Highlights d RNA condensates promote intermolecular RNA-RNA interactions d eIF4A limits the recruitment of RNAs to stress granules in cells d eIF4A reduces the formation of stress granules in cells d Recombinant eIF4A1 inhibits RNA condensation in vitro in an ATP-dependent manner

Stress granules (SGs) are ribonucleoprotein (RNP) assemblies that form in eukaryotic cells as a result of limited translation in response to stress. SGs form during viral infection and are thought to promote the antiviral response because many viruses encode inhibitors of SG assembly. However, the antiviral endoribonuclease RNase L also alters SG formation, whereby only small punctate SG-like bodies that we term RNase L–dependent bodies (RLBs) form during RNase L activation. How RLBs relate to SGs and their mode of biogenesis is unknown. Herein, using immunofluorescence, live-cell imaging, and MS-based analyses, we demonstrate that RLBs represent a unique RNP granule with a protein and RNA composition distinct from that of SGs in response to dsRNA lipofection in human cells. We found that RLBs are also generated independently of SGs and the canonical dsRNA-induced SG biogenesis pathway, because RLBs did not require protein kinase R, phosphorylation of eukaryotic translation initiation factor 2 subunit 1 (eIF2α), the SG assembly G3BP paralogs, or release of mRNAs from ribosomes via translation elongation. Unlike the transient interactions between SGs and P-bodies, RLBs and P-bodies extensively and stably interacted. However, despite both RLBs and P-bodies exhibiting liquid-like properties, they remained distinct condensates. Taken together, these observations reveal that RNase L promotes the formation of a unique RNP complex that may have roles during the RNase L–mediated antiviral response.

Ribonucleoprotein (RNP) granules are RNA-protein assemblies that are involved in multiple aspects of RNA metabolism and are linked to memory, development, and disease. Some RNP granules form, in part, through the formation of intermolecular RNA-RNA interactions. In vitro, such trans RNA condensation occurs readily, suggesting that cells require mechanisms to modulate RNA-based condensation. We assess the mechanisms of RNA condensation and how cells modulate this phenomenon. We propose that cells control RNA condensation through ATP-dependent processes, static RNA buffering, and dynamic post-translational mechanisms. Moreover, perturbations in these mechanisms can be involved in disease. This reveals multiple cellular mechanisms of kinetic and thermodynamic control that maintain the proper distribution of RNA molecules between dispersed and condensed forms. Ribonucleoprotein Granules Are Built via a Summation of Multivalent InteractionsEukaryotic cells contain a variety of ribonucleoprotein (RNP) granules (see Glossary). RNP granules are large non-membrane-bound assemblies of RNA and protein and are present in the nucleus and the cytosol. Examples of RNP granules include the nucleolus (the site of rRNA biogenesis), stress granules (SGs; which form from untranslating RNAs [1]), and neuronal granules (that are important for the transport and translation of synaptic mRNAs and synaptic plasticity [2]).RNP granules are members of a growing class of biological assemblies referred to as biomolecular condensates (reviewed in [3]). Biomolecular condensates are non-membranous assemblies that form through multivalent interactions between their components. Condensates differ from traditional assemblies in that the diverse and multivalent nature of the interactions allows condensates to be variable in their assembly and size and lack any unique stoichiometry or stereospecificity.RNP granules generally require a specific population of RNA for their formation and can be enriched for many RNAs. As examples, SGs and P-bodies (PBs) require a cytoplasmic population of untranslating RNAs, the nucleolus requires rRNA transcripts to maintain its organization [4], and nuclear paraspeckles require the NEAT1 long noncoding (lnc)RNA [5]. RNP granules also compartmentalize specific RNA-binding proteins (RBPs). For instance, distinct RBPs accumulate in SGs and PBs, although they can also share some components [6][7][8][9][10].RNP granules form from a summation of both protein-protein and RNA-RNA interactions between RNPs (Figure 1). Protein-protein interactions that promote RNP granule formation occur between RBPs bound to the RNA and can involve well-folded domains of RBPs [11]. For example, the G3BP1 protein can bind to mRNAs, and then through dimerization can increase the formation of SGs [12]. Many RNP granule proteins also contain intrinsically disordered Highlights Intermolecular RNA-RNA interactions contribute to the formation, content, and biophysical properties of many RNP granules. Cells utilize both genetically programmed and prom...

eTOC Blurb: The RNA helicase eIF4A limits stress granule formation by reducing RNA condensation. Highlights:•! RNA condensates promote intermolecular RNA-RNA interactions at their surfaces! •! eIF4A helicase activity limits the recruitment of RNAs to stress granules in cells! •! eIF4A helicase activity reduces the nucleation of stress granules in cells! •! Recombinant eIF4A1 inhibits the condensation of RNA in vitro in an ATP-dependent manner! Keywords: DEAD-box protein, RNA-RNA interactions, stress granule, ribonucleoprotein ! 2 SUMMARY:Stress granules are condensates of non-translating mRNAs and proteins involved in the stress response and neurodegenerative diseases. Stress granules are proposed to form in part through intermolecular RNA-RNA interactions, although the process of RNA condensation is not well understood. In vitro, we demonstrate that the minimization of surface free energy promotes the recruitment and interaction of RNAs on RNA or RNP condensate surfaces. We demonstrate that the ATPase activity of the DEAD-box RNA helicase eIF4A reduces RNA recruitment to RNA condensates in vitro and in cells, as well as limiting stress granule formation. This defines a new function for eIF4A, and potentially other RNA helicases, to limit thermodynamically favored intermolecular RNA-RNA interactions in cells, thereby allowing for proper RNP function.

Stress granules (SGs) are cytoplasmic RNA–protein aggregates formed in response to inhibition of translation initiation. SGs contribute to the stress response and are implicated in a variety of diseases, including cancer and some forms of neurodegeneration. Neurodegenerative diseases often involve chronic phosphorylation of eukaryotic initiation factor 2α (eIF2α), with deletions of eIF2α kinases or treatment with eIF2α kinase inhibitors being protective in some animal models of disease. However, how and why the integrated stress response (ISR) is activated in different forms of neurodegeneration remains unclear. Because neuroinflammation is common to many neurodegenerative diseases, we hypothesized that inflammatory factors contribute to ISR activation in a cell-nonautonomous manner. Using fluorescence microscopy and immunoblotting, we show here that the endogenously produced product of inflammation, 15-deoxy-Δ 12,14 -prostaglandin J2 (15-d-PGJ2), triggers eIF2α phosphorylation, thereby activating the ISR, repressing bulk translation, and triggering SG formation. Our findings define a mechanism by which inflammation activates the ISR in a cell-nonautonomous manner and suggest that inhibition of 15-d-PGJ2 production might be a useful therapeutic strategy in some neuroinflammatory contexts.