Paul Lasko

Autism-related deficits via dysregulated eIF4E-dependent translational control

Hyperconnectivity of neuronal circuits due to increased synaptic protein synthesis is thought to cause autism spectrum disorders (ASDs). The mammalian target of rapamycin (mTOR) is strongly implicated in ASDs by means of upstream signalling; however, downstream regulatory mechanisms are ill-defined. Here we show that knockout of the eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2)—an eIF4E repressor downstream of mTOR—or eIF4E overexpression leads to increased translation of neuroligins, which are postsynaptic proteins that are causally linked to ASDs. Mice that have the gene encoding 4E-BP2 (Eif4ebp2) knocked out exhibit an increased ratio of excitatory to inhibitory synaptic inputs and autistic-like behaviours (that is, social interaction deficits, altered communication and repetitive/stereotyped behaviours). Pharmacological inhibition of eIF4E activity or normalization of neuroligin 1, but not neuroligin 2, protein levels restores the normal excitation/inhibition ratio and rectifies the social behaviour deficits. Thus, translational control by eIF4E regulates the synthesis of neuroligins, maintaining the excitation-to-inhibition balance, and its dysregulation engenders ASD-like phenotypes.

The translational inhibitor 4E-BP is an effector of PI(3)K/Akt signalling and cell growth in Drosophila

The initiation factor 4E for eukaryotic translation (eIF4E) binds the messenger RNA 5′-cap structure and is important in the regulation of protein synthesis. Mammalian eIF4E activity is inhibited when the initiation factor binds to the translational repressors, the 4E-binding proteins (4E-BPS). Here we show that the Drosophila melanogaster 4E-BP (d4E-BP) is a downstream target of the phosphatidylinositol-3-OH kinase (PI(3)K) signal-transduction cascade, which affects the interaction of d4E-BP with eIF4E. Ectopic expression of a highly active d4E-BP mutant in wing-imaginal discs causes a reduction of wing size, brought about by a decrease in cell size and number. A marked reduction in cell size was also observed in post-mitotic cells. Expression of d4E-BP in the eye and wing together with PI(3)K or dAkt1, the serine/threonine kinase downstream of PI(3)K, resulted in suppression of the growth phenotype elicited by these kinases. Our results support a role for d4E-BP as an effector of cell growth.

Requirement for a noncoding RNA in Drosophila polar granules for germ cell establishment

In Drosophila embryos, germ cell formation is induced by specialized cytoplasm at the posterior of the egg, the pole plasm. Pole plasm contains polar granules, organelles in which maternally produced molecules required for germ cell formation are assembled. An untranslatable RNA, called Polar granule component (Pgc), was identified and found to be localized in polar granules. Most pole cells in embryos produced by transgenic females expressing antisense Pgc RNA failed to complete migration and to populate the embryonic gonads, and females that developed from these embryos often had agametic ovaries. These results support an essential role for Pgc RNA in germline development.

Postsynaptic translation affects the efficacy and morphology of neuromuscular junctions.

Long-term synaptic plasticity may be associated with structural rearrangements within the neuronal circuitry. Although the molecular mechanisms governing such activity-controlled morphological alterations are mostly elusive, polysomal accumulations at the base of developing dendritic spines and the activity-induced synthesis of synaptic components suggest that localized translation is involved during synaptic plasticity. Here we show that large aggregates of translational components as well as messenger RNA of the postsynaptic glutamate receptor subunit DGluR-IIA are localized within subsynaptic compartments of larval neuromuscular junctions of Drosophila melanogaster. Genetic models of junctional plasticity and genetic manipulations using the translation initiation factors eIF4E and poly(A)-binding protein showed an increased occurrence of subsynaptic translation aggregates. This was associated with a significant increase in the postsynaptic DGluR-IIA protein levels and a reduction in the junctional expression of the cell-adhesion molecule Fasciclin II. In addition, the efficacy of junctional neurotransmission and the size of larval neuromuscular junctions were significantly increased. Our results therefore provide evidence for a postsynaptic translational control of long-term junctional plasticity.

Self-association of the single-KH-domain family members Sam68, GRP33, GLD-1, and Qk1: role of the KH domain.

Sam68 is a member of a growing family of proteins that contain a single KH domain embedded in a larger conserved domain of approximately 170 amino acids. Loops 1 and 4 of this KH domain family are longer than the corresponding loops in other KH domains and contain conserved residues. KH domains are protein motifs that are involved in RNA binding and are often present in multiple copies. Here we demonstrate by coimmunoprecipitation studies that Sam68 self-associated and that cellular RNA was required for the association. Deletion studies demonstrated that the Sam68 KH domain loops 1 and 4 were required for self-association. The Sam68 interaction was also observed in Saccharomyces cerevisiae by the two-hybrid system. In situ chemical cross-linking studies in mammalian cells demonstrated that Sam68 oligomerized in vivo. These Sam68 complexes bound homopolymeric RNA and the SH3 domains of p59fyn and phospholipase Cgamma1 in vitro, demonstrating that Sam68 associates with RNA and signaling molecules as a multimer. The formation of the Sam68 complex was inhibited by p59fyn, suggesting that tyrosine phosphorylation regulates Sam68 oligomerization. Other Sam68 family members including Artemia salina GRP33, Caenorhabditis elegans GLD-1, and mouse Qk1 also oligomerized. In addition, Sam68, GRP33, GLD-1, and Qk1 associated with other KH domain proteins such as Bicaudal C. These observations indicate that the single KH domain found in the Sam68 family, in addition to mediating protein-RNA interactions, mediates protein-protein interactions.

Phosphorylation of eukaryotic translation initiation factor 4E is critical for growth.

ABSTRACT Eukaryotic translation initiation factor 4E (eIF4E) binds to the cap structure at the 5′ end of mRNAs and is a critical target for the control of protein synthesis. eIF4E is phosphorylated in many systems in response to extracellular stimuli, but biochemical evidence to date has been equivocal as to the biological significance of this modification. Here we use a genetic approach to this problem. We show that, in Drosophila melanogaster, homozygous eIF4E mutants arrest growth during larval development. In Drosophila eIF4EI, Ser251 corresponds to Ser209 of mammalian eIF4E, which is phosphorylated in response to extracellular signals. We find that, in vivo, eIF4EI Ser251 mutants cannot incorporate labeled phosphate. Furthermore, transgenic Drosophila organisms expressing eIF4ESer251Ala in an eIF4E mutant background have reduced viability. Escapers develop more slowly than control siblings and are smaller. These genetic data provide evidence that eIF4E phosphorylation is biologically significant and is essential for normal growth and development.

VASA mediates translation through interaction with a Drosophila yIF2 homolog.

The Drosophila gene vasa (vas) encodes an RNA-binding protein required for embryonic patterning and germ cell specification. In vas mutants, translation of several germline mRNAs is reduced. Here we show that VAS interacts directly with the Drosophila homolog of yeast translation initiation factor 2, encoded by a novel gene, dIF2. Embryos produced by vas/+; dIF2/+ females have pattern defects and fewer germline progenitor cells, indicating a functional interaction between endogenous vas and dIF2 activities. Mutations in other translation initiation factors do not enhance the vas phenotype, suggesting that dIF2 has a particular role in germ plasm function. We conclude that VAS regulates translation of germline mRNAs by specific interaction with dIF2, an essential factor conserved from bacteria to humans.

Nuclear Retention of MBP mRNAs in the Quaking Viable Mice

Quaking viable (qk(v)) mice fail to properly compact myelin in their central nervous systems. Although the defect in the qk(v) mice involves a mutation affecting the expression of the alternatively spliced qk gene products, their roles in myelination are unknown. We show that the QKI RNA binding proteins regulate the nuclear export of MBP mRNAs. Disruption of the QKI nucleocytoplasmic equilibrium in oligodendrocytes results in nuclear and perikaryal retention of the MBP mRNAs and lack of export to cytoplasmic processes, as it occurs in qk(v) mice. MBP mRNA export defect leads to a reduction in the MBP levels and their improper cellular targeting to the periphery. Our findings suggest that QKI participates in myelination by regulating the mRNA export of key protein components.

Drosophila Pgc protein inhibits P-TEFb recruitment to chromatin in primordial germ cells

Germ cells are the only cells that transmit genetic information to the next generation, and they therefore must be prevented from differentiating inappropriately into somatic cells. A common mechanism by which germline progenitors are protected from differentiation-inducing signals is a transient and global repression of RNA polymerase II (RNAPII)-dependent transcription. In both Drosophila and Caenorhabditis elegans embryos, the repression of messenger RNA transcription during germ cell specification correlates with an absence of phosphorylation of Ser 2 residues in the carboxy-terminal domain of RNAPII (hereafter called CTD), a critical modification for transcriptional elongation. Here we show that, in Drosophila embryos, a small protein encoded by polar granule component (pgc) is essential for repressing CTD Ser 2 phosphorylation in newly formed pole cells, the germline progenitors. Ectopic Pgc expression in somatic cells is sufficient to repress CTD Ser 2 phosphorylation. Furthermore, Pgc interacts, physically and genetically, with positive transcription elongation factor b (P-TEFb), the CTD Ser 2 kinase complex, and prevents its recruitment to transcription sites. These results indicate that Pgc is a cell-type-specific P-TEFb inhibitor that has a fundamental role in Drosophila germ cell specification. In C. elegans embryos, PIE-1 protein segregates to germline blastomeres, and is thought to repress mRNA transcription through interaction with P-TEFb. Thus, inhibition of P-TEFb is probably a common mechanism during germ cell specification in the disparate organisms C. elegans and Drosophila.

Localized Bicaudal‐C RNA encodes a protein containing a KH domain, the RNA binding motif of FMR1

The Bicaudal-C (Bic-C) gene of Drosophila melanogaster is required for correct targeting of the migrating anterior follicle cells and for specifying anterior position. Females lacking any wild type copies of Bic-C produce only eggshells open at the anterior end, because of the failure of the columnar follicle cells to migrate in the correct position at the nurse cell--oocyte boundary. Embryos which develop from eggs produced in females with only one wild type copy of Bic-C show defects in anterior patterning and an abnormal persistence of oskar RNA in anterior regions. We cloned Bic-C and found that, in ovaries, Bic-C RNA is expressed only in germline cells. Bic-C RNA is localized to the oocyte in early oogenesis, and later concentrates at its anterior cortex. The Bic-C protein includes five KH domains similar to those found in the human fragile-X protein FMR1. Alteration of a highly conserved KH domain codon by mutation abrogates in vivo Bic-C function. These results suggest roles for the Bic-C protein in localizing RNAs and in intercellular signaling.