Expansions of short nucleotide repeats produce several neurological and neuromuscular disorders including Huntington’s disease, muscular dystrophy and amyotrophic lateral sclerosis. A common pathological feature of these diseases is the accumulation of the repeat containing transcripts into aberrant foci in the nucleus. RNA foci, as well as the disease symptoms, only manifest above a critical number of nucleotide repeats, but the molecular mechanism governing foci formation above this characteristic threshold remains unresolved. Here, we show that repeat expansions create templates for multivalent base-pairing, which causes purified RNA to undergo a sol-gel transition at a similar critical repeat number as observed in the diseases. In cells, RNA foci form by phase separation of the repeat-containing RNA and can be dissolved by agents that disrupt RNA gelation in vitro. Analogous to protein aggregation disorders, our results suggest that the sequence-specific gelation of RNAs could be a contributing factor to neurological disease.

Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here, we describe a single molecule pull-down (SiMPull) assay that combines the principles of conventional pull-down assay with single molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signaling proteins found in cytosol, membrane, and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled down proteins are functional and are used, without further processing, for single molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analyzing protein assemblies in biological pathways.

During intracellular membrane trafficking and remodeling, protein complexes known as the ESCRTs interact with membranes and are required for budding processes directed away from the cytosol, including the budding of intralumenal vesicles to form multivesicular bodies, for the budding of some enveloped viruses, and for daughter cell scission in cytokinesis. Here we found that the ESCRT-III proteins CHMP2A and CHMP3 could assemble in vitro into helical tubular structures that expose their membrane interaction sites on the outside of the tubule while the AAA-type ATPase VPS4 could bind on the inside of the tubule and disassemble the tubes upon ATP hydrolysis. CHMP2A and CHMP3 co-polymerized in solution and their membrane targeting was cooperatively enhanced on planar lipid bilayers. Such helical CHMP structures could thus assemble within the neck of an inwardly-budding vesicle, catalyzing late steps in budding under the control of VPS4.