RNA Trafficking Between Membraneless Organelles at Single-Molecule Resolution in Live Cells

Weak, multivalent interactions between proteins and RNAs drive the formation of membraneless organelles via liquid-liquid phase separation (LLPS) of one set of biomolecules from others. Unlike membrane-enclosed organelles, membraneless organelles lack a physical boundary for restricting molecular movement and isolating the compartment from the cellular environment. Therefore, their formation and disassembly are highly dynamic and regulated in response to stimuli. Membraneless organelles have been recently found to be involved in cellular activities such as gene expression, metabolic pathways and signal transduction. Despite its biological importance, how LLPS organizes the intracellular milieu remains an open question. Membraneless organelles have been found to sort specific RNAs but the mechanism of RNA sorting (i.e., selective partitioning) into specific LLPS granules and its impact on RNA function is still not fully understood. We have found evidence that RNAs (mRNA, miRNA, lncRNA, etc.) are sorted into membraneless organelles based on their functionality. Our hypothesis is that RNA sorting ubiquitously relies on distinct RNA structural elements and the sorting controls the interaction kinetics between RNA and its downstream interaction partners. Here, we are aiming to expand our single-molecule fluorescence microscopy platform to test such hypothesis. Multiple single-molecule live-cell tracking tools are used to quantify partitioning of RNAs like the lncRNA MALAT1 in nuclear speckles, including the intracellular single molecule, high-resolution localization and counting (iSHiRLoC) method we developed and the bacteriophage-derived PP7-PCP labeling system. RNA partitioning is measured as a function of mutations or truncations on RNA to find structural elements that are responsible for sorting. Our long-term goal is to uncover the effects of RNA sorting on its interactions with functional downstream targets, which will unveil mechanisms for how LLPS regulates RNA function.