Single-molecule mass measurements uncover shifting RNA interactions during condensate phase transitions

Characterization of RNA Structure Dynamics with Trans-Factors Using Single-Molecule Optical Tweezers.

Potential structure induced effects of the hairpin motif in RNA to form the phase separated droplet like associate in different solution condition.

Reversible associations of RNAs among themselves, or with RNA binding proteins through the process of phase separation, are found to be important in many different cellular contexts, including cellular responses to stress, gene regulation, development, and disease. Short RNA repeat sequences, which are mostly linked with many repeat expansion neurodegenerative diseases, are found to undergo phase separation and yield protein-free biomolecular condensates in vitro and in cells. However, the physicochemical principles governing phase separation of RNAs considering both sequence and structural aspects, especially for short RNAs, remain elusive. It is intriguing, as well as challenging, to characterize the RNA phase behavior at a submolecular resolution. Based on atomistic enhanced sampling simulations, here we report potential dynamic structural effects in the mutual association properties of a tetra loop containing 14-mer hairpin RNA. We show that the folded and unfolded conformations of the hairpin fragment lead to different energetic barriers for the formation of an associated pair. Unfolded conformation leads to the formation of gel like energetically stable associated phases spontaneously; the folded hairpin motif is found to yield droplet like associated phases accompanying by cations in solution. Overall, our findings illustrate that dynamic association/dissociation is energetically more favorable for folded RNA hairpins, and the presence of additional salt in solution assists the formation of dense droplet like associated phases, while ionic concentration plays a critical role in the formation and stability of the droplet like associated phase. These observations open avenues for exploring structure-based phase separation mechanisms of RNAs in the context of RNA mediated functional biomolecular condensate formation within cells and designing RNA based novel biomaterials.

Aminoglycoside antibiotics drive RNA phase separation

Aminoglycoside antibiotics bind RNA with high affinity through networks of amine and hydroxyl contacts, yet whether this multivalent binding can drive macroscopic RNA phase transitions has never been tested. Here we show that aminoglycosides are a class of small-molecule RNA condensers, and we take neomycin B (neoB), an FDA-approved member of the family, as a representative drug through which to dissect the mechanism. NeoB induces concentration-dependent phase separation of poly(A), poly(U), and total E. coli RNA, and kanamycin, apramycin, and gentamicin condense RNA as well. Condensate size and density are tunable by pH and ionic strength, which independently modulate neoB protonation and screening of interdroplet repulsion. NeoB forms more stable condensates than spermine despite spermine’s larger effective charge at physiological pH, whereas the amine-free polyol fucitol fails to condense RNA. Molecular dynamics simulations attribute neoB’s greater efficiency to additional hydrogen bonds donated by its hydroxyl groups. The chemical complexity that aminoglycosides evolved for RNA recognition thus also drives a macroscopic RNA phase transition that may contribute to bactericidal activity and cellular toxicity.

Integrative structural biology of protein-RNA interactions in four dimensions.

Recent developments in integrative modeling of protein-RNA complexes are reviewed, highlighting advances in in-cell, 4D and condensate structural biology, and how these approaches shape the understanding of RNP assembly, regulation, and function in physiologically relevant environments are discussed.

Rho (𝛒) Analysis to Dissect RNA Folding and Assembly Pathways

The results establish ρ analysis as a general framework to probe RNA conformational pathways and function and uncover multiple folding pathways and modulation in pathway flux that are readily understood from the energetic properties of the constituent RNA motifs.

Single-Cell Imaging of Site-Specific RNA-Protein Interactions.

Detecting and quantifying specific RNA-protein interaction pairs under diverse physiological conditions is crucial for elucidating disease mechanisms and exploring potential therapeutic targets. However, it still remains a major challenge to visualize specific intracellular RNA-protein interactions at near-single-molecule resolution. In this work, we introduce an RNA-protein interaction visualization method by antibody-guided proximal in situ reverse-transcription detection (RAPID), to enable spatial localization and quantification of specific RNA-protein interaction pairs within single cells at near-single-molecule resolution. We demonstrate the high specificity and robustness of RAPID by validating the interaction between the heterogeneous nuclear ribonucleoprotein hnRNPC and the long noncoding RNA (lncRNA) MALAT1 in HeLa cells. Using RAPID, we found that SFPQ-NEAT1 interactions exhibited distinct spatial organization and dynamic changes upon cellular stimulation, indicating that the SFPQ-NEAT1 axis is closely associated with nuclear reorganization and stimulus-responsive gene regulation. RAPID, providing a new perspective on RNA-protein interactions, represents a novel analytical tool that facilitates further investigation of their roles in biology and disease.