mRNA structure determines specificity of a polyQ-driven phase separation

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Abstract

RNA promotes liquid-liquid phase separation (LLPS) to build membraneless compartments in cells. How distinct molecular compositions are established and maintained in these liquid compartments is unknown. Here, we report that secondary structure allows mRNAs to self-associate and determines whether an mRNA is recruited to or excluded from liquid compartments. The polyQ-protein Whi3 induces conformational changes in RNA structure and generates distinct molecular fluctuations depending on the RNA sequence. These data support a model in which structure-based, RNA-RNA interactions promote assembly of distinct droplets and protein-driven, conformational dynamics of the RNA maintain this identity. Thus, the shape of RNA can promote the formation and coexistence of the diverse array of RNA-rich liquid compartments found in a single cell.

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Selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis.

Matthew J. Smola, Greggory M Rice, Steven Busan … (2015)

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistries exploit small electrophilic reagents that react with 2'-hydroxyl groups to interrogate RNA structure at single-nucleotide resolution. Mutational profiling (MaP) identifies modified residues by using reverse transcriptase to misread a SHAPE-modified nucleotide and then counting the resulting mutations by massively parallel sequencing. The SHAPE-MaP approach measures the structure of large and transcriptome-wide systems as accurately as can be done for simple model RNAs. This protocol describes the experimental steps, implemented over 3 d, that are required to perform SHAPE probing and to construct multiplexed SHAPE-MaP libraries suitable for deep sequencing. Automated processing of MaP sequencing data is accomplished using two software packages. ShapeMapper converts raw sequencing files into mutational profiles, creates SHAPE reactivity plots and provides useful troubleshooting information. SuperFold uses these data to model RNA secondary structures, identify regions with well-defined structures and visualize probable and alternative helices, often in under 1 d. SHAPE-MaP can be used to make nucleotide-resolution biophysical measurements of individual RNA motifs, rare components of complex RNA ensembles and entire transcriptomes.

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RNA stores tau reversibly in complex coacervates

Xuemei Zhang, Yanxian Lin, Neil Eschmann … (2017)

Nonmembrane-bound organelles that behave like liquid droplets are widespread among eukaryotic cells. Their dysregulation appears to be a critical step in several neurodegenerative conditions. Here, we report that tau protein, the primary constituent of Alzheimer neurofibrillary tangles, can form liquid droplets and therefore has the necessary biophysical properties to undergo liquid-liquid phase separation (LLPS) in cells. Consonant with the factors that induce LLPS, tau is an intrinsically disordered protein that complexes with RNA to form droplets. Uniquely, the pool of RNAs to which tau binds in living cells are tRNAs. This phase state of tau is held in an approximately 1:1 charge balance across the protein and the nucleic acid constituents, and can thus be maximal at different RNA:tau mass ratios, depending on the biopolymer constituents involved. This feature is characteristic of complex coacervation. We furthermore show that the LLPS process is directly and sensitively tuned by salt concentration and temperature, implying it is modulated by both electrostatic interactions between the involved protein and nucleic acid constituents, as well as net changes in entropy. Despite the high protein concentration within the complex coacervate phase, tau is locally freely tumbling and capable of diffusing through the droplet interior. In fact, tau in the condensed phase state does not reveal any immediate changes in local protein packing, local conformations and local protein dynamics from that of tau in the dilute solution state. In contrast, the population of aggregation-prone tau as induced by the complexation with heparin is accompanied by large changes in local tau conformations and irreversible aggregation. However, prolonged residency within the droplet state eventually results in the emergence of detectable β-sheet structures according to thioflavin-T assay. These findings suggest that the droplet state can incubate tau and predispose the protein toward the formation of insoluble fibrils.

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Accurate detection of chemical modifications in RNA by mutational profiling (MaP) with ShapeMapper 2

Steven Busan, Kevin Weeks (2018)

Mutational profiling (MaP) enables detection of sites of chemical modification in RNA as sequence changes during reverse transcription (RT), subsequently read out by massively parallel sequencing. We introduce ShapeMapper 2, which integrates careful handling of all classes of adduct-induced sequence changes, sequence variant correction, basecall quality filters, and quality-control warnings to now identify RNA adduct sites as accurately as achieved by careful manual analysis of electrophoresis data, the prior highest-accuracy standard. MaP and ShapeMapper 2 provide a robust, experimentally concise, and accurate approach for reading out nucleic acid chemical probing experiments.