RNA is required for the maintenance of multiple cytoplasmic and nuclear membrane-less organelles

  1. Carolyn J. Decker
  2. James M. Burke
  3. Patrick K. Mulvaney
  4. Roy Parker

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Abstract

Numerous membrane-less organelles composed of a combination of RNA and proteins, referred to as RNP granules, are observed in the nucleus and cytoplasm of eukaryotic cells, including stress granules, processing bodies, Cajal bodies, and nuclear speckles. An unresolved issue is how frequently RNA molecules are required for the maintenance of RNP granules in either the nucleus or cytosol. To address this issue, we degraded intracellular RNA in either the cytosol or the nucleus by the activation of RNase L and examined the impact of RNA loss on several RNP granules. Strikingly, we find the majority of RNP granules, including stress granules, processing bodies, Cajal bodies, nuclear speckles and the nucleolus are altered by the degradation of their RNA components. In contrast, super-enhancer complexes and TIS granules were largely unaffected by widespread intracellular RNA degradation. This highlights a critical and widespread role of RNA in the organization of many, but not all, RNP granules.

SUMMARY

Decker et al. examine the role of RNA in maintaining membrane-less organelles composed of RNA and protein by activating RNase L in the cytoplasm or nucleus. Degradation of RNA alters the majority of membrane-less organelles indicating that RNA plays a widespread role in their organization.

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  1. Review Commons

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    The authors do not wish to provide a response at this time.

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    The authors examine the important and challenging question in current biology, the role of RNA in the structural maintenance of nuclear and cytoplasmic membrane-less organelles including stress granules, processing bodies, nucleolus, Cajal bodies, and nuclear speckles. Furthermore, the authors explored super-enhancer complexes involved in the regulation of gene expression. The authors used RNase L, an interferon-induced ribonuclease which, upon activation in the cytoplasm or targeted to the nucleus, degrades all RNAs within the cell. Then they took the quantitative approach to analyze the effect of RNA degradation on disassembly or reorganization of membrane-less organelles. Interestingly, the authors observed that RNAs present within nuclear organelles are susceptible to RNase IL degradation leading to their disassembly. In contrast, super-enhancer-containing eRNAs are largely unaffected.

    Major concerns

    Many studied organelles are challenging to see in many of the figures. Thus this reviewer encourages the authors to present clearer insets at higher magnification to illustrate what is being quantified, and then show that quantification in the central figure next to the immunofluorescent images.

    The amount of specific RNAs degradation after induction of RNase L for several assemblies should be analyzed by qRT-PCR and quantified. This will justify observations provided by microscopy on an individual cell basis. The main issue regards the connection between RNA and its role in the formation and structural integrity of nuclear organelles. There is consensus that these nuclear assemblies are built on specific nascent transcripts which act as a nucleation scaffold. If specific RNA synthesis is impaired, these assemblies collapse. The authors should discuss it. It would be relevant to mention two experimental works on this topics, DOI: 10.1038/ncb2140 and DOI: 10.1038/ncb2157 The study is limited to observed macroscopical changes in the appearances of assemblies. The authors must dig deeper and provide more conclusive results by several colocalizing components of these assemblies. It has been documented that the visualization of a selective marker for a specific assembly is not enough to prove its functionality/dysfunctionality but also the level of its disassembly. For example, in Figure 4A the authors should more convincible visualize nascent 47/45S pre-rRNA transcript to demonstrate that the nucleolus is built on ongoing pre-rRNA synthesis reflected by the tripartite nucleolar substructures. The loss of the GC component after rRNA depletion should be better presented with NPM1 colocalization.

    In Figure 4C, D the authors used the term "coilin assemblies". That's confusing for a reader. The Cajal body after activation of RNase L likely undergoes the structural rearrangement which cannot be justified only by the presence of rearranged coilin foci. The authors should colocalize them with at least one or two functional markers.

    Enhancer RNAs likely play the role in gene control rather than as a nucleation element to build nuclear assemblies. This should be discussed in the explanation of observed differences between MED1 foci and other assemblies.

    Significance

    Understanding changes in the nuclear and cellular organization that accompany and drive changes in the formation and maintenance of cellular structures is an essential and not well-understood topic. Thus, this manuscript is relevant. However, the presented data in this paper are based on a limited approach, and particularly their interpretation and presentation could be substantially improved. Consequently, the conclusions are not convincingly supported by published data. However, some open questions need to be addressed. Specific criticisms are outlined above.

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript by Decker et al. "RNA is required for the maintenance of multiple cytoplasmic and nuclear membrane-less organelles" investigates the structural role of RNA in membraneless organelles. The authors show that degradation of RNA in transient or constitutive membraneless organelles results in the altered formation and structure of many but not all organelles studied. The main assay is the activation of RNAseL activity by dsRNA which then destroys mRNA in the cell. The collected data leads the authors to highlight the possible roles of RNA in membraneless organelle formation and categorize the organelles: some relying more on the RNA-RNA interactions while others on protein-RNA or protein-protein interactions. The manuscript is well written and the data is sound.

    Major comments:

    The authors study the maintenance of organelles by RNA. For the transient ones, like stress granules (SG), it would be very interesting to see the formation/clearance kinetics with and without RNA. Also maybe using something other than dsRNA to trigger the formation. The idea being - if RNA is needed for SG maintenance, then the clearance kinetics with RNA would differ from that of the depleted RNA.

    The experiments were done in cells. It is known that core components of the organelles can form granule like structures in vitro without RNA. If it is possible to show that RNA presence improves the integrity in vitro, that would support the authors claim. For example studying SG maintenance with and without RNAseL using the previously developed SG extraction protocol.

    Minor comments:

    In the Figure 1a, it is not clear if the smaller granules are different from SGs as mentioned in the text, maybe using additional markers can make it clearer. Figure 3 and 4 requires quantification.

    Significance

    This is a solid paper that advances our understanding of membraneless organelle formation and dynamics. This field is of high general interest for the broader scientific community. My expertise is in the field of membraneless organelles.

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    Referee #1

    Evidence, reproducibility and clarity

    In the paper entitled. "RNA is required for the maintenance of multiple cytoplasmic and nuclear membrane-less organelles" Decker et al set out to test the rolw of RNA in maintaining the integrity of a variety of biomolecular condensates. To do this, they assess how multiple different assemblies in the cytoplasm and nucleus retain their structural integrity following RNAseL activation. They identified many condensates which are solubilized and have protein components redistributed following RNAse L activation and presumably subsequent RNA digestion. These experiments largely concur with previous findings from RNAseA treatment. The implication is that RNA rather than protein is the essential organizing component for most tested condensates. The manuscript is well written, and the data are convincing. It is my judgement that this is worthy of publication following a few additional experiments/clarifications.

    1. The authors identify condensates which are sensitive to or refractory to RNAaseL. It would be good if the authors more conclusively eliminate the possibility that remaining condensates contain specific residual antiviral RNAs and this is the reason why these condensates remain intact. Are any of these condensates enriched in anti-viral RNAs like IFNbeta following polyIC treatment by FISH, for example (PMID: 31494035)?
    2. Is there a particular protein feature, charge, IDR-types etc. which is common to solubilized versus not solubilized groups? What about dissolved and novel formed assemblies? A simple table comparing protein features in the three groups would suffice, with particular emphasis on RNA binding domains PMID: 32243832 and intrinsic disordered regions PMID: 24773235.
    3. Demonstrate that the RNAseL treatment is reversible (i.e. withdraw polyIC, particularly for a protein that ends up in a novel assembly) or remove the word maintenance from the title.
    4. Control for RNA-dependence of the activity. Try to dissolve a non-RNA dependent/enriched condensate with RNAseL. SPOP mutations (PMID: 30244836) might be interesting as both SPOP and RNAseL loss of function mutations (PMID: 11799394) are associated with prostate cancer.
    5. A caveat is that certain regions of condensates enriched in RNA may not be accessible to RNAseL protein. A way to address this might be to attempt to directly target the enzyme to a compartment that is deemed refractory to the activity (and inferred to not require RNA) via an inducible systsem (ie FKBP12/FK506)
    6. Overall, this paper would be greatly enhanced by including a more extensive discussion on the basic biological implications for these findings. Why are some condensates RNA dependent? What function(s) are common to these condensates? How does disruption of this lead to disease?

    Significance

    This work addresses the neglected role of RNA in structuring condensates throughout the cell. Despite the prevalence of RNA in many condensates and the enrichment of RNA-binding proteins in condensates, there is still a highly limited understanding of the structural roles RNA plays in their assembly s most work has been protein/IDR-centric. This work seeks to systematically assess the RNA-dependence of the assemblies.

  5. Version published to 10.1101/2021.08.16.456519v1 on bioRxiv