A major pathway of eukaryotic messenger RNA (mRNA) turnover begins with deadenylation, followed by decapping and 5′ to 3′ exonucleolytic decay. We provide evidence that mRNA decapping and 5′ to 3′ degradation occur in discrete cytoplasmic foci in yeast, which we call processing bodies (P bodies). First, proteins that activate or catalyze decapping are concentrated in P bodies. Second, inhibiting mRNA turnover before decapping leads to loss of P bodies; however, inhibiting turnover at, or after, decapping, increases the abundance and size of P bodies. Finally, mRNA degradation intermediates are localized to P bodies. These results define the flux of mRNAs between polysomes and P bodies as a critical aspect of cytoplasmic mRNA metabolism and a possible site for regulation of mRNA degradation.Messenger RNA turnover is an important step in regulating gene expression. In yeast, the major pathway of mRNA decay is initiated by shortening of the 3′ poly(adenosine) [poly(A)] tail, followed by removal of the cap by the decapping enzyme Dcp1p/Dcp2p, which in turn allows 5′ to 3′ exonucleolytic decay (1-8). Decapping is a key step in this pathway, because it permits the destruction of the mRNA and is a site of numerous control inputs (9).Several observations suggest that decapping occurs when the mRNA undergoes a transition from a translationally competent messenger ribonucleoprotein (mRNP) to an mRNP state destined for decay. For example, the translation initiation factor eIF4E, which binds the cap structure, is an inhibitor of decapping both in vitro and in vivo (10,11). Moreover, deadenylated mRNAs that interact with a complex of Lsm1-7 proteins, which activates decapping, are no longer bound by eIF4E or eIF4G (12).The hypothesis that mRNAs enter a non-translating state after deadenylation and before decapping is analogous to the storage of mRNA in numerous biological contexts where deadenylated mRNAs are translationally repressed before their later activation. Consistent with a mechanistic similarity between decapping and mRNA storage, Dhh1p, which is an activator of decapping in yeast (13), has homologs that are required for the translational repression and storage of maternal mRNAs in Xenopus, Drosophila, and Caenorhabditis (14-16). Such stored mRNAs are often localized in discrete cytoplasmic granules, which represent accumulations of translationally repressed mRNAs (15). This analogy suggested the possibility that Dhh1p

Recent results indicate that many untranslating mRNAs in somatic eukaryotic cells assemble into related mRNPs that accumulate in specific cytoplasmic foci referred to as P bodies. Transcripts associated with P body components can either be degraded or return to translation. Moreover, P bodies are also biochemically and functionally related to some maternal and neuronal mRNA granules. This suggests an emerging model of cytoplasmic mRNA function in which the rates of translation and degradation of mRNAs are influenced by a dynamic equilibrium between polysomes and the mRNPs seen in P bodies. Moreover, some mRNA-specific regulatory factors, including miRNAs and RISC, appear to repress translation and promote decay by recruiting P body components to individual mRNAs.

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.

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In eukaryotes, a specialized pathway of mRNA degradation termed nonsense-mediated decay (NMD) functions in mRNA quality control by recognizing and degrading mRNAs with aberrant termination codons. We demonstrate that NMD in yeast targets premature termination codon (PTC)-containing mRNA to P-bodies. Upf1p is sufficient for targeting mRNAs to P-bodies, whereas Upf2p and Upf3p act, at least in part, downstream of P-body targeting to trigger decapping. The ATPase activity of Upf1p is required for NMD after the targeting of mRNAs to P-bodies. Moreover, Upf1p can target normal mRNAs to P-bodies but not promote their degradation. These observations lead us to propose a new model for NMD wherein two successive steps are used to distinguish normal and aberrant mRNAs.

SUMMARYGermline-specific granules of unknown function are found in a wide variety of organisms, including C. elegans, where they are called P granules. P granules are cytoplasmic bodies in oocytes and early embryos. Throughout most of the C. elegans life cycle, however, P granules are associated with clusters of nuclear pore complexes (NPCs) on germ cell nuclei. We show that perinuclear P granules differ from cytoplasmic P granules in many respects, including structure, stability and response to metabolic changes. Our results suggest that nuclear-associated P granules provide a perinuclear compartment where newly exported mRNAs are collected prior to their release to the general cytoplasm. First, we show that mRNA export factors are highly enriched at the NPCs associated with P granules. Second, we discovered that the expression of high-copy transgenes could be induced in a subset of germ cells, and used this system to demonstrate that nascent mRNA traffics directly to P granules. P granules appear to sequester large amounts of mRNA in quiescent germ cells, presumably preventing translation of that mRNA. However, we did not find evidence that P granules normally sequester aberrant mRNAs, or mRNAs targeted for destruction by the RNAi pathway.

Virus-like particles (VLPs) have not been observed in Caenorhabditis germ cells, although nematode genomes contain low numbers of retrotransposon and retroviral sequences. We used electron microscopy to search for VLPs in various wild strains of Caenorhabditis, and observed very rare candidate VLPs in some strains, including the standard laboratory strain of C. elegans, N2. We identified the N2 VLPs as capsids produced by Cer1, a retrotransposon in the Gypsy/Ty3 family of retroviruses/retrotransposons. Cer1 expression is age and temperature dependent, with abundant expression at 15°C and no detectable expression at 25°C, explaining how VLPs escaped detection in previous studies. Similar age and temperature-dependent expression of Cer1 retrotransposons was observed for several other wild strains, indicating that these properties are common, if not integral, features of this retroelement. Retrotransposons, in contrast to DNA transposons, have a cytoplasmic stage in replication, and those that infect non-dividing cells must pass their genomic material through nuclear pores. In most C. elegans germ cells, nuclear pores are largely covered by germline-specific organelles called P granules. Our results suggest that Cer1 capsids target meiotic germ cells exiting pachytene, when free nuclear pores are added to the nuclear envelope and existing P granules begin to be removed. In pachytene germ cells, Cer1 capsids concentrate away from nuclei on a subset of microtubules that are exceptionally resistant to microtubule inhibitors; the capsids can aggregate these stable microtubules in older adults, which exhibit a temperature-dependent decrease in egg viability. When germ cells exit pachytene, the stable microtubules disappear and capsids redistribute close to nuclei that have P granule-free nuclear pores. This redistribution is microtubule dependent, suggesting that capsids that are released from stable microtubules transfer onto new, dynamic microtubules to track toward nuclei. These studies introduce C. elegans as a model to study the interplay between retroelements and germ cell biology.

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