fractions in their previous work, are now reduced. In my opinion, this would be more appropriately communicated as a correction to their previous work, not as a main Figure in another manuscript.

(3) The fold changes reported in Figure 7 (ranging between log2(-0.2) and log2(+0.25)) are all extremely small and in my opinion should not be used to derive claims such as "The loss of FMRP significantly affected the abundance and occupancy of FMRP-Clipped mRNAs in WT and FMR1-KO RG (Fig 7A, 7B), but not their enrichment between RG and RCs".

(4) Figure 8 / S8-1 - The authors show that ~2/3 of their reads stem from PCR duplicates, but that even after removing those, the majority of peaks remain unaltered. At the same time, Figure S8-1 shows the total number of peaks to be 615 compared with 1392 before duplicate removal. Can the authors comment on this discrepancy? In addition, the dataset with properly removed artefacts should be used for their main display item instead of the current Figure 8.

(5) Figure 9 / S9-1, the density of punctae in both WT and FMR1-KO actually increases after treatment of HHT or Anisomycin (Figure S9-1 B-C). Even if a large fraction would now be "resistant to run-off", there should not be an increase. While this effect is deemed not significant, a much smaller effect in Figure 9C is deemed significant. Can the authors explain this? Given how vastly different the sample sizes are (ranging from 23 neurites in Figures S9-1 to 5,171 neurites in Figure 9), the authors should (randomly) sample to the same size and repeat their statistical analysis again, to improve their credibility.

Reviewer #3 (Public review):

Summary: Li et al describe a set of experiments to probe the role of FMRP in ribosome stalling and RNA granule composition. The authors are able to recapitulate findings from a previous study performed in rats (this one is in mice).

Strengths:

1. The work addresses an important and challenging issue, investigating mechanisms that regulate stalled ribosomes, focusing on the role of FMRP. This is a complicated problem, given the heterogeneity of the granules and the challenges related to their purification. This work is a solid attempt at addressing this issue, which is widely understudied.

2. The interpretation of the results could be interesting, if supported by solid data. The idea that FMRP could control the formation and release of RNA granules, rather than the elongation by stalled ribosomes is of high importance to the field, offering a fresh perspective into translational regulation by FMRP.

3. The authors focused on recapitulating previous findings, published elsewhere (Anadolu et al., 2023) by the same group, but using rat tissue, rather than mouse tissue. Overall, they succeeded in doing so, demonstrating, among other findings, that stalled ribosomes are enriched in consensus mRNA motifs that are linked to FMRP. These interesting findings reinforce the role of FMRP in formation and stabilization of RNA granules. It would be nice to see extensive characterization of the mouse granules as performed in Figure 1 of Anadolu and colleagues, 2023.

4. Some of the techniques incorporated aid in creating novel hypotheses, such as the ribopuromycilation assay and the cryo-EM of granule ribosomes.

Weaknesses:

1. The RNA granule characterization needs to be more rigorous. Coomassie is not proper for this type of characterization, simply because protein weight says little about its nature. The enrichment of key proteins is not robust and seems to not reach significance in multiple instances, including S6 and UPF1. Furthermore, S6 is the only proxy used for ribosome quantification. Could the authors include at least 3 other ribosomal proteins (2 from small, 2 from large subunit)?

2. Page 12-13 - The Gene Ontology analysis is performed incorrectly. First, one should not rank genes by their RPKM levels. It is well known that housekeeping genes such as those related to actin dynamics, molecular transport and translation are highly enriched in sequencing datasets. It is usually more informative when significantly different genes are ranked by p adjust or log2 Fold Change, then compared against a background to verify enrichment of specific processes. However, the authors found no DEGs. I would suggest the removal of this analysis, incorporation of a gene set enrichment analyses (ranked by p adjust). I further suggest that the authors incorporate a dimensionality reduction analysis to demonstrate that the lack of significance stems from biology and not experimental artifacts, such as poor reproducibility across biological replicates.

Author response:

Reviewer #1 (Public review):

Summary:

The authors have investigated the role of FMRP in the formation and function of RNA granules in mouse brain/cultured hippocampal neurons. Most of their results indicate that FMRP does not have a role in the formation or function of RNA granules with specific mRNAs, but may have some role in distal RNA granules in neurons and their response to synaptic stimulation. This is an important work (though the results are mostly negative) in understanding the composition and function of neuronal RNA granules. The last part of the work in cultured neurons is disjointed from the rest of the manuscript, and the results are neither convincing nor provide any mechanistic insight.

Strengths:

(1) The study is quite thorough, the methods and analysis used are robust, and the conclusion and interpretation are diligent.

(2) The comparative study of Rat and Mouse RNA granules is very helpful for future studies.

(3) The conclusion that the absence of FMRP does not affect the RNA granule composition and many of its properties in the system the authors have chosen to study is well supported by the results.

(4) The difference in the response to DHPG stimulation concerning RNA granules described here is very interesting and could provide a basis for further studies, though it has some serious technical issues.

Weaknesses:

(1) The system used for the study (P5 mouse brain or DIV 8-10 cultured neuron) is surprising, as the majority of defects in the absence of FMRP are reported in later stages (P30+ brain and DIV 14+ neurons). It is important to test if the conclusions drawn here hold good at different developmental stages.

(2) The term 'distal granules' is very vague. Since there is no structural or biochemical characterization of these granules, it is difficult to understand how they are different from the proximal granules and why FMRP has an effect only on these granules.

(3) Since the manuscript does not find any effect of FMRP on neuronal RNA granules, it does not provide any new molecular insight with respect to the function of FMRP

Thank you for your comments and for pointing out the strengths of the manuscript. Unfortunately, we will not be able to respond to point #1. The protocol for purification of the ribosomes from RNA granules does not work in older brains (See Khandjian et al, 2004 PNAS 101:13357), presumably due to the presence of large concentrations of myelin. While it would be possible to repeat our results later in culture, we have no expectation that it would be different since we do observe DHPG induction of elongation dependent, initiation independent mGLUR-LTD in later cultures (Graber et al, 2017 J. Neuroscience 37:9116)..We will strengthen this caveat in the discussion that our results are only at a snapshot of development and that it is certainly possible that different results may be seen at different times. We agree with point 2 that ‘distal granules’ is a vague term. We will remove the term and clarify that we only quantified granules larger than 50 microns from the cell soma. We do not know if these granules are distinct. We would respectfully disagree with point #3 that the study does not provide molecular insight into the function of FMRP, as disproving that FMRP is important for stalling and determining the position of stalling removes a major hypothesis about the function of FMRP, and showing that something is not true, is at least to me, providing insight.

Reviewer #2 (Public review):

In the present manuscript, Li et al. use biochemical fractionation of "RNA granules" from P5 wildtype and FMR1 knock-out mouse brains to analyze their protein/RNA content, determine a single particle cryo-EM structure of contained ribosomes, and perform ribo-seq analysis of ribosome-protected RNA fragments (RPFs). The authors conclude from these that neither the composition of the ribosome granules, nor the state of their contained ribosomes, nor the mRNA positions with high ribosome occupancy change significantly. Besides minor changes in mRNA occupancy, the one change the authors identified is a decrease in puromycylated punctae in distal neurites of cultured primary neurons of the same mice, and their enhanced resistance to different pharmacological treatments. These results directly build on their earlier work (Anadolu et al., 2023) using analogous preparations of rat brains; the authors now perform a very similar study using WT and FMR1-KO mouse brains. This is an important topic, aiming to identify the molecular underpinnings of the FMRP protein, which is the basis of a major neurological disease. Unfortunately, several limitations of this study prevent it from being more convincing in its present form.

In order to improve this study, our main suggestions are as follows:

(1) The authors equate their biochemically purified "RG" fraction with their imaging-based detection of puromycin-positive punctae. They claim essentially no differences in RGs, but detect differences in the latter (mostly their abundance and sensitivity to DHPG/HHT/Aniso). In the discussion the authors acknowledge the inconsistency between these two modalities: "An inconsistency in our findings is the loss of distal RPM puncta coupled with an increase in the immunoreactivity for S6 in the RG." and "Thus, it may be that the RG is not simply made up of ribosomes from the large liquid-liquid phase RNA granules."

How can the authors be sure that they are analysing the same entities in both modalities? A more parsimonious explanation of their results would be that, while there might be some overlap, two different entities are analyzed. Much of the main message rests on this equivalence, and I believe the authors should show its validity.

(2) The authors show that increased nuclease digestion (and magnesium concentration) led to a reduction of their RPF sizes down to levels also seen by other researchers. Analyzing these now properly digested RPFs, the authors state that the CDS coverage and periodicity drastically improved, and that spurious enrichments of secretory mRNAs, which made up one of the major fractions in their previous work, are now reduced. In my opinion, this would be more appropriately communicated as a correction to their previous work, not as a main Figure in another manuscript.

(3) The fold changes reported in Figure 7 (ranging between log2(-0.2) and log2(+0.25)) are all extremely small and in my opinion should not be used to derive claims such as "The loss of FMRP significantly affected the abundance and occupancy of FMRP-Clipped mRNAs in WT and FMR1-KO RG (Fig 7A, 7B), but not their enrichment between RG and RCs".

(4) Figure 8 / S8-1 - The authors show that ~2/3 of their reads stem from PCR duplicates, but that even after removing those, the majority of peaks remain unaltered. At the same time, Figure S8-1 shows the total number of peaks to be 615 compared with 1392 before duplicate removal. Can the authors comment on this discrepancy? In addition, the dataset with properly removed artefacts should be used for their main display item instead of the current Figure 8.

(5) Figure 9 / S9-1, the density of punctae in both WT and FMR1-KO actually increases after treatment of HHT or Anisomycin (Figure S9-1 B-C). Even if a large fraction would now be "resistant to run-off", there should not be an increase. While this effect is deemed not significant, a much smaller effect in Figure 9C is deemed significant. Can the authors explain this? Given how vastly different the sample sizes are (ranging from 23 neurites in Figures S9-1 to 5,171 neurites in Figure 9), the authors should (randomly) sample to the same size and repeat their statistical analysis again, to improve their credibility.

Thank you for your comments. We agree with the issue in point #1 that the equivalence of RPM puncta with the RG fraction is an issue and while we believe that we show in a number of ways that the two are related (anisomycin-resistant puromycylation, puromyclation only at high concentrations consistent with the hybrid state, etc), we would respectfully disagree that our main message results from the equivalence of the RPM-labeled RNA granules in neurites and the ribosomes isolated by sedimentation. We will make this point clearer in our revision. For point #2, we agree that the changes with increased nuclease is somewhat out of place in a narrative sense, but it is clearly relevant to this work. Whether or not one sees this as a ‘correction’ or an interesting point will depend on a better characterization of the structures of the stalled polysomes. My personal view is that the nuclease resistance of cleavage near the RNA entrance site is quite interesting. Since we reproduce our results with a similar nuclease treatment in mice, as reported in our previous publication, I believe the comparison could be of interest in the future and would like to retain it. We agree with point #3 and will temper these claims in our revised version. For point #4, we will determine more carefully why the number of peaks differs and switch the main and supplemental figures. We apologize for the typo in the figure legend in Figure 9, 171, not 5171. The box plot line shows the median not the average and the data is clearly skewed such that the median and average are different (i.e. there is a two-fold decrease in the average density of distal puncta between WT and FMRP, but the average density is actually slightly decreased with HHT and A, although the median increases slightly. We will now report the results in distinct modalities to clarify this, and we will reexamine the statistics to better address the skewed distribution of values in the revised version.

Summary:

Li et al describe a set of experiments to probe the role of FMRP in ribosome stalling and RNA granule composition. The authors are able to recapitulate findings from a previous study performed in rats (this one is in mice).

Strengths:

(1) The work addresses an important and challenging issue, investigating mechanisms that regulate stalled ribosomes, focusing on the role of FMRP. This is a complicated problem, given the heterogeneity of the granules and the challenges related to their purification. This work is a solid attempt at addressing this issue, which is widely understudied.

(2) The interpretation of the results could be interesting, if supported by solid data. The idea that FMRP could control the formation and release of RNA granules, rather than the elongation by stalled ribosomes is of high importance to the field, offering a fresh perspective into translational regulation by FMRP.

(3) The authors focused on recapitulating previous findings, published elsewhere (Anadolu et al., 2023) by the same group, but using rat tissue, rather than mouse tissue. Overall, they succeeded in doing so, demonstrating, among other findings, that stalled ribosomes are enriched in consensus mRNA motifs that are linked to FMRP. These interesting findings reinforce the role of FMRP in formation and stabilization of RNA granules. It would be nice to see extensive characterization of the mouse granules as performed in Figure 1 of Anadolu and colleagues, 2023.

(4) Some of the techniques incorporated aid in creating novel hypotheses, such as the ribopuromycilation assay and the cryo-EM of granule ribosomes.

Weaknesses:

(1) The RNA granule characterization needs to be more rigorous. Coomassie is not proper for this type of characterization, simply because protein weight says little about its nature. The enrichment of key proteins is not robust and seems to not reach significance in multiple instances, including S6 and UPF1. Furthermore, S6 is the only proxy used for ribosome quantification. Could the authors include at least 3 other ribosomal proteins (2 from small, 2 from large subunit)?

(2) Page 12-13 - The Gene Ontology analysis is performed incorrectly. First, one should not rank genes by their RPKM levels. It is well known that housekeeping genes such as those related to actin dynamics, molecular transport and translation are highly enriched in sequencing datasets. It is usually more informative when significantly different genes are ranked by p adjust or log2 Fold Change, then compared against a background to verify enrichment of specific processes. However, the authors found no DEGs. I would suggest the removal of this analysis, incorporation of a gene set enrichment analyses (ranked by p adjust). I further suggest that the authors incorporate a dimensionality reduction analysis to demonstrate that the lack of significance stems from biology and not experimental artifacts, such as poor reproducibility across biological replicates.

Thank you for your comments on the strengths of the manuscript. We agree with point #1 that the mouse RNA granule characterization needs to be more rigorous and we plan to accomplish this in our revised version. Similarly, we will incorporate the additional statistical analysis suggested by the reviewer in a revised version.