Summary In eukaryotic cells, diverse stresses trigger coalescence of RNA-binding proteins into stress granules. In vitro, stress-granule-associated proteins can demix to form liquids, hydrogels, and other assemblies lacking fixed stoichiometry. Observing these phenomena has generally required conditions far removed from physiological stresses. We show that poly(A)-binding protein (Pab1 in yeast), a defining marker of stress granules, phase-separates and forms hydrogels in vitro upon exposure to physiological stress conditions. Other RNA-binding proteins depend upon low-complexity regions (LCRs) or RNA for phase separation, whereas Pab1’s LCR is not required for demixing, and RNA inhibits it. Based on unique evolutionary patterns, we create LCR mutations which systematically tune its biophysical properties and Pab1 phase separation in vitro and in vivo. Mutations which impede phase separation reduce organism fitness during prolonged stress. Poly(A)-binding protein thus acts as a physiological stress sensor, exploiting phase separation to precisely mark stress onset, a broadly generalizable mechanism.

Highlights d Stress granule formation requires RNA-binding nodes with high network connectivity d Capping of nodes by ligands lacking connectivity prevents condensation d Protein disorder and RNA-binding specificity play nonessential, modulatory roles d Competition of RNP networks for connecting nodes controls multiphase organization

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Among numerous microscopically visible nuclear substructures, the nucleolus is the most prominent and represents a functionally and biophysically distinct body or compartment. The nucleolus is, in fact, so prominent that it drew the attention of early biologists over 200 years ago, in light microscopy studies by Fontana, Valentin and Wagner 1. Following these early descriptions came the understanding of the functional importance of the nucleolus, including its primary role as the site for the initial steps of ribosome biogenesis, including RNA polymerase I (Pol I)-driven transcription, processing and modification of ribosomal RNA (rRNA) and the assembly of rRNA-containing complexes 2 (Fig. 1). These processes involve several hundred protein transacting factors and small nucleolar RNAs 3 , which serve to guide the specificity of rRNA chemical modifications, pre-rRNA folding and cleavage. Once precursor subunits are released from the nucleolar structure, they undergo further maturation in the nucleoplasm and cytoplasm prior to becoming fully functional ribosomal subunits, ready to engage in translating mRNA into protein. This nucleolar function is accompanied by organization of the nucleolus into distinct subcompartments. In mammalian cells, nucleoli display three layers, nested like Russian dolls, where successive steps of ribosome production take place, starting

Summary Heat causes protein misfolding and aggregation, and in eukaryotic cells triggers aggregation of proteins and RNA into stress granules. We have carried out extensive proteomic studies to quantify heat-triggered aggregation and subsequent disaggregation in budding yeast, identifying more than 170 endogenous proteins aggregating within minutes of heat shock in multiple subcellular compartments. We demonstrate that these aggregated proteins are not misfolded and destined for degradation. Stable-isotope labeling reveals that even severely aggregated endogenous proteins are disaggregated without degradation during recovery from shock, contrasting with the rapid degradation observed for exogenous thermolabile proteins. Although aggregation likely inactivates many cellular proteins, in the case of a heterotrimeric aminoacyl-tRNA synthetase complex, the aggregated proteins remain active with unaltered fidelity. We propose that most heat-induced aggregation of mature proteins reflects the operation of an adaptive, autoregulatory process of functionally significant aggregate assembly and disassembly that aids cellular adaptation to thermal stress.