Understanding the pathophysiology of Fragile X Syndrome and its extensive overlaps with autism offers the hope of gaining insights into the molecular basis of cognition and behavior. Learning and memory almost certainly involve changes in long-term synaptic strength and require new protein synthesis. This activity-dependent protein synthesis must be finely tuned, as exemplified by cases of autism caused by mutations in proteins that normally limit translation (Kelleher and Bear, 2008
), and by FXS, as FMRP has been hypothesized to inhibit translation of mRNAs encoding “plasticity-related proteins” (Bear et al., 2004
). Here we provide a molecular basis for the overlap between FXS, autism and defects in synaptic plasticity by first showing that many transcripts bound by FMRP encode plasticity-related proteins, and then by demonstrating that FMRP represses their translation by stalling ribosomal translocation. These results are set apart from previous studies in their overlay of unbiased, genome-wide, direct biochemical assays of endogenous interactions (Licatalosi and Darnell, 2010
); while they cannot describe FMRP function on every individual candidate transcript, they provide a statistically robust model for the predominant action of FMRP in translational regulation (Figure S7C
Our data support a model in which FMRP acts to stall ribosomal translocation during elongation as part of a complex containing target mRNAs and multiple ribosomes. This complex is relatively resistant to MN treatment, similar to mRNA-associated stacked ribosomes stalled by the action of the signal recognition particle (SRP), which represses translation as transcripts encoding secreted and transmembrane proteins are transported to the ER (Wolin and Walter, 1988
). It is increasingly recognized that a large fraction of cellular mRNA may be translated within subcellular domains (Martin and Ephrussi, 2009
), and our data are consistent with FMRP playing a role in controlling such processes.
Translational repression by ribosome stalling and stacking may confer several advantages to FMRP in regulating neuronal translation. Stalling of ribosomes may permit translocation to sites of axonal or dendritic protein synthesis (Krichevsky and Kosik, 2001
). Pre-loading of mRNAs with ribosomes would permit very rapid protein synthesis in response to synaptic activation, as seen for example in response to local application of BDNF (Aakalu et al., 2001
). The stalling of a single ribosome almost immediately slows protein production, while recycled ribosomes continue to re-initiate and restore the “loaded” state () (Wolin and Walter, 1988
). Stalled ribosomes may also protect mRNA from degradation during transport or storage. Although the biochemical mechanism by which FMRP stalls ribosomes remains to be determined, it is likely to be dynamic, as it can be acutely reversed by RNA decoys in run-off assays. Such reversibility could be mediated in vivo
by FMRP phosphorylation, which has been hypothesized to regulate FMRP’s association with apparently stalled polyribosomes in mouse fibroblasts (Ceman et al., 2003
), by FMRP degradation (Hou et al., 2006
), or other means. We suggest that agents such as antibiotics that slow ribosomal translocation may restore the brake on translation that is lost in FXS, and may be of worthy of clinical consideration.
In neurons, translational inhibition by ribosomal stalling is associated with NMDAR activation, eEF2 phosphorylation, and other unknown factors (Sutton et al., 2007
) which may include FMRP. Moreover, FMRP may inhibit translation in response to activation of mACh or BDNF receptors, as there are synaptic plasticity defects in these pathways in Fmr1
null mice (Lauterborn et al., 2007
; Volk et al., 2007
). A small fraction of FMRP is reported to inhibit translation initiation (Napoli et al., 2008
), suggesting additional mechanisms for FMRP action are likely to exist. However, our data indicate that in the brain at steady-state, the majority of FMRP is associated with stalled ribosomal complexes.
While much attention has been paid to the role of FMRP in regulating Hebbian synaptic plasticity through the control of local translation, the bulk of the protein is in the cell body (Christie et al., 2009
). Moreover, FMRP regulates many mRNAs that are probably not localized to the synapse: for example, Bsn
mRNAs encode proteins that are synthesized in the cell body and transported to the synapse as a complex (Shapira et al., 2003
). These observations suggest that FMRP may play a role in homeostatic synaptic plasticity (Turrigiano, 2008
). In this model, FMRP would act to repress the translation stimulated by neuronal activity to generate a feedback loop limiting neuronal responses to activity at the level of the neuron rather than a specific synapse.
In contrast to HITS-CLIP studies that have yielded interaction “maps” of regulatory sites involved in position-dependent alternative splicing and polyadenylation (reviewed in (Darnell, 2010
)), FMRP HITS-CLIP revealed a different and unexpected mode of protein-RNA interaction. We did not find peaks of RNA tags indicating specific high affinity binding sites for FMRP, such as those suggested by previous in vitro
FMRP RNA selection experiments (G-quadruplex or kissing complex motifs; (Darnell et al., 2001
; Darnell et al., 2005a
; Schaeffer et al., 2001
)). We considered the possibility that FMRP might interact with high affinity binding sites prior to its association with polyribosomes and subsequently redistribute on the same target transcripts, such that FMRP crosslinking in polyribosomes does not reflect the initial sites of transcript interaction. However, to date we have found no biochemical evidence to support such a model, and using the RNABob motif-finding program (Darnell et al., 2001
), we found that bioinformatically predicted G-quadruplex motifs are no more abundant in the set of 842 robust FMRP target mRNAs than in an equivalent set of 842 non-targets (controlled for neuronal expression and length; 1112 vs. 1068, or 43% vs. 39% of targets versus controls, respectively). Indeed, previous studies have shown that the RGG box is not necessary for polyribosome association (Darnell et al., 2005b
) and G-quadruplex RNA ligands cannot compete FMRP off polyribosomes (Darnell et al., 2005a
). This leaves the question of how FMRP associates with a specific set of mRNAs unanswered. In addition to the possibility that FMRP is binding to occult RNA motifs, FMRP may be recruited to its targets via a protein-protein interaction.
The proteins encoded by FMRP target mRNAs indicate a high level of control over the balance of activity-dependent translation in synaptic plasticity. First, mGluR5 and the NMDAR subunits, the only glutamate receptors known to mediate protein-synthesis dependent long-term synaptic plasticity, are themselves FMRP targets, as are many other protein components of their macromolecular complexes at the synapse. These findings are consistent with the finding that mGluR and NMDAR-dependent synaptic plasticity are altered in FXS mouse models (Harlow et al., 2010
; Pilpel et al., 2009
). Second, FMRP regulates the expression of components of the ERK and mTOR signal transduction pathways that convert receptor activity into translational output. Finally, FMRP controls the expression of many downstream pre- and post-synaptic structural, scaffolding, catalytic, receptor and channel proteins that are likely to be final determinants of changes in synaptic strength.
FMRP target mRNAs are a valuable dataset for considering pharmacologic therapy for FXS. mGluR5 inhibitors have shown some clinical efficacy, and NMDAR antagonists may also be worthy of clinical consideration. FMRP also targets ERK1 and the mTOR inhibitors Pten, Nf1 and Tsc2, proteins closely linked to autism, supporting the possibility that pharmacologic agents acting on the mTOR and ERK pathways may be clinically relevant for FXS and autism (Hoeffer and Klann, 2010
; Kelleher and Bear, 2008
). However, FMRP also targets the PI3K-enhancer PIKE (Centg1), and PIKE overexpression in FMRP null mice results in elevated PI3K signaling to the mTOR pathway (Sharma et al., 2010
), indicating the need for care in translating these findings into therapy.
The molecular basis for the overlap in symptoms between Fragile X Syndrome and autism is poorly understood. The overlap between FMRP targets and the current list of autism susceptibility genes and loci is extensive and sheds light on common pathways, supporting the hypothesis that synaptic dysfunction is critical to the development of autistic features common to both disorders (Kelleher and Bear, 2008
). We find that FMRP targets fall into several functional categories, as do ASD candidate genes (Sebat et al., 2007
; Pinto et al., 2010
) and the overlap is enriched in several inter-related functional categories, including synaptic cell adhesion molecules, the NMDAR complex, the mTOR pathway, and regulators of the small GTPases.
The FMRP target list also provides a valuable tool for focusing attention on specific gene candidates within multigenic loci for ASD. These include the three most common syndromic duplications linked to ASD, each containing between 13–28 protein-coding genes, and each locus harbors at least one FMRP target gene (Table S5
). Moreover, two well-studied candidate genes present in the 17p11.2 and 15q11-13 duplications, Rai1
, harbored FMRP CLIP tags in 6/7 and 3/7 experiments, respectively; their extremely low expression levels in brain polyribosomes may have precluded their inclusion on the statistically robust FMRP target list. Although the FMRP target set disproportionately overlaps amplified versus deleted ASD-related CNVs, they include genes whose loss results in autism. This is consistent with the gene balance hypothesis, which posits that the same phenotype can arise from under- or overexpression of dosage-sensitive proteins because either disrupts stoichiometry of the same complex (Conrad and Antonarakis, 2007
). It seems likely that FMRP tightly controls the synthesis of dosage-sensitive genes in neurons, and the overlap of FMRP targets with genes whose loss of function leads to ASD is highly significant in this regard. Taken together, the relationship between FMRP target transcripts and genes linked to the ASDs, particularly overexpressed genes, provides a new connection between loss-of-function of FMRP and the development of autistic symptoms in patients.