A Proxitome-RNA-capture Approach Reveals that Processing Bodies Repress Co-Regulated Hubs

Abstract

Cellular condensates are usually ribonucleoprotein assemblies with liquid- or solid-like properties. Because they lack a delineating membrane, the compositional determination of condensates is laborious. Here we set up a pipeline for proximity-biotinylation-dependent capture of RNA to investigate the RNA composition of the condensate in Arabidopsis known as the processing bodies (PBs). Using this pipeline together with in situ protein-protein interaction and RNA detection, in silico, and high-resolution imaging approaches, we studied PBs under normal and heat stress conditions. The composition of PBs in RNAs is much more dynamic than that of the total transcriptome. RNAs involved in cell wall development and regeneration, hormonal signaling, secondary metabolism/defense, and RNA metabolism were enriched in PBs. RNA binding proteins and liquid-to-solid phase transitions modulated specificity of RNA recruitment in PBs. Surprisingly, RNAs were sometimes recruited together with their encoded proteins. In PBs RNAs follow distinct fates, with small liquid-like PBs modulating RNA decay while larger ones storage. The size and properties of PBs are regulated by the actin polymerization cAMP receptor (SCAR)-WASP family verprolin homologous (WAVE) complex. SCAR/WAVE modulates signaling by shuttling RNAs between PBs and the translational machinery adjusting the ethylene signaling pathway. Heat stress leads to the storage of immunity-related RNAs in PBs by reducing PBs dynamics, suggesting why processes such as immunity malfunction under heat stress. In summary, we provide a method to identify RNAs in condensates which allowed us to reveal a mechanism for RNA fate regulation.