Abstract
Processing bodies (PBs) are cytoplasmic mRNP granules that assemble via liquid-liquid phase separation and are implicated in the decay or storage of mRNAs. How PB assembly is regulated in cells remains unclear. Previously, we identified the ATPase activity of the DEAD-box protein Dhh1 as a key regulator of PB dynamics and demonstrated that Not1, an activator of the Dhh1 ATPase and member of the CCR4-NOT deadenylase complex inhibits PB assembly in vivo (Mugler et al., 2016). Here, we show that the PB component Pat1 antagonizes Not1 and promotes PB assembly via its direct interaction with Dhh1. Intriguingly, in vivo PB dynamics can be recapitulated in vitro, since Pat1 enhances the phase separation of Dhh1 and RNA into liquid droplets, whereas Not1 reverses Pat1-Dhh1-RNA condensation. Overall, our results uncover a function of Pat1 in promoting the multimerization of Dhh1 on mRNA, thereby aiding the assembly of large multivalent mRNP granules that are PBs.
Highlights
Cells are often subjected to severe environmental fluctuations such as nutrient deficiency, temperature changes or osmotic shock
Processing bodies (PBs) are cytoplasmic mRNP granules that assemble via liquid–liquid phase separation and are implicated in the decay or storage of mRNAs
We recently showed that Dhh1 undergoes phase separation in vitro, and controls PB dynamics in vivo via its RNA-stimulated ATPase activity whereas the ATPase activator Not1 dissolves Dhh1 droplets in vitro and inhibits PB formation in vivo
Summary
Cells are often subjected to severe environmental fluctuations such as nutrient deficiency, temperature changes or osmotic shock. PB formation occurs via a plethora of multivalent protein–RNA interactions in a redundant fashion (Rao and Parker, 2017), there are key players whose deletion drastically attenuates PB assembly (Decker et al, 2007; Pilkington and Parker, 2008; Scheller et al, 2007) One such component is the highly abundant DEAD-box ATPase Dhh (DDX6 in humans). Whether additional factors assist Dhh in promoting PB formation remains to be determined and is the focus of this study One such candidate is the eukaryotic PB component Pat, an evolutionarily conserved multidomain RNA binding protein that, like Dhh, functions in both translational repression and mRNA decay (Pilkington and Parker, 2008; Teixeira and Parker, 2007). Our in vivo and in vitro data suggests that during stress Pat antagonizes the inhibitory effect of Not on PB assembly and promotes the multimerization of Dhh on mRNA, leading to the formation of PBs
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