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

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.

Multi-ciliated cells (MCCs) use polarized fields of undulating cilia (ciliary array) to produce fluid flow that is essential for many biological processes. Cilia are positioned by microtubule scaffolds called basal bodies (BBs) that are arranged within a spatially complex 3-dimensional geometry (3D). Here, we develop a robust and automated computational image analysis routine to quantify 3D BB organization in the ciliate, Tetrahymena thermophila. Using this routine, we generate the first morphologically constrained 3D reconstructions of Tetrahymena cells and elucidate rules that govern the kinetics of MCC organization. We demonstrate the interplay between BB duplication and cell size expansion through the cell cycle. In mutant cells, we identify a potential BB surveillance mechanism that balances large gaps in BB spacing by increasing the frequency of closely spaced BBs in other regions of the cell. Finally, by taking advantage of a mutant predisposed to BB disorganization, we locate the spatial domains that are most prone to disorganization by environmental stimuli. Collectively, our analyses reveal the importance of quantitative image analysis to understand the principles that guide the 3D organization of MCCs.