The cytoplasmic polyadenylation element-binding protein (CPEB) has been characterized in Xenopus laevis as a translational regulator. During the early development, it behaves first as an inhibitor and later as an activator of translation. In mammals, its closest homologue is CPEB1 for which two isoforms, short and long, have been described. Here we describe an additional isoform with a different RNA recognition motif, which is differentially expressed in the brain and ovary. We show that all CPEB1 isoforms are found associated with two previously described cytoplasmic structures, stress granules and dcp1 bodies. This association requires the RNA binding ability of the protein, whereas the Aurora A phosphorylation site is dispensable. Interestingly, the rck/p54 DEAD box protein, which is known as a CPEB partner in Xenopus and clam, and as a component of dcp1 bodies in mammals, is also present in stress granules. Both stress granules and dcp1 bodies are involved in mRNA storage and/or degradation, although so far no link has been made between the two, in terms of neither morphology nor protein content. Here we show that transient CPEB1 expression induces the assembly of stress granules, which in turn recruit dcp1 bodies. This dynamic connection between the two structures sheds new light on the compartmentalization of mRNA metabolism in the cytoplasm.
In mammals, repression of translation during stress is associated with the assembly of stress granules in the cytoplasm, which contain a fraction of arrested mRNA and have been proposed to play a role in their storage. Because physical contacts are seen with GW bodies, which contain the mRNA degradation machinery, stress granules could also target arrested mRNA to degradation. Here we show that contacts between stress granules and GW bodies appear during stress-granule assembly and not after a movement of the two preassembled structures. Despite this close proximity, the GW body proteins, which in some conditions relocalize in stress granules, come from cytosol rather than from adjacent GW bodies. It was previously reported that several proteins actively traffic in and out of stress granules. Here we investigated the behavior of mRNAs. Their residence time in stress granules is brief, on the order of a minute, although stress granules persist over a few hours after stress relief. This short transit reflects rapid return to cytosol, rather than transfer to GW bodies for degradation. Accordingly, most arrested mRNAs are located outside stress granules. Overall, these kinetic data do not support a direct role of stress granules neither as storage site nor as intermediate location before degradation.
Stress granules are cytoplasmic ribonucleoprotein granules formed following various stresses that inhibit translation. They are thought to help protecting untranslated mRNAs until stress relief. Stress granules are frequently seen adjacent to P-bodies, which are involved in mRNA degradation and storage. We have previously shown in live cells that stress granule assembly often takes place in the vicinity of pre-existing P-bodies, suggesting that these two compartments are structurally related. Here we provide the first ultrastructural characterization of stress granules in eukaryotic cells by electron microscopy. Stress granules resulting from oxidative stress, heat-shock or protein overexpression are loosely organised fibrillo-granular aggregates of a moderate electron density, whereas P-bodies are denser and fibrillar. By in situ hybridization at the electron microscopic level, we show that stress granules are enriched in poly(A)+ mRNAs, although these represent a minor fraction of the cellular mRNAs. Finally, we show that, despite close contact with P-bodies, both domains remain structurally distinct and do not interdigitate.
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