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Dynamic Recruitment of Single RNAs to Processing Bodies Depends on RNA Functionality

Pitchiaya, Sethuramasundaram, Mourao, Marcio D.A., Jalihal, Ameya P., Xiao, Lanbo, Jiang, Xia, Chinnaiyan, Arul M., Schnell, Santiago, Walter, Nils G.
Molecular cell 2019 v.74 no.3 pp. 521-533.e6
enzymes, fluorescence microscopy, granules, messenger RNA, microRNA, models, monitoring, non-coding RNA, ribonucleoproteins, separation, translation (genetics)
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 3′ versus 5′ placement of miRNA target sites significantly affect the PB localization dynamics of mRNAs. Using computational modeling and supporting experimental approaches, we show that partitioning in the PB phase attenuates mRNA silencing, suggesting that physiological mRNA turnover occurs predominantly outside of PBs. Instead, our data support a PB role in sequestering unused miRNAs for surveillance and provide a framework for investigating the dynamic assembly of RNP granules by phase separation at single-molecule resolution.