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Research

We study the molecular biology of mRNA translational control by cytoplasmic polyadenylation and how this process influences interesting biological phenomena including early animal development, cellular senescence/growth control, neuron synaptic plasticity, learning, and memory, and neurologic disease.

Translational control by 3’ end formation

Many inactive mRNAs have short poly(A) tails and only when the tails are elongated does translation ensue. A key factor that regulates polyadenylation-induced translation is the RNA binding protein CPEB (Cytoplasmic Polyadenylation Element Binding Protein). CPEB binds specific 3’UTR cis elements in mRNAs and recruits unusual poly(A) polymerases and translation factors that extend poly(A) tails in the cytoplasm and promote translation.

One poly(A) polymerase also monoadenylates and stabilizes specific miRNAs, adding an important and unexpected layer of translational control by 3’ end formation.

Early animal development

Maternal (masked) mRNAs in oocytes have short poly(A) tails, which are elongated when the cells re-enter the meiotic divisions and prepare for fertilization. This cytoplasmic polyadenylation induces translation, and the biochemistry of these events in most easily studied in oocytes of the frog Xenopus. The importance of CPEB for germ cell development is demonstrated by the observation that meiosis does not proceed beyond the pachytene stage in CPEB knockout mice.

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Figure 1. The mRNA binding protein CPEB associates with the UUUUUAU cytoplasmic polyadenylation element (CPE) in 3’ UTRs. CPEB also binds the non-canonical poly(A) polymerase Gld2, the deadenylase PARN, the AAUAAA poly(A) cleavage site factors CPSF, the scaffold protein symplekin, and the translation inhibitor factor maskin. PARN activity removes the poly(A) tail as soon as it is added by Gld2. Maskin represses translation buy binding the cap-binding factor eIF4E. In response to environmental cues, the kinase Aurora A phosphorylates CPEB, which leads to the expulsion of PARN and Gld2-catalyzed cytoplasmic polyadenylation. The newly elongated poly(A) tail is bound by poly(A) binding protein (PABP), which in turn binds eIF4G and helps it bind eIF4E in place of maskin. eIF4G, via eIF3, positions the 40S ribosomal subunit on the 5’ end of the mRNA where it begins to scan for an AUG initiation codon.

 

Senescence and growth control

Senescence is a mechanism cells employ to exit the cell cycle as a guard against malignant transformation. Primary mouse cells that lack the CPEB gene do not senesce but instead are immortal. Similarly, primary human cells from which CPEB is depleted also bypass senescence and exhibit the Warburg Effect, an alteration in bioenergetics often employed by cancer cells to survive in an inhospitable environment.

CPEB knockout mice display defects in the polarity of mammary epithelial cells, which is recapitulated in vitro with mouse mammary cells depleted of CPEB. This loss of polarity is due to the mis-localization of the mRNA encoding ZO-1, a tight junction protein. When CPEB-depleted mammary cells lose polarity, they undergo an epithelial to mesenchyme transition (EMT), which often presages enhanced metastatic potential. Indeed, CPEB-depleted mammary cells are highly metastatic.

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Figure 2. Mouse mammary epithelial cells grown in suspension form a polarized 3 dimensional architecture with a central cavity and a basal external surface. ZO-1, a tight junction protein (red), is apically localized. In CPEB-depleted cells, the polarity is lost and no central cavity is formed; ZO-1 staining is randomly distributed. The blue (DAPI) staining identifies nuclei.

 

Synaptic plasticity, learning and memory, and neuronal metabolism

CPEB and the cytoplasmic polyadenylation complex reside at postsynaptic sites of neurons in the mammalian central nervous system. In dendrites, they control local mRNA polyadenylation-induced translation in response to synaptic stimulation. Synaptic plasticity, the ability of synapses to undergo long-lasting biochemical and morphological changes in response to stimulation, forms the underlying basis of learning and memory. CPEB knockout mice are defective for synaptic plasticity and hippocampal-dependent memory formation. Hippocampal neurons depleted of other components of the cytoplasmic polyadenylation complex with lentivirus-based shRNAs also display defects in synaptic plasticity, indicating that polyadenylation-induced translation forms an essential mechanism to control translation and higher cognitive function.

Neurons derived from CPEB knockout mice have alterations in metabolism in that ATP production by mitochondria is compromised. This deficit in ATP reduces dendrite arborization and is observed in both neurons cultured in vitro and neurons expressing an shRNA for CPEB in vivo.

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Figure 3. Cultured mouse hippocampal neurons form neurite extensions with filipodia extending from growth cones. The CPEB-associated translational control protein neuroguidin forms puncta in the neurites and filipodia.

 

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Figure 4. Rat hippocampus was injected with lentivirus expressing shRNA for the unusual poly(A) polymerase Gld2 as well as GFP. The sectioned hippocampus shows GFP fluorescence in the dentate gyrus region of the hippocampus.

 

Neurologic disease

The Fragile X Syndrome (FXS) is the most common heritable form of mental retardation and the most common monogenic form of autism. FXS is caused by a triplet repeat expansion in and transcriptional silencing of the FMR1 gene. FMR1 encodes FMRP, an RNA binding protein that normally represses translation in the brain. In the absence of FMRP, aberrantly high translation likely causes FXS in both humans and a mouse model. Restoration of normal translation occurs in FMRP/CPEB double knockout mice. Moreover, rescue of synapse function and learning and memory also occurs in FMRP/CPEB double knockout mice, suggesting that CPEB might be a novel therapeutic to reverse FXS.

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