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Highlights d R-rich DPRs sequester NPM1 into large, soluble phaseseparation-inhibited complexes d NPM1 sequestration dissolves droplets in vitro and delocalizes nucleolar NPM1 in cells d poly(PR) entraps rRNA in static puncta in vitro and in nucleoli d poly(PR) interactions disrupt nucleolar organization in a length-dependent manner

Membrane-less organelles are cellular structures which arise through the phenomenon of phase separation. This process enables compartmentalization of specific sets of macromolecules (e.g., proteins, nucleic acids), thereby regulating cellular processes by increasing local concentration, and modulating the structure and dynamics of their constituents. Understanding the connection between structure, material properties and function of membrane-less organelles requires inter-disciplinary approaches, which address length and timescales that span several orders of magnitude (e.g., Ångstroms to micrometer, picoseconds to hours). In this review, we discuss the wide variety of methods that have been applied to characterize the morphology, rheology, structure and dynamics of membrane-less organelles and their components, in vitro and in live cells.

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Enteroviruses use a type I IRES structure to facilitate protein synthesis and promote genome replication. Type I IRES elements require auxiliary host proteins to organize RNA structure for 40S ribosomal subunit assembly. Heterogeneous nuclear ribonucleoprotein A1 stimulates Enterovirus 71 (EV71) translation in part through specific interactions with its stem loop II (SLII) IRES domain. Here, we determined a conjoined NMR-SAXS structure of the EV71 SLII domain and a mutant that significantly attenuates viral replication by abrogating hnRNP A1 interactions. Native SLII adopts a locally compact structure wherein stacking interactions in a conserved 5′-AUAGC-3′ bulge preorganize the adjacent helices at nearly orthogonal orientations. Mutating the bulge sequence to 5′-ACCCC-3′ ablates base stacking in the loop and globally reorients the SLII structure. Biophysical titrations reveal that the 5′-AUAGC-3′ bulge undergoes a conformational change to assemble a functional hnRNP A1-RNA complex. Importantly, IRES mutations that delete the bulge impair viral translation and completely inhibit replication. Thus, this work provides key details into how an EV71 IRES structure adapts to hijack a cellular protein and it suggests the SLII domain is a potential target for antiviral therapy.