Proteomic Changes Resulting from Gene Copy Number Variations in Cancer Cells

Along the transformation process, cells accumulate DNA aberrations, including mutations, translocations, amplifications, and deletions. Despite numerous studies, the overall effects of amplifications and deletions on the end point of gene expression—the level of proteins—is generally unknown. Here we use large-scale and high-resolution proteomics combined with gene copy number analysis to investigate in a global manner to what extent these genomic changes have a proteomic output and therefore the ability to affect cellular transformation. We accurately measure expression levels of 6,735 proteins and directly compare them to the gene copy number. We find that the average effect of these alterations on the protein expression is only a few percent. Nevertheless, by using a novel algorithm, we find the combined impact that many of these regional chromosomal aberrations have at the protein level. We show that proteins encoded by amplified oncogenes are often overexpressed, while adjacent amplified genes, which presumably do not promote growth and survival, are attenuated. Furthermore, regulation of biological processes and molecular complexes is independent of general copy number changes. By connecting the primary genome alteration to their proteomic consequences, this approach helps to interpret the data from large-scale cancer genomics efforts

An shRNA-Based Screen of Splicing Regulators Identifies SFRS3 as a Negative Regulator of IL-1β Secretion

The generation of diversity and plasticity of transcriptional programs are key components of effective vertebrate immune responses. The role of Alternative Splicing has been recognized, but it is underappreciated and poorly understood as a critical mechanism for the regulation and fine-tuning of physiological immune responses. Here we report the generation of loss-of-function phenotypes for a large collection of genes known or predicted to be involved in the splicing reaction and the identification of 19 novel regulators of IL-1β secretion in response to E. coli challenge of THP-1 cells. Twelve of these genes are required for IL-1β secretion, while seven are negative regulators of this process. Silencing of SFRS3 increased IL-1β secretion due to elevation of IL-1β and caspase-1 mRNA in addition to active caspase-1 levels. This study points to the relevance of splicing in the regulation of auto-inflammatory diseases

Biosorption and Biomineralization of U(VI) by the Marine Bacterium Idiomarina loihiensis MAH1: Effect of Background Electrolyte and pH

The main goal of this study is to compare the effects of pH, uranium concentration, and background electrolyte (seawater and NaClO4 solution) on the speciation of uranium(VI) associated with the marine bacterium Idiomarina loihiensis MAH1. This was done at the molecular level using a multidisciplinary approach combining X-ray Absorption Spectroscopy (XAS), Time-Resolved Laser-Induced Fluorescence Spectroscopy (TRLFS), and High Resolution Transmission Electron Microscopy (HRTEM). We showed that the U(VI)/bacterium interaction mechanism is highly dependent upon pH but also the nature of the used background electrolyte played a role. At neutral conditions and a U concentration ranging from 5·10−4 to 10−5 M (environmentally relevant concentrations), XAS analysis revealed that uranyl phosphate mineral phases, structurally resembling meta-autunite [Ca(UO2)2(PO4)2 2–6H2O] are precipitated at the cell surfaces of the strain MAH1. The formation of this mineral phase is independent of the background solution but U(VI) luminescence lifetime analyses demonstrated that the U(VI) speciation in seawater samples is more intricate, i.e., different complexes were formed under natural conditions. At acidic conditions, pH 2, 3 and 4.3 ([U] = 5·10−4 M, background electrolyte = 0.1 M NaClO4), the removal of U from solution was due to biosorption to Extracellular Polysaccharides (EPS) and cell wall components as evident from TEM analysis. The LIII-edge XAS and TRLFS studies showed that the biosorption process observed is dependent of pH. The bacterial cell forms a complex with U through organic phosphate groups at pH 2 and via phosphate and carboxyl groups at pH 3 and 4.3, respectively. The differences in the complexes formed between uranium and bacteria on seawater compared to NaClO4 solution demonstrates that the actinide/microbe interactions are influenced by the three studied factors, i.e., the pH, the uranium concentration and the chemical composition of the solution.This work was funded by the grants CGL2009-09760 and CGL2012-36505 (Ministerio de Ciencia e Innovación), and RNM 3943 (Junta de Andalucía), Spain

Nitrogen and Carbon Isotopic Dynamics of Subarctic Soils and Plants in Southern Yukon Territory and its Implications for Paleoecological and Paleodietary Studies

We examine here the carbon and nitrogen isotopic compositions of bulk soils (8 topsoil and 7 subsoils, including two soil profiles) and five different plant parts of 79 C3 plants from two main functional groups: herbs and shrubs/subshrubs, from 18 different locations in grasslands of southern Yukon Territory, Canada (eastern shoreline of Kluane Lake and Whitehorse area). The Kluane Lake region in particular has been identified previously as an analogue for Late Pleistocene eastern Beringia. All topsoils have higher average total nitrogen δ15N and organic carbon δ13C than plants from the same sites with a positive shift occurring with depth in two soil profiles analyzed. All plants analyzed have an average whole plant δ13C of −27.5 ± 1.2 ‰ and foliar δ13C of ±28.0 ± 1.3 ‰, and average whole plant δ15N of −0.3 ± 2.2 ‰ and foliar δ15N of ±0.6 ± 2.7 ‰. Plants analyzed here showed relatively smaller variability in δ13C than δ15N. Their average δ13C after suitable corrections for the Suess effect should be suitable as baseline for interpreting diets of Late Pleistocene herbivores that lived in eastern Beringia. Water availability, nitrogen availability, spacial differences and intra-plant variability are important controls on δ15N of herbaceous plants in the study area. The wider range of δ15N, the more numerous factors that affect nitrogen isotopic composition and their likely differences in the past, however, limit use of the modern N isotopic baseline for vegetation in paleodietary models for such ecosystems. That said, the positive correlation between foliar δ15N and N content shown for the modern plants could support use of plant δ15N as an index for plant N content and therefore forage quality. The modern N isotopic baseline cannot be applied directly to the past, but it is prerequisite to future efforts to detect shifts in N cycling and forage quality since the Late Pleistocene through comparison with fossil plants from the same region

Rad51 and DNA-PKcs are involved in the generation of specific telomere aberrations induced by the quadruplex ligand 360A that impair mitotic cell progression and lead to cell death

Functional telomeres are protected from non-homologous end-joining (NHEJ) and homologous recombination (HR) DNA repair pathways. Replication is a critical period for telomeres because of the requirement for reconstitution of functional protected telomere conformations, a process that involves DNA repair proteins. Using knockdown of DNA-PKcs and Rad51 expression in three different cell lines, we demonstrate the respective involvement of NHEJ and HR in the formation of telomere aberrations induced by the G-quadruplex ligand 360A during or after replication. HR contributed to specific chromatid-type aberrations (telomere losses and doublets) affecting the lagging strand telomeres, whereas DNA-PKcs-dependent NHEJ was responsible for sister telomere fusions as a direct consequence of G-quadruplex formation and/or stabilization induced by 360A on parental telomere G strands. NHEJ and HR activation at telomeres altered mitotic progression in treated cells. In particular, NHEJ-mediated sister telomere fusions were associated with altered metaphase-anaphase transition and anaphase bridges and resulted in cell death during mitosis or early G1. Collectively, these data elucidate specific molecular and cellular mechanisms triggered by telomere targeting by the G-quadruplex ligand 360A, leading to cancer cell death

Systematic Two-Hybrid and Comparative Proteomic Analyses Reveal Novel Yeast Pre-mRNA Splicing Factors Connected to Prp19

Prp19 is the founding member of the NineTeen Complex, or NTC, which is a spliceosomal subcomplex essential for spliceosome activation. To define Prp19 connectivity and dynamic protein interactions within the spliceosome, we systematically queried the Saccharomyces cerevisiae proteome for Prp19 WD40 domain interaction partners by two-hybrid analysis. We report that in addition to S. cerevisiae Cwc2, the splicing factor Prp17 binds directly to the Prp19 WD40 domain in a 1∶1 ratio. Prp17 binds simultaneously with Cwc2 indicating that it is part of the core NTC complex. We also find that the previously uncharacterized protein Urn1 (Dre4 in Schizosaccharomyces pombe) directly interacts with Prp19, and that Dre4 is conditionally required for pre-mRNA splicing in S. pombe. S. pombe Dre4 and S. cerevisiae Urn1 co-purify U2, U5, and U6 snRNAs and multiple splicing factors, and dre4Δ and urn1Δ strains display numerous negative genetic interactions with known splicing mutants. The S. pombe Prp19-containing Dre4 complex co-purifies three previously uncharacterized proteins that participate in pre-mRNA splicing, likely before spliceosome activation. Our multi-faceted approach has revealed new low abundance splicing factors connected to NTC function, provides evidence for distinct Prp19 containing complexes, and underscores the role of the Prp19 WD40 domain as a splicing scaffold

Metabolism-dependent bioaccumulation of uranium by Rhodosporidium toruloides isolated from the flooding water of a former uranium mine

Remediation of former uranium mining sites represents one of the biggest challenges worldwide that have to be solved in this century. During the last years, the search of alternative strategies involving environmentally sustainable treatments has started. Bioremediation, the use of microorganisms to clean up polluted sites in the environment, is considered one the best alternative. By means of culture-dependent methods, we isolated an indigenous yeast strain, KS5 (Rhodosporidium toruloides), directly from the flooding water of a former uranium mining site and investigated its interactions with uranium. Our results highlight distinct adaptive mechanisms towards high uranium concentrations on the one hand, and complex interaction mechanisms on the other. The cells of the strain KS5 exhibit high a uranium tolerance, being able to grow at 6 mM, and also a high ability to accumulate this radionuclide (350 mg uranium/g dry biomass, 48 h). The removal of uranium by KS5 displays a temperature- and cell viability-dependent process, indicating that metabolic activity could be involved. By STEM (scanning transmission electron microscopy) investigations, we observed that uranium was removed by two mechanisms, active bioaccumulation and inactive biosorption. This study highlights the potential of KS5 as a representative of indigenous species within the flooding water of a former uranium mine, which may play a key role in bioremediation of uranium contaminated sites.This work was supported by the Bundesministerium für Bildung und Forschung grand nº 02NUK030F (TransAqua). Further support took place by the ERDF-co-financed Grants CGL2012-36505 and 315 CGL2014-59616R, Ministerio de Ciencia e Innovación, Spain

A global reference for human genetic variation

The 1000 Genomes Project set out to provide a comprehensive description of common human genetic variation by applying whole-genome sequencing to a diverse set of individuals from multiple populations. Here we report completion of the project, having reconstructed the genomes of 2,504 individuals from 26 populations using a combination of low-coverage whole-genome sequencing, deep exome sequencing, and dense microarray genotyping. We characterized a broad spectrum of genetic variation, in total over 88 million variants (84.7 million single nucleotide polymorphisms (SNPs), 3.6 million short insertions/deletions (indels), and 60,000 structural variants), all phased onto high-quality haplotypes. This resource includes >99% of SNP variants with a frequency of >1% for a variety of ancestries. We describe the distribution of genetic variation across the global sample, and discuss the implications for common disease studies.We thank the many people who were generous with contributing their samples to the project: the African Caribbean in Barbados; Bengali in Bangladesh; British in England and Scotland; Chinese Dai in Xishuangbanna, China; Colombians in Medellin, Colombia; Esan in Nigeria; Finnish in Finland; Gambian in Western Division – Mandinka; Gujarati Indians in Houston, Texas, USA; Han Chinese in Beijing, China; Iberian populations in Spain; Indian Telugu in the UK; Japanese in Tokyo, Japan; Kinh in Ho Chi Minh City, Vietnam; Luhya in Webuye, Kenya; Mende in Sierra Leone; people with African ancestry in the southwest USA; people with Mexican ancestry in Los Angeles, California, USA; Peruvians in Lima, Peru; Puerto Ricans in Puerto Rico; Punjabi in Lahore, Pakistan; southern Han Chinese; Sri Lankan Tamil in the UK; Toscani in Italia; Utah residents (CEPH) with northern and western European ancestry; and Yoruba in Ibadan, Nigeria. Many thanks to the people who contributed to this project: P. Maul, T. Maul, and C. Foster; Z. Chong, X. Fan, W. Zhou, and T. Chen; N. Sengamalay, S. Ott, L. Sadzewicz, J. Liu, and L. Tallon; L. Merson; O. Folarin, D. Asogun, O. Ikpwonmosa, E. Philomena, G. Akpede, S. Okhobgenin, and O. Omoniwa; the staff of the Institute of Lassa Fever Research and Control (ILFRC), Irrua Specialist Teaching Hospital, Irrua, Edo State, Nigeria; A. Schlattl and T. Zichner; S. Lewis, E. Appelbaum, and L. Fulton; A. Yurovsky and I. Padioleau; N. Kaelin and F. Laplace; E. Drury and H. Arbery; A. Naranjo, M. Victoria Parra, and C. Duque; S. Däkel, B. Lenz, and S. Schrinner; S. Bumpstead; and C. Fletcher-Hoppe. Funding for this work was from the Wellcome Trust Core Award 090532/Z/09/Z and Senior Investigator Award 095552/Z/11/Z (P.D.), and grants WT098051 (R.D.), WT095908 and WT109497 (P.F.), WT086084/Z/08/Z and WT100956/Z/13/Z (G.M.), WT097307 (W.K.), WT0855322/Z/08/Z (R.L.), WT090770/Z/09/Z (D.K.), the Wellcome Trust Major Overseas program in Vietnam grant 089276/Z.09/Z (S.D.), the Medical Research Council UK grant G0801823 (J.L.M.), the UK Biotechnology and Biological Sciences Research Council grants BB/I02593X/1 (G.M.) and BB/I021213/1 (A.R.L.), the British Heart Foundation (C.A.A.), the Monument Trust (J.H.), the European Molecular Biology Laboratory (P.F.), the European Research Council grant 617306 (J.L.M.), the Chinese 863 Program 2012AA02A201, the National Basic Research program of China 973 program no. 2011CB809201, 2011CB809202 and 2011CB809203, Natural Science Foundation of China 31161130357, the Shenzhen Municipal Government of China grant ZYC201105170397A (J.W.), the Canadian Institutes of Health Research Operating grant 136855 and Canada Research Chair (S.G.), Banting Postdoctoral Fellowship from the Canadian Institutes of Health Research (M.K.D.), a Le Fonds de Recherche duQuébec-Santé (FRQS) research fellowship (A.H.), Genome Quebec (P.A.), the Ontario Ministry of Research and Innovation – Ontario Institute for Cancer Research Investigator Award (P.A., J.S.), the Quebec Ministry of Economic Development, Innovation, and Exports grant PSR-SIIRI-195 (P.A.), the German Federal Ministry of Education and Research (BMBF) grants 0315428A and 01GS08201 (R.H.), the Max Planck Society (H.L., G.M., R.S.), BMBF-EPITREAT grant 0316190A (R.H., M.L.), the German Research Foundation (Deutsche Forschungsgemeinschaft) Emmy Noether Grant KO4037/1-1 (J.O.K.), the Beatriu de Pinos Program grants 2006 BP-A 10144 and 2009 BP-B 00274 (M.V.), the Spanish National Institute for Health Research grant PRB2 IPT13/0001-ISCIII-SGEFI/FEDER (A.O.), Ewha Womans University (C.L.), the Japan Society for the Promotion of Science Fellowship number PE13075 (N.P.), the Louis Jeantet Foundation (E.T.D.), the Marie Curie Actions Career Integration grant 303772 (C.A.), the Swiss National Science Foundation 31003A_130342 and NCCR “Frontiers in Genetics” (E.T.D.), the University of Geneva (E.T.D., T.L., G.M.), the US National Institutes of Health National Center for Biotechnology Information (S.S.) and grants U54HG3067 (E.S.L.), U54HG3273 and U01HG5211 (R.A.G.), U54HG3079 (R.K.W., E.R.M.), R01HG2898 (S.E.D.), R01HG2385 (E.E.E.), RC2HG5552 and U01HG6513 (G.T.M., G.R.A.), U01HG5214 (A.C.), U01HG5715 (C.D.B.), U01HG5718 (M.G.), U01HG5728 (Y.X.F.), U41HG7635 (R.K.W., E.E.E., P.H.S.), U41HG7497 (C.L., M.A.B., K.C., L.D., E.E.E., M.G., J.O.K., G.T.M., S.A.M., R.E.M., J.L.S., K.Y.), R01HG4960 and R01HG5701 (B.L.B.), R01HG5214 (G.A.), R01HG6855 (S.M.), R01HG7068 (R.E.M.), R01HG7644 (R.D.H.), DP2OD6514 (P.S.), DP5OD9154 (J.K.), R01CA166661 (S.E.D.), R01CA172652 (K.C.), P01GM99568 (S.R.B.), R01GM59290 (L.B.J., M.A.B.), R01GM104390 (L.B.J., M.Y.Y.), T32GM7790 (C.D.B., A.R.M.), P01GM99568 (S.R.B.), R01HL87699 and R01HL104608 (K.C.B.), T32HL94284 (J.L.R.F.), and contracts HHSN268201100040C (A.M.R.) and HHSN272201000025C (P.S.), Harvard Medical School Eleanor and Miles Shore Fellowship (K.L.), Lundbeck Foundation Grant R170-2014-1039 (K.L.), NIJ Grant 2014-DN-BX-K089 (Y.E.), the Mary Beryl Patch Turnbull Scholar Program (K.C.B.), NSF Graduate Research Fellowship DGE-1147470 (G.D.P.), the Simons Foundation SFARI award SF51 (M.W.), and a Sloan Foundation Fellowship (R.D.H.). E.E.E. is an investigator of the Howard Hughes Medical Institute