Biochemical purification and functional characterization of the She RNP complex from S. cerevisiae

Asymmetric mRNA localization is a widely used mechanism to sort cell fate determinants in development. In the budding yeast Saccharomyces cerevisae ASH1 (for 'asymmetric synthesis of HO') mRNA localization to the tip of the growing bud leads to targeting of Ash1p to the daughter cell nucleus prior to cytokinesis and is a prerequisite for proper mating type switching. In a genetic screen 5 SHE (for 'Swi5p-dependent HO expression') genes have been isolated, coding for proteins required for the RNA localization process. SHE1 is equivalent to MYO4, a locus that encodes a member of the class V unconventional myosins. The finding that a motor protein is required for ASH1 mRNA targeting suggested a cytoskeleton-based, active transport mechanism. Functional characterization of the SHE genes in turn established a working model for the 'core She machinery' which implies She1p / Myo4p as the ATP-dependent motor protein, She2p as the ASH1 mRNA-binding protein, and She3p as adapter protein connecting She2p to She1p / Myo4p. She4p has been suggested to function in myosin assembly, whereas She5p is required for cell cycle regulated remodeling of the actin cytoskeleton. In a shared project with C. Kruse we could show that Myo4p trafficking is regulated by the formation of a robust She RNP, and relies on She2p and RNA association. In addition to the She proteins, accessory factors such as Loc1p or Khd1p have been suggested to function in ASH1 mRNA localization though they have not been identified in the genetic screen. Thus, in order to allow a detailed characterization of the ASH1-She RNP ('ribo-nucleoprotein') complex, I initiated a biochemical purification. In order to enrich for the She RNP two myo4p mutants were generated in the myosin ATPase, a domain required for Myo4p force generation. Localization studies reveal that the mutants do not transport ASH1 mRNA anymore to the bud tip but instead accumulate in an intermediate, 'frozen' state in the cytoplasm. Affinity purification was carried out based on the TAP ('tandem affinity purification') protocol, using two alternative bait proteins (She2-TAP or Myo4-TAP). In either case I could purify the core She machinery together with ASH1 mRNA. Further analysis of the She RNP by gel filtration experiments revealed a peak fraction with a molecular weight of approximately 4.5 MDa. Within this fraction I could identify Myo4p, She2p and ASH1 mRNA, arguing for the integrity of a single RNP. In addition to She1-3p mass-spectrometric analysis identified a number of so far unknown proteins, including the kinase Gin4p and the translation inhibitor Eap1p. Eap1p has been of outstanding interest since a systematic RNA localization assay with ash1 mutants that contained premature stop codons inserted at various positions within the coding sequence have revealed severe localization defects, indicating that translation (and translational regulation) is required for correct localization. Eap1p has been characterized as an inhibitor of translation initiation and belongs to the family of 'eukaryotic initiation factor 4E-binding proteins', eIF 4E-BPs. Members of this protein family share a common sequence motif which mediates association with eIF 4E, thereby blocking initiation of translation. Initial studies with D eap1 yeast strains have revealed a defective partial accumulation of Ash1p in mother cell nuclei, whereas ASH1 mRNA localization to the bud tip and total Ash1p levels remained unaffected. This observation prompted me to introduce a new model for ASH1 mRNA localization, including the regulation of translation initiation during cytoplasmic She RNP trafficking to the bud

All-weather avalanche activity monitoring from space?

Information on avalanche activity or on non-activity on local and regional scale is of great value for avalanche warning services, traffic authorities and experts responsible for safety in communities or ski resorts. In particular during bad weather condition, such information is available only very limited or not at all. The aim of ESA IAP feasibility study "Improved Alpine Avalanche Forecast Service" was to investigate existing technology to overcome this gap. Of particular interest were radar-based techniques that have the potential to operate independently of daylight and weather conditions

Ribonucleoprotein-dependent localization of the yeast class V myosin Myo4p

Class V myosins are motor proteins with functions in vesicle transport, organelle segregation, and RNA localization. Although they have been extensively studied, only little is known about the regulation of their spatial distribution. Here we demonstrate that a GFP fusion protein of the budding yeast class V myosin Myo4p accumulates at the bud cortex and is a component of highly dynamic cortical particles. Bud-specific enrichment depends on Myo4p's association with its cargo, a ribonucleoprotein complex containing the RNA-binding protein She2p. Cortical accumulation of Myo4p at the bud tip can be explained by a transient retention mechanism that requires SHE2 and, apparently, localized mRNAs bound to She2p. A mutant She2 protein that is unable to recognize its cognate target mRNA, ASH1, fails to localize Myo4p. Mutant She2p accumulates inside the nucleus, indicating that She2p shuttles between the nucleus and cytoplasm and is exported in an RNA-dependent manner. Consistently, inhibition of nuclear mRNA export results in nuclear accumulation of She2p and cytoplasmic Myo4p mislocalization. Loss of She2p can be complemented by direct targeting of a heterologous lacZ mRNA to a complex of Myo4p and its associated adaptor She3p, suggesting that She2p's function in Myo4p targeting is to link an mRNA to the motor complex

A Novel Multiplex Cell Viability Assay for High-Throughput RNAi Screening

Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%–58.7±14.4% when two indicators were combined and 40–48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost

The Compartmentalized Bacteria of the Planctomycetes-Verrucomicrobia-Chlamydiae Superphylum Have Membrane Coat-Like Proteins

Compartmentalized bacteria have proteins that are structurally related to eukaryotic membrane coats, and one of these proteins localizes at the membrane of vesicles formed inside bacterial cells

Climate in Svalbard 2100

publishedVersio

Hepatitis C strategy in Scotland

This report was commissioned by the Norwegian Environment Agency in order to provide basic information for use in climate change adaptation in Svalbard. It includes descriptions of historical, as well as projections for the future climate development in the atmosphere, hydrosphere, cryosphere and ocean, and it includes effects on the physical nature e.g. on permafrost and various types of landslides and avalanches. The projections for the future climate are based on results in the IPCCs fifth assessment report. The report is to a large degree an assessment of existing literature and model results. New results from atmosphere, ocean and hydrological models are, however, also presented. The report may be downloaded from the Norwegian Centre for Climate Service’s web portal www.klimaservicesenter.no