The Groucho/TLE/Grg family of transcriptional co-repressors

The Groucho family of co-repressor proteins are essential for development and may also have a role in some human cancers

Syncrip/hnRNP Q is required for activity-induced Msp300/Nesprin-1 expression and new synapse formation.

Memory and learning involve activity-driven expression of proteins and cytoskeletal reorganization at new synapses, requiring posttranscriptional regulation of localized mRNA a long distance from corresponding nuclei. A key factor expressed early in synapse formation is Msp300/Nesprin-1, which organizes actin filaments around the new synapse. How Msp300 expression is regulated during synaptic plasticity is poorly understood. Here, we show that activity-dependent accumulation of Msp300 in the postsynaptic compartment of the Drosophila larval neuromuscular junction is regulated by the conserved RNA binding protein Syncrip/hnRNP Q. Syncrip (Syp) binds to msp300 transcripts and is essential for plasticity. Single-molecule imaging shows that msp300 is associated with Syp in vivo and forms ribosome-rich granules that contain the translation factor eIF4E. Elevated neural activity alters the dynamics of Syp and the number of msp300:Syp:eIF4E RNP granules at the synapse, suggesting that these particles facilitate translation. These results introduce Syp as an important early acting activity-dependent regulator of a plasticity gene that is strongly associated with human ataxias

Differential Axial Requirements for Lunatic Fringe and Hes7 Transcription during Mouse Somitogenesis

Vertebrate segmentation is regulated by the “segmentation clock”, which drives cyclic expression of several genes in the caudal presomitic mesoderm (PSM). One such gene is Lunatic fringe (Lfng), which encodes a modifier of Notch signalling, and which is also expressed in a stripe at the cranial end of the PSM, adjacent to the newly forming somite border. We have investigated the functional requirements for these modes of Lfng expression during somitogenesis by generating mice in which Lfng is expressed in the cranial stripe but strongly reduced in the caudal PSM, and find that requirements for Lfng activity alter during axial growth. Formation of cervical, thoracic and lumbar somites/vertebrae, but not sacral and adjacent tail somites/vertebrae, depends on caudal, cyclic Lfng expression. Indeed, the sacral region segments normally in the complete absence of Lfng and shows a reduced requirement for another oscillating gene, Hes7, indicating that the architecture of the clock alters as segmentation progresses. We present evidence that Lfng controls dorsal-ventral axis specification in the tail, and also suggest that Lfng controls the expression or activity of a long-range signal that regulates axial extension

Imp/IGF2BP levels modulate individual neural stem cell growth and division through myc mRNA stability.

The numerous neurons and glia that form the brain originate from tightly controlled growth and division of neural stem cells, regulated systemically by important known stem cell-extrinsic signals. However, the cell-intrinsic mechanisms that control the distinctive proliferation rates of individual neural stem cells are unknown. Here, we show that the size and division rates of Drosophila neural stem cells (neuroblasts) are controlled by the highly conserved RNA binding protein Imp (IGF2BP), via one of its top binding targets in the brain, myc mRNA. We show that Imp stabilises myc mRNA leading to increased Myc protein levels, larger neuroblasts, and faster division rates. Declining Imp levels throughout development limit myc mRNA stability to restrain neuroblast growth and division, and heterogeneous Imp expression correlates with myc mRNA stability between individual neuroblasts in the brain. We propose that Imp-dependent regulation of myc mRNA stability fine-tunes individual neural stem cell proliferation rates

Integrin-Linked Kinase Is a Functional Mn2+-Dependent Protein Kinase that Regulates Glycogen Synthase Kinase-3β (GSK-3β) Phosphorylation

Integrin-linked kinase (ILK) is a highly evolutionarily conserved, multi-domain signaling protein that localizes to focal adhesions, myofilaments and centrosomes where it forms distinct multi-protein complexes to regulate cell adhesion, cell contraction, actin cytoskeletal organization and mitotic spindle assembly. Numerous studies have demonstrated that ILK can regulate the phosphorylation of various protein and peptide substrates in vitro, as well as the phosphorylation of potential substrates and various signaling pathways in cultured cell systems. Nevertheless, the ability of ILK to function as a protein kinase has been questioned because of its atypical kinase domain.Here, we have expressed full-length recombinant ILK, purified it to >94% homogeneity, and characterized its kinase activity. Recombinant ILK readily phosphorylates glycogen synthase kinase-3 (GSK-3) peptide and the 20-kDa regulatory light chains of myosin (LC(20)). Phosphorylation kinetics are similar to those of other active kinases, and mutation of the ATP-binding lysine (K220 within subdomain 2) causes marked reduction in enzymatic activity. We show that ILK is a Mn-dependent kinase (the K(m) for MnATP is approximately 150-fold less than that for MgATP).Taken together, our data demonstrate that ILK is a bona fide protein kinase with enzyme kinetic properties similar to other active protein kinases

Genetic and molecular studies of early embryogenesis in Drosophila

The Drosophila embryo is patterned by a complex interplay of zygotically expressed genes and maternally supplied components, a large number of which have been identified. However, many maternal components are encoded by essential zygotic genes whose maternal effects are not amenable to conventional genetic analysis. Investigation of such genes requires the generation of homozygous mutant germ cells in chimeric females, and analysis of their embryos. The recent development of techniques which allow the efficient generation of germline clones has made the screening of zygotic lethal mutations for maternal effects more feasible. I have generated a collection of X-linked zygotic lethal mutations and used FLP recombinase catalysed mitotic recombination to look for maternal effects affecting segmentation. Two mutations have been recovered which have maternal effect phenotypes similar to those of the pair-rule segmentation genes. The leprechaun mutation affects oogenesis, so fertile females are very rare, preventing straightforward phenotypic analysis of the segmentation phenotype. Attempts to generate a rescuing duplication are described. The second mutation, stunted (sun), initially gave rise to a segmentation cuticle phenotype. Subsequent attempts to reproduce the phenotype were unsuccessful as it was masked by a severe reduction in the amount of cuticle secreted, a phenotype characteristic of the neurogenic genes. Detailed analysis revealed that the primary lesion affects neither segmentation or neurogenesis. Rather, sun+ is required for cellularisation of the syncytial blastoderm and for the localisation of actin to 'caps' above the syncytial nuclei. Cloning of a candidate gene for stunted revealed a predicted protein product limited homology to cyclins. In addition to searching for novel segmentation genes, potential proteinprotein interactions of the segmentation gene hairy's protein product were also investigated, and a model is presented for its mode of action as a transcriptional repressor.</p

Intracellular message localisation in Drosophila melanogaster

The blastoderm embryo of Drosophila melanogaster consists of a unicellular syncytium with a large number of peripheral nuclei. The cytoplasm surrounding each peripheral nucleus is compartmentalised into apical periplasm above each nucleus and basal periplasm below it. The expression of different genes in the syncytial blastoderm is crucial for the genetic control of development. The pair-rule genes are involved in controlling the pattern of metamerisation of the embryo. Pair-rule mRNAs are expressed in alternate metameres, in a pattern of stripes. Within each stripe, mRNA is found in the apical periplasm of the syncytial blastoderm.By analysing the distribution of mRNA of a number of hybrid constructs, I show that the 3' untranslated part of three pair-rule genes are required for the apical localisation of their transcripts. A 1.2kb region in the 3' end of fushi tarazu (ftz), a 700bp region in the 3' end of hairy (h) and a 160bp fragment of the 3' untranslated part of the even-skipped (eve) pair-rule gene are shown to contain apical localisation signals.I show that the mechanism of apical localisation is unlikely to involve a cytoplasmic process and that the 3' untranslated part of the bicoid (bed) gene contains sequences necessary for apical localisation. I propose that apical localisation involves a nuclear mechanism which exports mRNA from the apical side of the nuclear membrane. I demonstrate that apical localisation is achieved by an RNA-mediated process and not by a DNA-mediated mechanism.Finally, I demonstrate that the intracellular localisation of transcripts encoding cytoplasmic proteins influences the distribution of the protein in the periplasm. I propose that the function of apical localisation is to limit the diffusion of pair-rule proteins so that the pattern of protein expression resembles precisely the transcriptional domain.</p

A Conserved Motif in Goosecoid Mediates Groucho-Dependent Repression in Drosophila Embryos

8 pages, 5 figures, 1 table.-- PMID: 10022895 [PubMed].-- PMCID: PMC84001.Surprisingly small peptide motifs can confer critical biological functions. One example is the WRPW tetrapeptide present in the Hairy family of transcriptional repressors, which mediates recruitment of the Groucho (Gro) corepressor to target promoters. We recently showed that Engrailed (En) is another repressor that requires association with Gro for its function. En lacks a WRPW motif; instead, it contains another short conserved sequence, the En homology region 1 (eh1)/GEH motif, that is likely to play a role in tethering Gro to the promoter. Here, we characterize a repressor domain from the Goosecoid (Gsc) developmental regulator that includes an eh1/GEH-like motif. We demonstrate that this domain (GscR) mediates efficient repression in Drosophila blastoderm embryos and that repression by GscR requires Gro function. GscR and Gro interact in vitro, and the eh1/GEH motif is necessary and sufficient for the interaction and for in vivo repression. Because WRPW- and eh1/GEH-like motifs are present in different proteins and in many organisms, the results suggest that interactions between short peptides and Gro represent a widespread mechanism of repression. Finally, we investigate whether Gro is part of a stable multiprotein complex in the nucleus. Our results indicate that Gro does not form stable associations with other proteins but that it may be able to assemble into homomultimeric complexes.G.J. was supported by the EC Human Capital and Mobility Programme, the Imperial Cancer Research Fund, and EMBO. This work was supported by the Imperial Cancer Research Fund and a grant from the Howard Hughes Medical Institute through the International Research Scholars Program.Peer reviewe