(NAS Colloquium) Molecular Kinesis in Cellular Function and Plasticity (2002)

Joel D.Richter^*

Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Biotech 4, Room 330, 377 Plantation Street, Worcester, MA 01605

Early metazoan development is programmed by maternal mRNAs inherited by the egg at the time of fertilization. These mRNAs are not translated en masse at any one time or at any one place, but instead their expression is regulated both temporally and spatially. Recent evidence has shown that one maternal mRNA, cyclin B1, is concentrated on mitotic spindles in the early Xenopus embryo, where its translation is controlled by CPEB (cytoplasmic polyadenylation element binding protein), a sequence-specific RNA binding protein. Disruption of the spindle-associated translation of this mRNA results in a morphologically abnormal mitotic apparatus and inhibited cell division. Mammalian neurons, particularly in the synapto-dendritic compartment, also contain localized mRNAs such as that encoding a-CaMKII. Here, synaptic activation drives local translation, an event that is involved in synaptic plasticity and possibly long-term memory storage. Synaptic translation of a-CaMKII mRNA also appears to be controlled by CPEB, which is enriched in the postsynaptic density. Therefore, CPEB-controlled local translation may influence such seemingly disparate processes as the cell cycle and synaptic plasticity.

Many cells are remarkably polar. Neurons of the central nervous system, for example, have multiple extensions from the cell body, typically one axon and many dendrites. It stands to reason that this cellular polarity is dictated by the region-specific deposition of proteins and perhaps mRNAs. Vertebrate oocytes, whose radial symmetry would suggest a lack of morphological polarity, are actually characterized by considerable molecular polarity. Consider Xenopus oocytes, which sort many proteins and mRNAs to different locations, particularly along the animal-vegetal axis. This molecular asymmetry is inherited by the fertilized egg and is essential for the establishment of the body plan. Neurons and eggs both contain mRNAs whose translation is regulated both temporally and spatially. Although a number of factors mediate sequence-specific translation in these two cell types, one that has a central role is CPEB, the cytoplasmic polyadenylation element binding protein. In the synapto-dendritic compartment of mammalian hippocampal neurons, CPEB appears to stimulate the translation of a-CaMKII mRNA, which is essential for synaptic plasticity and long-term memory storage. In blastomeres of the developing Xenopus embryo, the control of cyclin B1 mRNA translation on mitotic spindles by CPEB is necessary for the integrity of the mitotic apparatus and for cell division. For both cell types, then, local translational control by CPEB mediates key biological functions.

One characteristic of early metazoan development is the mobilization of stored mRNAs into polysomes. In many cases, the stored mRNAs have relatively short poly (A) tails that are elongated at a time that is coincident with translational activation. During oocyte maturation, when oocytes re-enter the meiotic divisions after prolonged prophase I arrest, polyadenylation is stimulated by two cis-acting sequences in the 3' untranslated regions of responding mRNAs. The first is the hexanucleotide AAUAAA, which is also necessary for nuclear pre-mRNA polyadenylation, and the second is the cytoplasmic polyadenylation element (CPE), which has the general structure of UUUUUAU (1). The CPE is bound by the phospho-protein CPEB (2–4), and the hexanucleotide AAUAAA is bound by CPSF (cleavage and polyadenylation specificity factor), a group of factors that also promote nuclear pre-mRNA polyadenylation (5, 6). CPEB and CPSF, plus poly(A) polymerase (7, 8), comprise the core cytoplasmic polyadenylation complex.

The identification of the core factors does not explain how cytoplasmic polyadenylation is initiated, nor does it explain the mechanism of translational dormancy or activation. An analysis of the early signaling events of Xenopus oocyte maturation revealed the stimulus for polyadenylation. Progesterone binding to an as-yet-unidentified surface-associated receptor leads to a transient but essential decrease in cAMP. This decrease is soon followed by the activation of Eg2, a member of the Aurora family of protein kinases (9). Active Eg2 phosphorylates CPEB on a single residue (4), which causes it (CPEB) to bind and recruit CPSF into an active cytoplasmic polyadenylation complex (10). By analogy with nuclear pre-mRNA polyadenylation, it is CPSF that recruits poly(A) polymerase to the end of the mRNA.

Before oocyte maturation, mRNAs are actively repressed by the CPE, that is, by the same sequence that activates translation by promoting cytoplasmic polyadenylation. The mechanism by which the CPE could be bifunctional was indicated by experiments of de Moor and Richter (11), who demonstrated that efficient CPE-mediated repression requires a 5' cap (i.e., ^7mG). This finding suggested that a factor that interacts with the CPE (i.e., CPEB) also could bind the cap (or cap binding proteins), which might limit access of the 5' end of the mRNA to initiation factors (for review of initiation factors, see ref. 12). Although there was no evidence that CPEB interacts with the cap, a CPEB-interacting protein was found to contain a peptide sequence that mediates its interaction with eIF4E, the cap binding factor (13). This factor, termed maskin, interacts with eIF4E in such a way as to preclude an association of eIF4E with eIF4G, thereby preventing the 40s ribosomal subunit from being correctly positioned on the 5' end of the mRNA. Because at least some eIF4E dissociates from maskin during oocyte maturation (and is coincident with polyadenylation), newly “liberated” eIF4E then is free to bind eIF4G and initiate translation. Consequently, maskin appears to belong to a class of proteins known as eIF4EBPs (14), which modulate cap-dependent trans-

lation by transiently interacting with eIF4E. Unlike the case with other known eIF4EBPs, however, maskin-mediated translational control is mRNA-specific because of its interaction with CPEB.

At a late stage of oocyte maturation, after the activation of M-phase promoting factor (a heterodimer of cdc2 and cyclin B), ˜90% of the CPEB is destroyed (3). That which remains stable is highly localized to the cortex of the animal pole, which in the embryo will give rise to the ectoderm. After fertilization, CPEB remains concentrated in animal pole blastomeres. Within these cells CPEB, as well as maskin, is localized to the mitotic apparatus (15). At metaphase, these proteins are found along the length of the spindles, although there is a greater concentration of them toward the centrosomes. At prophase and prometaphase, the proteins are concentrated on centrosomes. Although the CPEB-activating kinase Eg2 also is found specifically on centrosomes, other proteins involved in polyadenylation-induced translation [poly(A) polymerase, CPSF, eIF4E], which although not particularly concentrated on the mitotic apparatus, are still coincident with it. These results, plus the observation that cyclin B1 mRNA is colocalized with CPEB on spindles, suggest that local polyadenylation-induced translation could take place on or near the mitotic apparatus (15).

CPEB amino acid residues 168–211, which contain a PEST protein-protein interaction domain, mediate the interaction of this protein with microtubules in vitro and with centrosomes in vivo (15). When injected into embryos, a CPEB protein lacking these residues has little effect on the synthesis and oscillation of cyclin B1 protein during the cell cycle. However, this deletion mutant CPEB protein induces the “delocalization” of cyclin B1 mRNA and protein from mitotic spindles. The result of this delocalization is inhibited cell division and a malformation of the mitotic apparatus, which includes tripolar spindles, spindles detached from centrosomes, and multiple centrosomes. These data indicate that not only is regulated cyclin mRNA translation important for cell division in embryos, but that the critical translational event occurs in association with mitotic spindles. This finding implies that an important cell division-promoting activity of cyclin B1 protein must be directed to spindles. It is worth noting that cyclin protein is also present on the spindles of Drosophila embryos (16) and HeLa cells (17), where it also may have an essential function.

In the central nervous system, a single neuron may receive input signals from thousands of different cells. A dendrite that receives a signal from a given axon establishes a “tag” at the point of reception (i.e., the synapse), which distinguishes this stimulated synapse from the many others that are not stimulated (18). This tag establishes a history or memory of the stimulated synapse. Thus, synapses are considered to be “plastic” because their response to activation is influenced by their stimulation history. Two forms of synaptic plasticity, the long-lasting phase of long-term potentiation and long-term depression, require new protein synthesis but not new mRNA synthesis (refs. 19–21; see also ref. 22). These observations, as well as others demonstrating that many of the components of the protein synthesis machinery, including mRNAs, are present in dendrites, suggest that local translational control by synaptic activation could underlie, at least partially, synaptic plasticity (23, 24).

In mammals, CPEB was first thought to be relatively restricted to germ cells (25). However, subsequent studies showed it to also be present in the hippocampus, the portion of the brain that is responsible for long-term memory. Further analysis demonstrated that CPEB resides in the dendritic layer of the hippocampus, at synapses in cultured hippocampal neurons, and in the postsynaptic density of biochemically fractionated synapses (26). The presence of CPEB at synapses suggested a mechanism of translational control that could influence synaptic strength. It therefore became important to identify the synapto-dendritic mRNA(s) whose translation might be regulated by CPEB.

The gene encoding a-CaMKII is necessary for long-term potentiation (27), a-CaMKII mRNA is present in dendrites (28), and a-CaMKII protein levels increase upon synaptic stimulation (29, 30). These observations, plus the further revelation that the 3' untranslated region of a-CaMKII mRNA contains a CPE (26), suggested that this molecule could be a substrate for CPEB activity and undergo polyadenylation-induced translation. Because CPEB is present in the visual cortex as well as in the hippocampus, the effect of synaptic activity on CPEB-mediated translation could be tested by using dark-reared rats. In this paradigm, light exposure elicits massive synaptic activation in the visual cortex of rats raised in the dark. In such animals, light stimulation induced a-CaMKII mRNA polyadenylation and translational activation (26). Thus, CPEB may control local translation of this (and possibly other) mRNAs in the postsynaptic region and, by extension, synaptic plasticity.

Although there are clear biological consequences of local CPEB-mediated translational control, many particulars remain obscure. For example, why must cyclin mRNA apparently be translated on spindles to effect cell division? If cyclin mRNA polyadenylation-induced translation is under cycle control, as suggested by the data of Groisman et al. (15), then what are the essential upstream signaling events? Is Eg2-mediated CPEB phosphorylation under cell cycle control, or is cytoplasmic polyadenylation, like nuclear polyadenylation, controlled at the level of poly(A) polymerase phosphorylation (31)? In the brain, many questions remain to be explored, such as whether CPEB is activated by Eg2-catalyzed phosphorylation, and most importantly, whether a CPEB knockout mouse would have impaired synaptic plasticity. Finally, the data of Groisman et al. (15) indicate that not only does CPEB regulate translation on spindles, but that it is also involved in localizing mRNA to the mitotic apparatus. Because several CPE-containing mRNAs are localized in dendrites (28), CPEB might influence this process in neurons as well.

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