Recent experiments have defined cytoplasmic foci, referred to as processing bodies (P-bodies), wherein mRNA decay factors are concentrated and where mRNA decay can occur. However, the physical nature of P-bodies, their relationship to translation, and possible roles of P-bodies in cellular responses remain unclear. We describe four properties of yeast P-bodies that indicate that P-bodies are dynamic structures that contain nontranslating mRNAs and function during cellular responses to stress. First, in vivo and in vitro analysis indicates that P-bodies are dependent on RNA for their formation. Second, the number and size of P-bodies vary in response to glucose deprivation, osmotic stress, exposure to ultraviolet light, and the stage of cell growth. Third, P-bodies vary with the status of the cellular translation machinery. Inhibition of translation initiation by mutations, or cellular stress, results in increased P-bodies. In contrast, inhibition of translation elongation, thereby trapping the mRNA in polysomes, leads to dissociation of P-bodies. Fourth, multiple translation factors and ribosomal proteins are lacking from P-bodies. These results suggest additional biological roles of P-bodies in addition to being sites of mRNA degradation.

Eukaryotic cells contain nontranslating messenger RNA concentrated in P-bodies, which are sites where the mRNA can be decapped and degraded. We present evidence that mRNA molecules within yeast P-bodies can also return to translation. First, inhibiting delivery of new mRNAs to P-bodies leads to their disassembly independent of mRNA decay. Second, P-bodies decline in a translation initiation-dependent manner during stress recovery. Third, reporter mRNAs concentrate in P-bodies when translation initiation is blocked and resume translation and exit P-bodies when translation is restored. Fourth, stationary phase yeast have large P-bodies containing mRNAs that reenter translation when growth resumes. The reciprocal movement of mRNAs between polysomes and Pbodies is likely to be important in the control of mRNA translation and degradation. Moreover, the presence of related proteins in P-bodies and maternal mRNA storage granules suggests this mechanism is widely adapted for mRNA storage.A key aspect of the regulation of eukaryotic gene expression is the control of mRNA translation and degradation, which often occurs by decapping, followed by 5′ to 3′ decay (1). Translation and mRNA degradation via decapping are tightly linked. To decap, an mRNA exits translation and assembles into a translationally repressed messenger ribonucleoprotein (mRNP) lacking translation initiation factors and containing the decapping enzyme and several accessory proteins (1,2). These translationally repressed mRNPs accumulate within P-bodies (also referred to as GW or Dcp bodies) (3-8), where decapping can occur (9,10). The formation of a P-body mRNP is also important for control of translational repression (2,11,12). An unresolved issue is whether P-bodies can store mRNAs and later release them to reenter translation.To determine whether mRNAs in P-bodies were committed to decapping, we examined Pbodies in dcp1Δ cells where decapping is blocked (1) after 10 min of cycloheximide treatment, which prevents mRNAs from exiting translation and entering P-bodies (7,9,10,13). We directly observed P-body proteins by using green fluorescent protein (GFP)-tagged versions of the components Dcp2p and Dhh1p, whose presence in P-bodies is dependent on RNA, thus also providing an indirect manner of observing P-body mRNAs (7). We observed that P-bodies in dcp1Δ (Fig. 1A) and xrn1Δ cells (fig. S1) declined after cycloheximide treatment, suggesting that mRNPs can exit P-bodies in the absence of decapping and 5′ to 3′ degradation.

Processing bodies (P-bodies) are cytoplasmic RNA granules that contain translationally repressed messenger ribonucleoproteins (mRNPs) and messenger RNA (mRNA) decay factors. The physical interactions that form the individual mRNPs within P-bodies and how those mRNPs assemble into larger P-bodies are unresolved. We identify direct protein interactions that could contribute to the formation of an mRNP complex that consists of core P-body components. Additionally, we demonstrate that the formation of P-bodies that are visible by light microscopy occurs either through Edc3p, which acts as a scaffold and cross-bridging protein, or via the “prionlike” domain in Lsm4p. Analysis of cells defective in P-body formation indicates that the concentration of translationally repressed mRNPs and decay factors into microscopically visible P-bodies is not necessary for basal control of translation repression and mRNA decay. These results suggest a stepwise model for P-body assembly with the initial formation of a core mRNA–protein complex that then aggregates through multiple specific mechanisms.