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Loss of the RNA-binding protein FMRP (fragile X mental retardation protein) leads to fragile X syndrome, the most common form of inherited mental retardation. Although some of the messenger RNA targets of this protein, including FMR1, have been ascertained, many have yet to be identified. We have found that Xenopus elongation factor 1A (EF-1A) mRNA binds tightly to recombinant human FMRP in vitro. Binding depended on protein determinants located primarily in the C-terminal end of hFMRP, but the hnRNP K homology domain influenced binding as well. When hFMRP was expressed in cultured cells, it dramatically reduced endogenous EF-1A protein expression but had no effect on EF-1A mRNA levels. In contrast, the translation of several other mRNAs, including those coding for dynamin and constitutive heat shock 70 protein, was not affected by the hFMRP expression. Most importantly, EF-1A mRNA and hFMR1 mRNA were coimmunoprecipitated with hFMRP. Finally, in fragile X lymphoblastoid cells in which hFMRP is absent, human EF-1A protein but not its corresponding mRNA is elevated compared with normal lymphoblastoid cells. These data suggest that hFMRP binds to EF-1A mRNA and also strongly argue that FMRP negatively regulates EF-1A expression in vivo.The loss of a normal cellular protein, FMRP, 1 causes fragile X syndrome, one of the most common forms of mental retardation (MR). FMRP is a RNA-binding protein that contains two hnRNP K-homology (KH) binding domains and an arginineglycine-rich region that resembles an RGG box (1, 2). Several studies indicate that both the KH 2 domain and the arginineglycine-rich region likely play a role in RNA binding (1, 3-6), the latter interaction being mediated by a G quartet (7). FMRP associates with polyribosomes via a mRNP particle (8, 9), and it has been proposed to regulate gene expression post-transcriptionally (5, 10 -14). Mammalian FMRPs inhibit mRNA translation in vitro at nanomolar concentrations in both rabbit reticulocyte lysates (15) and in microinjected Xenopus oocytes (16). These data suggest that translational repression may be an in vivo function of FMRP. Indeed, the Drosophila homolog of FMRP, dFMR1, was found to bind and negatively regulate futsch mRNA (17).Recent studies have begun to delineate the mRNAs that mammalian FMRPs interact with in vivo. These studies have taken one of two forms. On the one hand, potential FMRP target mRNAs have been identified solely on the basis of their ability to bind to purified recombinant FMRP (15, 16) or cellfree produced FMRP (1, 3). Notwithstanding, it has not been determined whether any of these mRNAs bind to FMRP in vivo. On the other hand, mRNAs, including FMR1 mRNA, which associate with FMRP-containing mRNPs have also been isolated from cultured cells (10, 18). However, although these messages require FMRP in the mRNP for their association, it has not been demonstrated that they bind solely to it. Using the former methodology, we isolated a subset of mRNAs derived from normal adult brain that bind human FMRP (hFMRP) in vitro (3). Duri...

The induction of a long-term hyperexcitability (LTH) in vertebrate nociceptive sensory neurons (SNs) after nerve injury is an important contributor to neuropathic pain in humans, but the signaling cascades that induce this LTH have not been identified. In particular, it is not known how injuring an axon far from the cell soma elicits changes in gene expression in the nucleus that underlie LTH. The nociceptive SNs of Aplysia (ap) develop an LTH with electrophysiological properties after axotomy similar to those of mammalian neurons and are an experimentally useful model to examine these issues. We cloned an Aplysia PKG (cGMP-dependent protein kinase; protein kinase G) that is homologous to vertebrate type-I PKGs and found that apPKG is activated at the site of injury in the axon after peripheral nerve crush. The active apPKG is subsequently retrogradely transported to the somata of the SNs, but apPKG activity does not appear in other neurons whose axons are injured. In the soma, apPKG phosphorylates apMAPK (Aplysia mitogen-activated protein kinase), resulting in its entry into the nucleus. Surprisingly, studies using recombinant proteins in vivo and in vitro indicate that apPKG directly phosphorylates the threonine moiety in the T-E-Y activation site of apMAPK when the -Y-site contains a phosphate. We used inhibitors of nitric oxide synthase, soluble guanyl cyclase, or PKG after nerve injury, and found that each prevented the appearance of the LTH. Moreover, blocking apPKG activation prevented the nuclear import of apMAPK. Consequently, the nitric oxide-PKG-MAPK pathway is a potential target for treatment of neuropathic pain.

No abstract

Sensory neurons (SNs) of Aplysia are widely used to study the molecular correlates of learning. Among these is the activation of an Aplysia (ap) MAPK that phosphorylates the transcription factor apC/EBPbeta. Because crushing the axons of the SNs induces changes similar to learning, we tested the hypothesis that apMAPK is a point of convergence on the pathways for learning and injury. One event in common is long-term hyperexcitability (LTH), and LTH was induced in the SNs after intrasomatic injection of active vertebrate extracellular signal-regulated kinase 1 (ERK1; as an apMAPK surrogate). Nerve crush activated an axoplasmic kinase at the site of injury that phosphorylated apC/EBPbeta. Surprisingly, this was not apMAPK, but a kinase that was recognized by antibodies to vertebrate ERKs and to doubly phosphorylated, activated ERKs. The activated kinase was transported to the cell body and nucleus and its arrival was concurrent with an injury-induced increase in apC/EBPbeta mRNA and protein. We call this retrogradely transported kinase RISK-1. RISK-1 initiated the binding of apC/EBPbeta to the ERE enhancer site in vitro and an increase in ERE-binding was detected in injured neurons containing active RISK-1. Thus, Aplysia neurons contain two MAPK homologues, one of which is a late acting retrogradely transported injury signal.