Drosophila CPEB Orb2A Mediates Memory Independent of Its RNA-Binding Domain

SummaryLong-term memory and synaptic plasticity are thought to require the synthesis of new proteins at activated synapses. The CPEB family of RNA binding proteins, including Drosophila Orb2, has been implicated in this process. The precise mechanism by which these molecules regulate memory formation is however poorly understood. We used gene targeting and site-specific transgenesis to specifically modify the endogenous orb2 gene in order to investigate its role in long-term memory formation. We show that the Orb2A and Orb2B isoforms, while both essential, have distinct functions in memory formation. These two isoforms have common glutamine-rich and RNA-binding domains, yet Orb2A uniquely requires the former and Orb2B the latter. We further show that Orb2A induces Orb2 complexes in a manner dependent upon both its glutamine-rich region and neuronal activity. We propose that Orb2B acts as a conventional CPEB to regulate transport and/or translation of specific mRNAs, whereas Orb2A acts in an unconventional manner to form stable Orb2 complexes that are essential for memory to persist

Aurora A phosphorylation of TACC3/maskin is required for centrosome-dependent microtubule assembly in mitosis

Centrosomes act as sites of microtubule growth, but little is known about how the number and stability of microtubules emanating from a centrosome are controlled during the cell cycle. We studied the role of the TACC3–XMAP215 complex in this process by using purified proteins and Xenopus laevis egg extracts. We show that TACC3 forms a one-to-one complex with and enhances the microtubule-stabilizing activity of XMAP215 in vitro. TACC3 enhances the number of microtubules emanating from mitotic centrosomes, and its targeting to centrosomes is regulated by Aurora A–dependent phosphorylation. We propose that Aurora A regulation of TACC3 activity defines a centrosome-specific mechanism for regulation of microtubule polymerization in mitosis

The expression, localisation and interactome of pigeon CRY2

Cryptochromes (CRY) are highly conserved signalling molecules that regulate circadian rhythms and are candidate radical pair based magnetoreceptors. Birds have at least four cryptochromes (CRY1a, CRY1b, CRY2, and CRY4), but few studies have interrogated their function. Here we investigate the expression, localisation and interactome of clCRY2 in the pigeon retina. We report that clCRY2 has two distinct transcript variants, clCRY2a, and a previously unreported splice isoform, clCRY2b which is larger in size. We show that clCRY2a mRNA is expressed in all retinal layers and clCRY2b is enriched in the inner and outer nuclear layer. To define the localisation and interaction network of clCRY2 we generated and validated a monoclonal antibody that detects both clCRY2 isoforms. Immunohistochemical studies revealed that clCRY2a/b is present in all retinal layers and is enriched in the outer limiting membrane and outer plexiform layer. Proteomic analysis showed clCRY2a/b interacts with typical circadian molecules (PER2, CLOCK, ARTNL), cell junction proteins (CTNNA1, CTNNA2) and components associated with the microtubule motor dynein (DYNC1LI2, DCTN1, DCTN2, DCTN3) within the retina. Collectively these data show that clCRY2 is a component of the avian circadian clock and unexpectedly associates with the microtubule cytoskeleton

Genotype, phenotype and disease severity reflected by serum LysoGb3 levels in patients with Fabry disease

Background: Fabry disease (FD) is a rare X-linked lysosomal storage disease caused by mutations in the α-galactosidase A (GLA) gene causing deficiency of α-galactosidase A which results in progressive glycosphingolipid accumulation, especially globotriaosylceramide (Gb3), in body liquids and lysosomes. In a large cohort of FD patients, we aimed to establish genotype/phenotype relations as indicated by serum LysoGb3 (deacylated Gb3). / Methods: In 69 consecutive adult FD patients (males: n = 28 (41%)) with a GLA-mutation confirmed diagnosis, we conducted a multidisciplinary clinical characterization during their routine annual examinations, and measured serum LysoGb3 levels by high-sensitive electrospray ionization liquid chromatography tandem mass spectrometry. / Results: Serum levels of LysoGb3 were significantly higher in Classic compared with Later-Onset phenotype and higher in the latter compared with controls, both in males (52 [40–83] vs 9.5 [4.5–20] vs 0.47 [0.41–0.61] ng/ml, P < 0.001) and in females (9.9 [7.9–14] vs 4.9 [1.6–4.9] vs 0.41 [0.33–0.48] ng/ml, P < 0.001), respectively. Multivariate linear regression analysis showed that LysoGb3 levels were independently associated with, serum creatinine (β = 0.09, 95%CI 0.04–0.13, P < 0.001) and the presence of cardiomyopathy (β = 25, 95%CI 9.8–41, P = 0.002). LysoGb3 levels were higher in males with frame-shift and nonsense mutations than in males with missense mutations (84 [72–109] vs 41 [37–52] ng/ml, P = 0.002). / Conclusion: LysoGb3 relates to disease severity, enzyme replacement response, and to the genotype severity in males. LysoGb3 supports identifying patients at risk who require intensive monitoring and treatment. LysoGb3 appears to be one marker of metabolic phenotyping of FD

Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand

We have constructed an artificial ligand for the hepatocyte-specific asialoglycoprotein receptor for the purpose of generating a synthetic delivery system for DNA. This ligand has a tetra-antennary structure, containing four terminal galactose residues on a branched carrier peptide. The carbohydrate residues of this glycopeptide were introduced by reductive coupling of lactose to the alpha- and epsilon-amino groups of the two N-terminal lysines on the carrier peptide. The C-terminus of the peptide, containing a cysteine separated from the branched N-terminus by a 10 amino acid spacer sequence, was used for conjugation to 3-(2-pyridyldithio)propionate-modified polylysine via disulfide bond formation. Complexes containing plasmid DNA bound to these galactose-polylysine conjugates have been used for asialoglycoprotein receptor-mediated transfer of a luciferase gene into human (HepG2) and murine (BNL CL.2) hepatocyte cell lines. Gene transfer was strongly promoted when amphipathic peptides with pH-controlled membrane-disruption activity, derived from the N-terminal sequence of influenza virus hemagglutinin HA-2, were also present in these DNA complexes. Thus, we have essentially borrowed the small functional domains of two large proteins, asialoglycoprotein and hemagglutinin, and assembled them into a supramolecular complex to generate an efficient gene-transfer system

Absolute quantification of cohesin, CTCF and their regulators in human cells.

The organisation of mammalian genomes into loops and topologically associating domains (TADs) contributes to chromatin structure, gene expression and recombination. TADs and many loops are formed by cohesin and positioned by CTCF. In proliferating cells, cohesin also mediates sister chromatid cohesion, which is essential for chromosome segregation. Current models of chromatin folding and cohesion are based on assumptions of how many cohesin and CTCF molecules organise the genome. Here we have measured absolute copy numbers and dynamics of cohesin, CTCF, NIPBL, WAPL and sororin by mass spectrometry, fluorescence-correlation spectroscopy and fluorescence recovery after photobleaching in HeLa cells. In G1-phase, there are similar to 250,000 nuclear cohesin complexes, of which similar to 160,000 are chromatin-bound. Comparison with chromatin immunoprecipitation-sequencing data implies that some genomic cohesin and CTCF enrichment sites are unoccupied in single cells at any one time. We discuss the implications of these findings for how cohesin can contribute to genome organisation and cohesion

Österreichisches Projekt Grundlagen zur Züchtung, Vermehrung und Sorten-/Saatgutprüfung für den Biolandbau: Ergebnisübersicht

In the Austrian research project “Basic principles for breeding, multiplication and variety testing for organic agriculture” (from 2004 until 2008) novel methods for the assessment of the suitability of seed and cultivars for organic farming were developed and evaluated by an interdisciplinary co-operation of researchers, breeders and variety testers. Organic farming requires specific combinations of crop plant characters, especially seed health and resistance against seed-borne diseases. Another crucial feature is the competitive ability against weeds. Early development was found to significantly increase the competitiveness of different crop plants. Genotypic variation in characters necessary for an efficient utilisation of below-ground resources was also investigated, e.g. interrelations between root development and drought stress tolerance and between mycorrhiza formation and nutrient use efficiency. Based on the results obtained in various cultivar trials, it can be concluded that genotypes suitable for organic growing conditions may be pre-selected from early breeding material of conventional breeding programmes. The selected breeding material must subsequently be rigorously tested on organically managed fields starting from the first yield trials at the latest. The project results were communicated to farmers and advisors during field days and excursions. They will be applied in the breeding of new cultivars

PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay

Lesson from the Stoichiometry Determination of the Cohesin Complex: A Short Protease Mediated Elution Increases the Recovery from Cross-Linked Antibody-Conjugated Beads

Affinity purification of proteins using antibodies coupled to beads and subsequent mass spectrometric analysis has become a standard technique for the identification of protein complexes. With the recent transfer of the isotope dilution mass spectrometry principle (IDMS) to the field of proteomics, quantitative analysesssuch as the stoichiometry determination of protein complexesshave become achievable. Traditionally proteins were eluted from antibody-conjugated beads using glycine at low pH or using diluted acids such as HCl, TFA, or FA, but elution was often found to be incomplete. Using the cohesin complex and the anaphase promoting complex/cyclosome (APC/C) as examples, we show that a short 15-60 min predigestion with a protease such as LysC (modified on-bead digest termed protease elution) increases the elution efficiency 2- to 3-fold compared to standard acid elution protocols. While longer incubation periodssas performed in standard on-bead digestionsled to partial proteolysis of the cross-linked antibodies, no or only insignificant cleavage was observed after 15-60 min protease mediated elution. Using the protease elution method, we successfully determined the stoichiometry of the cohesin complex by absolute quantification of the four core subunits using LC-SRM analysis and 19 reference peptides generated with the EtEP strategy. Protease elution was 3-fold more efficient compared to HCl elution, but measurements using both elution techniques are in agreement with