Joel D. Richter

FMRP Stalls Ribosomal Translocation on mRNAs Linked to Synaptic Function and Autism

FMRP loss of function causes Fragile X syndrome (FXS) and autistic features. FMRP is a polyribosome-associated neuronal RNA-binding protein, suggesting that it plays a key role in regulating neuronal translation, but there has been little consensus regarding either its RNA targets or mechanism of action. Here, we use high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) to identify FMRP interactions with mouse brain polyribosomal mRNAs. FMRP interacts with the coding region of transcripts encoding pre- and postsynaptic proteins and transcripts implicated in autism spectrum disorders (ASD). We developed a brain polyribosome-programmed translation system, revealing that FMRP reversibly stalls ribosomes specifically on its target mRNAs. Our results suggest that loss of a translational brake on the synthesis of a subset of synaptic proteins contributes to FXS. In addition, they provide insight into the molecular basis of the cognitive and allied defects in FXS and ASD and suggest multiple targets for clinical intervention.

Regulation of cap-dependent translation by eIF4E inhibitory proteins.

Eukaryotic messenger RNAs contain a modified guanosine, termed a cap, at their 5′ ends. Translation of mRNAs requires the binding of an initiation factor, eIF4E, to the cap structure. Here, we describe a family of proteins that through a shared sequence regulate cap-dependent translation. The biological importance of this translational regulation is immense, and affects such processes as cell growth, development, oncogenic transformation and perhaps even axon pathfinding and memory consolidation.

Translational control by CPEB: a means to the end

The regulated translation of messenger RNA is essential for cell-cycle progression, establishment of the body plan during early development, and modulation of key activities in the central nervous system. Cytoplasmic polyadenylation, which is one mechanism of controlling translation, is driven by CPEB — a highly conserved, sequence-specific RNA-binding protein that binds to the cytoplasmic polyadenylation element, and modulates translational repression and mRNA localization. What are the features and functions of this multifaceted protein?

CPEB-Mediated Cytoplasmic Polyadenylation and the Regulation of Experience-Dependent Translation of α-CaMKII mRNA at Synapses

Long-term changes in synaptic efficacy may require the regulated translation of dendritic mRNAs. While the basis of such regulation is unknown, it seemed possible that some features of translational control in development could be recapitulated in neurons. Polyadenylation-induced translation of oocyte mRNAs requires the cis-acting CPE sequence and the CPE-binding protein CPEB. CPEB is also present in the dendritic layers of the hippocampus, at synapses in cultured neurons, and in postsynaptic densities of adult brain. alpha-CaMKII mRNA, which is localized in dendrites and is necessary for synaptic plasticity and LTP, contains two CPEs. These CPEs are bound by CPEB and mediate polyadenylation-induced translation in injected Xenopus oocytes. In the intact brain, visual experience induces alpha-CaMKII mRNA polyadenylation and translation, suggesting that this process likely occurs at synapses.

CPEB is a specificity factor that mediates cytoplasmic polyadenylation during Xenopus oocyte maturation

The translational activation of several maternal mRNAs during Xenopus oocyte maturation is stimulated by cytoplasmic poly(A) elongation, which requires the uridine-rich cytoplasmic polyadenylation element (CPE) and the hexanucleotide AAUAAA. Here, we have enriched a CPE-binding protein (CPEB) by single-step RNA affinity chromatography, have obtained a CPEB cDNA, and have assessed the role of CPEB in cytoplasmic polyadenylation. The 62 kDa CPEB contains two RNA recognition motifs, and within this region, it is 62% identical to orb, an oocyte-specific RNA-binding protein from Drosophila. CPEB mRNA and protein are abundant in oocytes and are not detected in embryos beyond the gastrula stage. During oocyte maturation, CPEB is phosphorylated at a time that corresponds with the induction of polyadenylation. Immunodepletion of CPEB from polyadenylation-proficient egg extracts renders them incapable of adenylating exogenous RNA. Partial restoration of polyadenylation in depleted extracts is achieved by the addition of CPEB, thus demonstrating that this protein is required for cytoplasmic polyadenylation.

Human let-7a miRNA blocks protein production on actively translating polyribosomes.

MicroRNAs (miRNAs) regulate gene expression at a post-transcriptional level through base-pairing to 3′ untranslated regions (UTRs) of messenger RNAs. The mechanism by which human let-7a miRNA regulates mRNA translation was examined in HeLa cells expressing reporter mRNAs containing the Caenorhabditis elegans lin-41 3′ UTR. let-7a miRNA strongly repressed translation, yet the majority of control and lin-41–bearing RNAs sedimented with polyribosomes in sucrose gradients; these polyribosomes, together with let-7a miRNA and the miRISC protein AGO, were released from those structures by puromycin. RNA containing the lin-41 3′ UTR and an iron response element in the 5′ UTR sedimented with polysomes when cells were incubated with iron, but showed ribosome run-off when the iron was chelated. These data indicate that let-7a miRNA inhibits actively translating polyribosomes. Nascent polypeptide coimmunoprecipitation experiments further suggest that let-7a miRNA interferes with the accumulation of growing polypeptides.

Symplekin and xGLD-2 Are Required for CPEB-Mediated Cytoplasmic Polyadenylation

Cytoplasmic polyadenylation-induced mRNA translation is a hallmark of early animal development. In Xenopus oocytes, where the molecular mechanism has been defined, the core factors that control this process include CPEB, an RNA binding protein whose association with the CPE specifies which mRNAs undergo polyadenylation; CPSF, a multifactor complex that interacts with the near-ubiquitous polyadenylation hexanucleotide AAUAAA; and maskin, a CPEB and eIF4E binding protein whose regulation of initiation is governed by poly(A) tail length. Here, we define two new factors that are essential for polyadenylation. The first is symplekin, a CPEB and CPSF binding protein that serves as a scaffold upon which regulatory factors are assembled. The second is xGLD-2, an unusual poly(A) polymerase that is anchored to CPEB and CPSF even before polyadenylation begins. The identification of these factors has broad implications for biological process that employ polyadenylation-regulated translation, such as gametogenesis, cell cycle progression, and synaptic plasticity.

CPEB controls the cytoplasmic polyadenylation of cyclin, Cdk2 and c-mos mRNAs and is necessary for oocyte maturation in Xenopus.

Cytoplasmic polyadenylation is a key mechanism controlling maternal mRNA translation in early development. In most cases, mRNAs that undergo poly(A) elongation are translationally activated; those that undergo poly(A) shortening are deactivated. Poly(A) elongation is regulated by two cis‐acting sequences in the 3′‐untranslated region (UTR) of responding mRNAs, the polyadenylation hexanucleotide AAUAAA and the U‐rich cytoplasmic polyadenylation element (CPE). Previously, we cloned and characterized the Xenopus oocyte CPE binding protein (CPEB), showing that it was essential for the cytoplasmic polyadenylation of B4 RNA. Here, we show that CPEB also binds the CPEs of G10, c‐mos, cdk2, cyclins A1, B1 and B2 mRNAs. We find that CPEB is necessary for polyadenylation of these RNAs in egg extracts, suggesting that this protein is required for polyadenylation of most RNAs during oocyte maturation. Our data demonstrate that the complex timing and extent of polyadenylation are partially controlled by CPEB binding to multiple target sites in the 3′ UTRs of responsive mRNAs. Finally, injection of CPEB antibody into oocytes not only inhibits polyadenylation in vivo, but also blocks progesterone‐induced maturation. This is due to inhibition of polyadenylation and translation of c‐mos mRNA, suggesting that CPEB is critical for early development.

Making synaptic plasticity and memory last: mechanisms of translational regulation

Synaptic transmission in neurons is a measure of communication at synapses, the points of contact between axons and dendrites. The magnitude of synaptic transmission is a reflection of the strength of these synaptic connections, which in turn can be altered by the frequency with which the synapses are stimulated, the arrival of stimuli from other neurons in the appropriate temporal window, and by neurotrophic factors and neuromodulators. The ability of synapses to undergo lasting biochemical and morphological changes in response to these types of stimuli and neuromodulators is known as synaptic plasticity, which likely forms the cellular basis for learning and memory, although the relationship between any one form synaptic plasticity and a particular type of memory is unclear. RNA metabolism, particularly translational control at or near the synapse, is one process that controls long-lasting synaptic plasticity and, by extension, several types of memory formation and consolidation. Here, we review recent studies that reflect the importance and challenges of investigating the role of mRNA translation in synaptic plasticity and memory formation.

N-methyl-D-aspartate receptor signaling results in Aurora kinase-catalyzed CPEB phosphorylation and αCaMKII mRNA polyadenylation at synapses

Activity‐dependent local translation of dendritic mRNAs is one process that underlies synaptic plasticity. Here, we demonstrate that several of the factors known to control polyadenylation‐induced translation in early vertebrate development [cytoplasmic polyadenylation element‐binding protein (CPEB), maskin, poly(A) polymerase, cleavage and polyadenylation specificity factor (CPSF) and Aurora] also reside at synaptic sites of rat hippocampal neurons. The induction of polyadenylation at synapses is mediated by the N‐methyl‐D‐aspartate (NMDA) receptor, which transduces a signal that results in the activation of Aurora kinase. This kinase in turn phosphorylates CPEB, an essential RNA‐binding protein, on a critical residue that is necessary for polyadenylation‐induced translation. These data demonstrate a remarkable conservation of the regulatory machinery that controls signal‐induced mRNA translation, and elucidates an axis connecting the NMDA receptor to localized protein synthesis at synapses.