A pre-ribosomal RNA interaction network involving snoRNAs and the Rok1 helicase
Katherine E. Sloan
Markus T. Bohnsack
- Ribosome biogenesis in yeast requires 75 small nucleolar RNAs (snoRNAs) and a myriad of cofactors for processing, modification, and folding of the ribosomal RNAs (rRNAs). For the 19 RNA helicases implicated in ribosome synthesis, their sites of action and molecular functions have largely remained unknown. Here, we have used UV cross-linking and analysis of cDNA (CRAC) to reveal the pre-rRNA binding sites of the RNA helicase Rok1, which is involved in early small subunit biogenesis. Several contact sites were identified in the 18S rRNA sequence, which interestingly all cluster in the “foot” region of the small ribosomal subunit. These include a major binding site in the eukaryotic expansion segment ES6, where Rok1 is required for release of the snR30 snoRNA. Rok1 directly contacts snR30 and other snoRNAs required for pre-rRNA processing. Using cross-linking, ligation and sequencing of hybrids (CLASH) we identified several novel pre-rRNA base-pairing sites for the snoRNAs snR30, snR10, U3, and U14, which cluster in the expansion segments of the 18S rRNA. Our data suggest that these snoRNAs bridge interactions between the expansion segments, thereby forming an extensive interaction network that likely promotes pre-rRNA maturation and folding in early pre-ribosomal complexes and establishes long-range rRNA interactions during ribosome synthesis.
Reprogramming of tumor cells : signaling events and phenotypes
Chul Min Yang
- Cancer is a disease characterized by uncontrolled cell growth and the capacity to disseminate to distant organs. The properties of cancers are caused by genetic and epigenetic alterations when compared to their normal counterparts. Genetic mutations occur in oncogenes and tumor suppressor genes and are the initial drivers of cellular transformation (Lengauer et al., 1998; Vogelstein and Kinzler, 2004). In addition, epigenetic alterations, which influence the expression of oncogenes and tumor suppressor genes independently from sequence alterations, are also involved in the transformation process (Esteller and Herman, 2001; Sharma et al., 2010). Genetic alterations and epigenetic regulatory signals cooperate in tumor etiology. Glioblastoma multiforme (GBM) is a frequent and aggressive malignant brain tumor in humans. The median survival of GBM patients is about 15 months after diagnosis. Like in other cancers, genetic and epigenetic alterations can be detected in GBM. Genetic alterations in GBM affect cell growth, apoptosis, angiogenesis, and invasion; however, epigenetic alterations in GBM also affect the expression of oncogenes or tumor suppresser genes that increase tumor malignancy (Nagarajan and Costello, 2009).
Reprogramming is a cellular process in which somatic cells can be induced to assume the properties of less differentiated stem cells. This process can be mediated through epigenetic modifications of the genome of somatic cells by the action of four defined transcription factors (Oct4, Sox2, Klf4 and Myc) or by the action of the miR 302/367 cluster (Anokye-Danso et al., 2011; Takahashi and Yamanaka, 2006; Takahashi et al., 2007) and result in the generation of induced pluripotent stem cells (iPS cells). Reprogramming of somatic cells by the miR 302/367 cluster can generate nontumorigenic iPS cells through the inhibition of the epithelial to mesenchymal transition (EMT), cell cycle regulatory genes and epigenetic modifiers (Lin and Ying, 2013).
Polyploidy in haloarchaea: advantages for growth and survival
- The investigated haloarchaeal species, Halobacterium salinarum, Haloferax mediterranei, and H. volcanii, have all been shown to be polyploid. They contain several replicons that have independent copy number regulation, and most have a higher copy number during exponential growth phase than in stationary phase. The possible evolutionary advantages of polyploidy for haloarchaea, most of which have experimental support for at least one species, are discussed. These advantages include a low mutation rate and high resistance toward X-ray irradiation and desiccation, which depend on homologous recombination. For H. volcanii, it has been shown that gene conversion operates in the absence of selection, which leads to the equalization of genome copies. On the other hand, selective forces might lead to heterozygous cells, which have been verified in the laboratory. Additional advantages of polyploidy are survival over geological times in halite deposits as well as at extreme conditions on earth and at simulated Mars conditions. Recently, it was found that H. volcanii uses genomic DNA as genetic material and as a storage polymer for phosphate. In the absence of phosphate, H. volcanii dramatically decreases its genome copy number, thereby enabling cell multiplication, but diminishing the genetic advantages of polyploidy. Stable storage of phosphate is proposed as an alternative driving force for the emergence of DNA in early evolution. Several additional potential advantages of polyploidy are discussed that have not been addressed experimentally for haloarchaea. An outlook summarizes selected current trends and possible future developments.
The reconstruction of evolutionary patterns from daphnia resting egg banks
- In this study I analysed past and recent Daphnia populations from Lake Constance and Greifensee. Herefore, I first established a set of microsatellite markers applicable to European Hyalodaphnia species (chapter 1). Primers were also identified for species specific fragment lengths. 32 markers were then available to characterize the resting egg banks of Daphnia galeata and D. hyalina. Chapter 2 presents the reconstruction of the taxonomic composition in these two ecologically different lakes. This part of my work shows that the eutrophication that occurred in both lakes in the mid of the last century has strongly influenced the Daphnia populations. In both lakes Daphnia galeata established and hybridized with the indigenous D. hyalina. Interspecific hybridization resulted in introgression on the mitochondrial and nuclear level. In chapter 3 resting eggs from the sediments of the 1960s, 1970s, 1980s, 1990s and 2000s were characterized with microsatellite markers. The aim was to specify the extent of interspecific hybridization and nuclear introgression assuming that the genetic exchange between both species has an impact on their adaptation to their habitat. In life history experiments D. galeata and D. galeata x hyalina clones hatched from different time periods showed significant differential responses to food quality. Therefore, the question had to be answered how the Daphnia resting egg bank and the planktonic population are connected. In chapter 4 hatching experiments were conducted to bridge this gap of scientific knowledge in the life cycle of cyclic parthenogenetic waterfleas. Only D. galeata individuals were able to establish a clonal lineage after maturity. All observed recombinant individuals did not reproduce at all or firstly went through another sexual phase of reproduction i.e. produced resting eggs. In order to compare the findings of chapter 4 with the taxon composition of the recent planktonic population of Daphnia in Lake Constance, samples were taken over one season (between May 2005 and September 2006). During the season, the taxonomic composition of Daphnia changes severely with D. galeata being most abundant during the warm season and D. hyalina in the cold season. Moreover, some individuals were detected, that did not follow this pattern. With mitochondrial analysis those individuals were identified as mitochondrial introgressants and processed to life history experiments. Significant differences in the somatic growth rate under different temperatures (5°C, 12.5°C and 20°C) were related to the origin of the mitochondrial genome rather than the nuclear taxonomic assignment of the individual.
The findings of this study show that all organisms exposed to rapid ecological changes and their microevolutionary reaction to those.
Are In Vitro Methods for the Detection of Endocrine Potentials in the Aquatic Environment Predictive for In Vivo Effects? Outcomes of the Projects SchussenAktiv and SchussenAktivplus in the Lake Constance Area, Germany
- Many studies about endocrine pollution in the aquatic environment reveal changes in the reproduction system of biota. We analysed endocrine activities in two rivers in Southern Germany using three approaches: (1) chemical analyses, (2) in vitro bioassays, and (3) in vivo investigations in fish and snails. Chemical analyses were based on gas chromatography coupled with mass spectrometry. For in vitro analyses of endocrine potentials in water, sediment, and waste water samples, we used the E-screen assay (human breast cancer cells MCF-7) and reporter gene assays (human cell line HeLa-9903 and MDA-kb2). In addition, we performed reproduction tests with the freshwater mudsnail Potamopyrgus antipodarum to analyse water and sediment samples. We exposed juvenile brown trout (Salmo trutta f. fario) to water downstream of a wastewater outfall (Schussen River) or to water from a reference site (Argen River) to investigate the vitellogenin production. Furthermore, two feral fish species, chub (Leuciscus cephalus) and spirlin (Alburnoides bipunctatus), were caught in both rivers to determine their gonadal maturity and the gonadosomatic index. Chemical analyses provided only little information about endocrine active substances, whereas the in vitro assays revealed endocrine potentials in most of the samples. In addition to endocrine potentials, we also observed toxic potentials (E-screen/reproduction test) in waste water samples, which could interfere with and camouflage endocrine effects. The results of our in vivo tests were mostly in line with the results of the in vitro assays and revealed a consistent reproduction-disrupting (reproduction tests) and an occasional endocrine action (vitellogenin levels) in both investigated rivers, with more pronounced effects for the Schussen river (e.g. a lower gonadosomatic index). We were able to show that biological in vitro assays for endocrine potentials in natural stream water reasonably reflect reproduction and endocrine disruption observed in snails and field-exposed fish, respectively.
Function of flotillins in Alzheimer disease and apoptosis
Bincy Anu John
- Alzheimer’s disease (AD) is a common, age associated neurodegenerative disease that manifests as progressive dementia and is characterized by accumulation of the amyloid beta (Aβ) peptide which is a processing product of a transmembrane protein termed Alzheimer Amyloid Precursor Protein (APP). The Aβ peptide is generated by a sequential proteolytic processing of APP by two distinct proteases that are termed β- and γ-secretase. The β-secretase, also called BACE-1 or memapsin 2, belongs to the family of aspartyl proteases. BACE-1 evidently cleaves APP in an acidic endosomal compartment after endocytosis of APP, thereby facilitating Aβ peptide generation.
Sorting of transmembrane proteins is generally controlled by sorting signals in the cytoplasmic domains of the cargo proteins. The short cytoplasmic tail of BACE-1 with 23 amino acids contains a sorting signal of the acidic cluster, di-leucine (ACDL) type. The two Leu residues in this determinant are important for the clathrin mediated endocytosis of BACE-1, whereas the acidic residues together with the Leu are required for the endosomal sorting and recycling of BACE-1 back to the plasma membrane. The ACDL motif binds to the members of the GGA (Golgi-localized γ ear-containg ARF- binding proteins) family (GGA1-GGA3) that are involved in the sorting of BACE-1.
One of the major aims of this study was to address the role of flotillins in the intracellular sorting of BACE-1. This study shows that flotillin-1 directly binds to the di-leucine motif in the cytoplasmic tail of BACE-1, whereas flotillin-2 only shows an association mediated by flotillin-1. Flotillin-1 competes with GGA2 for the binding to BACE-1 tail, and thus influences the endosomal sorting of BACE-1. Importantly, depletion of flotillins results in an altered localization of the wildtype BACE-1, whereas the plasma membrane resident Leu to Ala (LLAA) mutant is not affected. Flotillin knockdown results in an accumulation of BACE-1, implicating reduced degradation and enhanced stability of this protease. Thus, flotillins appear to be important for the cellular targeting of BACE-1 and also influence the amyloidogenic processing of APP, as demonstrated by an increase in the amyloidogenic C-99 processing fragments.
When flotillin depleted cells were subjected to apoptotic stresses including Aβ25-35 synthetic peptide (inducer of the extrinsic apoptosis pathway) or several chemotherapeutic agents (staurosporine, brefeldin A, doxorubicin, carboplatin and paclitaxel: intrinsic apoptosis pathway) and cytotoxicity was determined, various apoptotic markers were activated in flotillin depleted cells. Caspase-3 and GGA3 are well accepted apoptosis markers and an enhanced caspase-3 cleavage was detected upon STS induced apoptosis in SH-SY5Y, HeLa, and HaCaT cell lines and increased GGA3 cleavage was observed in MCF7 cell line.
One of the major reasons for the apoptotic sensitivity in the absence of flotillins was a PI3K/Akt signaling defect. Neuroblastoma cells depleted of flotillins showed diminished levels of total Akt, phospho-Akt and phospho-ERK upon STS induced apoptosis. Since PI3K/Akt was the primary survival pathway affected upon STS induced apoptosis, ectopic expression of Akt in neuroblastoma cell line reduced caspase-3 cleavage and retarded apoptosis.
The direct downstream target of Akt is FOXO3a, whose localization was investigated in flotillin depleted cells. A major proportion of FOXO3a was localized in the nucleus of flotillin knockdown cells, implicating that FOXOs are active in these cells and subsequently trigger the transcription of death genes. Strikingly, an essential anti-apoptotic molecule and a major cancer target, Mcl-1, was inherently downregulated in flotillin knockdown cells. Mcl-1 is a chief member of the Bcl-2 family as it plays a pivotal role in cell survival and it is a critical protein in cancer therapeutics as suppression of Mcl-1 protein can curtail the survival and growth of tumorous cells.
Neuroblastoma cells were rescued from undergoing permanent damage due to STS induced apoptosis by overexpression of anti-apoptotic Bcl-2. Phorbol esters are well known PKC activators, and pre-treatment of neuroblastoma cells with phorbol esters along with staurosporine reduced caspase-3 cleavage.
These results demonstrate that absence of flotillins can sensitize cellular systems to apoptosis induction. The two main characteristics of cancer cells include resistance to apoptosis and unresponsiveness to chemotherapeutic agents. It is a well established fact that impaired apoptosis is central to tumour development. This study implicates that the downregulation of flotillin function can trigger cellular susceptibility and enhances apoptosis in response to conventional chemotherapeutic agents. Therefore, flotillins can serve as vital regulators in providing a more rational approach in molecular-targeted therapies for receding cancer growth and survival.
Ordnung des Fachbereichs Biowissenschaften der Johann Wolfgang Goethe-Universität für den Masterstudiengang Ökologie und Evolution mit dem Abschluss "Master of Science" (M.Sc.) vom 16. Juni 2009; zuletzt geändert am 4. Juni 2012 : genehmigt durch das Präsidium am 27. April 2012 ; hier: Änderung der Wahlpflichtmodule vom 24. Februar 2014 ; genehmigt durch das Präsidium am 25. März 2014
Ordnung der Fachbereiche Medizin, Biowissenschaften, Biochemie, Chemie und Pharmazie sowie Psychologie und Sportwissenschaften der Johann Wolfgang Goethe-Universität für den Masterstudiengang Interdisciplinary Neuroscience mit dem Abschluss "Master of Science" (M.Sc.) vom 30. März 2009 in der Fassung vom 22. April 2009 : genehmigt durch das Präsidium am 11. Oktober 2011 ; hier: Änderung der Wahlpflichtmodule vom 20. Januar 2014, genehmigt durch das Präsidium am 25. März 2014
Range-Wide Latitudinal and Elevational Temperature Gradients for the World's Terrestrial Birds: Implications under Global Climate Change
Frank A. La Sorte
Stuart H. M. Butchart
- Species' geographical distributions are tracking latitudinal and elevational surface temperature gradients under global climate change. To evaluate the opportunities to track these gradients across space, we provide a first baseline assessment of the steepness of these gradients for the world's terrestrial birds. Within the breeding ranges of 9,014 bird species, we characterized the spatial gradients in temperature along latitude and elevation for all and a subset of bird species, respectively. We summarized these temperature gradients globally for threatened and non-threatened species and determined how their steepness varied based on species' geography (range size, shape, and orientation) and projected changes in temperature under climate change. Elevational temperature gradients were steepest for species in Africa, western North and South America, and central Asia and shallowest in Australasia, insular IndoMalaya, and the Neotropical lowlands. Latitudinal temperature gradients were steepest for extratropical species, especially in the Northern Hemisphere. Threatened species had shallower elevational gradients whereas latitudinal gradients differed little between threatened and non-threatened species. The strength of elevational gradients was positively correlated with projected changes in temperature. For latitudinal gradients, this relationship only held for extratropical species. The strength of latitudinal gradients was better predicted by species' geography, but primarily for extratropical species. Our findings suggest threatened species are associated with shallower elevational temperature gradients, whereas steep latitudinal gradients are most prevalent outside the tropics where fewer bird species occur year-round. Future modeling and mitigation efforts would benefit from the development of finer grain distributional data to ascertain how these gradients are structured within species' ranges, how and why these gradients vary among species, and the capacity of species to utilize these gradients under climate change.
The Complexity of Vesicle Transport Factors in Plants Examined by Orthology Search
- Vesicle transport is a central process to ensure protein and lipid distribution in eukaryotic cells. The current knowledge on the molecular components and mechanisms of this process is majorly based on studies in Saccharomyces cerevisiae and Arabidopsis thaliana, which revealed 240 different proteinaceous factors either experimentally proven or predicted to be involved in vesicle transport. In here, we performed an orthologue search using two different algorithms to identify the components of the secretory pathway in yeast and 14 plant genomes by using the ‘core-set’ of 240 factors as bait. We identified 4021 orthologues and (co-)orthologues in the discussed plant species accounting for components of COP-II, COP-I, Clathrin Coated Vesicles, Retromers and ESCRTs, Rab GTPases, Tethering factors and SNAREs. In plants, we observed a significantly higher number of (co-)orthologues than yeast, while only 8 tethering factors from yeast seem to be absent in the analyzed plant genomes. To link the identified (co-)orthologues to vesicle transport, the domain architecture of the proteins from yeast, genetic model plant A. thaliana and agriculturally relevant crop Solanum lycopersicum has been inspected. For the orthologous groups containing (co-)orthologues from yeast, A. thaliana and S. lycopersicum, we observed the same domain architecture for 79% (416/527) of the (co-)orthologues, which documents a very high conservation of this process. Further, publically available tissue-specific expression profiles for a subset of (co-)orthologues found in A. thaliana and S. lycopersicum suggest that some (co-)orthologues are involved in tissue-specific functions. Inspection of localization of the (co-)orthologues based on available proteome data or localization predictions lead to the assignment of plastid- as well as mitochondrial localized (co-)orthologues of vesicle transport factors and the relevance of this is discussed.