Cellular RNAs often colocalize with cytoplasmic, membrane-less ribonucleoprotein (RNP) granules enriched for RNA processing enzymes, termed processing bodies (PBs). Here, we track the dynamic localization of individual miRNAs, mRNAs and long non-coding RNAs (lncRNAs) to PBs using intracellular single-molecule fluorescence microscopy. We find that unused miRNAs stably bind to PBs, whereas functional miRNAs, repressed mRNAs and lncRNAs both transiently and stably localize within either the core or periphery of PBs, albeit to different extents. Consequently, translation potential and positioning of cis-regulatory elements significantly impact PB-localization dynamics of mRNAs. Using computational modeling and supporting experimental approaches we show that phase separation into large PBs attenuates mRNA silencing, suggesting that physiological mRNA turnover predominantly occurs outside of PBs. Instead, our data support a role for PBs in sequestering unused miRNAs to regulate their surveillance and provides a framework for investigating the dynamic assembly of RNP granules by phase separation at single-molecule resolution.

Dynamic recruitment of single RNAs to processing bodies depends on RNA functionality