(12,37,39) It has been suggested, by extension, that P-bodies must also be liquid droplets, especially considering the frequent occurrence of low-complexity domains (LCDs) in P-body components. (38) Liquid droplet formation has been reconstituted using intrinsically disordered regions (IDRs) and protein fragments, low-complexity sequences, or SLiMs from RNA-binding and RNA granule associated proteins. (31−34) The physical basis of LLPS has attracted a great deal of attention in recent years because of the critical role that proper messenger ribonucleoprotein (mRNP) assembly plays in pathogenesis (33,35−37) and in stress responses. At the same time, in vitro studies have shown the propensity of RNA-binding proteins and low-sequence-complexity proteins to undergo LLPS either alone or in the presence of RNA. Many membraneless RNP granules, including Cajal bodies, nucleoli, and mammalian stress granules, have recently been described as having properties of liquid droplets (reviewed in refs (12,29,and30)). (26,27) The function of P-bodies in mRNA decay, therefore, is still an open question, largely due to the challenge of directly visualizing mRNA degradation in diffraction-limited structures within living cells, (28) as well as the difficulty of biochemically purifying labile liquid droplets from cells. (25) An alternative, though not necessarily mutually exclusive model, has thus emerged positing that P-bodies are storage sites for translationally repressed mRNAs and inactive mRNA decay enzymes, which undergo LLPS (vide infra) as a result of the dense network of protein–protein interactions that form when mRNA decay factors accumulate on polysome-free transcripts. (24) More recently, mRNA decay has been observed despite a lack of P-bodies in yeast strains lacking functional edc3 and lsm4 genes. However, it was subsequently demonstrated that macroscopically observable P-bodies are not required for mRNA decay to occur (22,23) and that mRNAs can recycle from P-bodies to translating polysomes. P-bodies were discovered during the investigation of the localization of proteins associated with the 5′-to-3′ mRNA decay pathway, and the additional observation of mRNA decay intermediates in these structures led to the initial hypothesis that P-bodies were cellular sites of mRNA decay. (21) Therefore, despite the nonmembrane-bounded nature of these RNP granules, each has a unique molecular composition that is likely related to its function. Similarly, while P-bodies and GW-bodies, which are associated with miRNA/siRNA silencing, were originally conflated, AGO2 and GW182 were found to localize to P-bodies only in metazoans, (16−20) and GW-bodies have more recently been shown to colocalize with multivesicular bodies, not P-bodies, in higher eukaryotes as well. For example, stress granules and P-bodies share some protein components, they can come into contact with each other, and both can be induced by cellular stress (15) however, stress granules uniquely contain translation initiation factors. (12) Despite their similarities, each of these RNP granules is distinct in its molecular composition and function. These RNP granules are conserved in eukaryotes and bear similarities to other RNP granules, such as Cajal bodies, nucleoli, and stress granules, in that they depend on complex networks of protein–RNA interactions, low-complexity protein sequences, and liquid–liquid phase separation (LLPS) for their formation. Processing bodies (P-bodies) are cytoplasmic ribonucleoprotein (RNP) granules comprised primarily of mRNAs in complex with proteins associated with translation repression and 5′-to-3′ mRNA decay.
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