Angus I Lamond

University of Dundee, Dundee, Scotland, United Kingdom

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Publications (214)1978.42 Total impact

  • Tony Ly, Aki Endo, Angus I Lamond
    eLife Sciences 01/2015; 4. · 8.52 Impact Factor
  • Saskia Hutten, Samuel Swift, Angus I Lamond
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    ABSTRACT: Fluorescence microscopy is a powerful technique that has become central in the study of the structure and function of biological specimens. This is due in large part to its specificity and versatility. Although an understanding of structure-typically through high-resolution imaging of fixed material-has proved an important tool to understanding function, fluorescence microscopy also offers a mechanism to interrogate cells in the living state, providing a means to explore dynamic process within the specimen over long time periods at high temporal resolution. The cell nucleus is a highly compartmented environment whose components are often highly motile and in a constant state of flux. The ability to monitor the dynamic behavior of nuclear bodies by live-cell imaging provides the researcher with important information regarding underlying mechanistic processes relating to their formation and maintenance. Two techniques have proved particularly valuable to our study of cellular dynamics and molecular mobility, namely, time-lapse imaging and fluorescence recovery after photobleaching (FRAP). Time-lapse microscopy allows for qualitative and quantitative analysis of a wide range of events at the cellular and subcellular level. FRAP provides a mechanism to study the mobility of a population of proteins in a range of conditions within discrete areas of the biological specimen. Therefore, fluorescence microscopy is unique in its ability to provide data at high temporal resolution and in such exquisite detail.
    Methods in molecular biology (Clifton, N.J.) 01/2015; 1262:55-67. · 1.29 Impact Factor
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    ABSTRACT: In vitro, assembly of box C/D small nucleolar ribonucleoproteins (snoRNPs) involves the sequential recruitment of core proteins to snoRNAs. In vivo, however, assembly factors are required (NUFIP, BCD1, and the HSP90-R2TP complex), and it is unknown whether a similar sequential scheme applies. In this paper, we describe systematic quantitative stable isotope labeling by amino acids in cell culture proteomic experiments and the crystal structure of the core protein Snu13p/15.5K bound to a fragment of the assembly factor Rsa1p/NUFIP. This revealed several unexpected features: (a) the existence of a protein-only pre-snoRNP complex containing five assembly factors and two core proteins, 15.5K and Nop58; (b) the characterization of ZNHIT3, which is present in the protein-only complex but gets released upon binding to C/D snoRNAs; (c) the dynamics of the R2TP complex, which appears to load/unload RuvBL AAA(+) adenosine triphosphatase from pre-snoRNPs; and (d) a potential mechanism for preventing premature activation of snoRNP catalytic activity. These data provide a framework for understanding the assembly of box C/D snoRNPs. © 2014 Bizarro et al.
    The Journal of Cell Biology 11/2014; 207(4):463-480. · 9.69 Impact Factor
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    ABSTRACT: Eukaryotic pre-mRNA splicing is an essential step in gene expression for all genes that contain introns. In contrast with transcription and translation, few well-characterized chemical inhibitors are available with which to dissect the splicing process, particularly in cells. Therefore, the identification of specific, small molecules that either inhibit, or modify, pre-mRNA splicing would be valuable for research and potentially also for therapeutic applications. We have screened a highly curated library of 71,504 drug-like small molecules using a high throughput in vitro splicing assay. This identified 10 new compounds that both inhibit pre-mRNA splicing in vitro and modify splicing of endogenous pre-mRNA in cells. One of these splicing modulators, DDD00107587 (termed "madrasin" i.e., (2-((7methoxy-4-methylquinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one RNA splicing inhibitor), was studied in more detail. Madrasin interferes with the early stages of spliceosome assembly and stalls spliceosome assembly at the A complex. Madrasin is cytotoxic at higher concentrations, while at lower concentrations it induces cell cycle arrest, promotes a specific reorganization of subnuclear protein localization and modulates splicing of multiple pre-mRNAs in both HeLa and HEK293 cells.
    Journal of Biological Chemistry 10/2014; · 4.60 Impact Factor
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    ABSTRACT: The study of replication of viruses that require high bio-secure facilities can be accomplished with less stringent containment using non-infectious 'replicon' systems. The FMDV replicon system (pT7rep) reported by Mclnerney and co-workers (2000) was modified by the replacement of sequences encoding chloramphenicol acetyl-transferase (CAT) with those encoding a functional L proteinase (L(pro)) linked to a bi-functional fluorescent/antibiotic resistance fusion protein (green fluorescent protein/puromycin resistance, [GFP-PAC]). Cells were transfected with replicon-derived transcript RNA and GFP fluorescence quantified. Replication of transcript RNAs was readily detected by fluorescence, whilst the signal from replication-incompetent forms of the genome was >2 fold lower. Surprisingly, a form of the replicon lacking the L(pro) showed a significantly stronger fluorescence signal, but appeared with slightly delayed kinetics. Replication can, therefore, be quantified simply by live-cell imaging and image analyses, providing a rapid and facile alternative to RT-qPCR or CAT assays.
    Journal of Virological Methods 09/2014; · 1.88 Impact Factor
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    ABSTRACT: Technological advances have enabled the analysis of cellular protein and RNA levels with unprecedented depth and sensitivity, allowing for an unbiased re-evaluation of gene regulation during fundamental biological processes. Here, we have chronicled the dynamics of protein and mRNA expression levels across a minimally perturbed cell cycle in human myeloid leukemia cells using centrifugal elutriation combined with mass spectrometry-based proteomics and RNA-Seq, avoiding artificial synchronization procedures. We identify myeloid-specific gene expression and variations in protein abundance, isoform expression and phosphorylation at different cell cycle stages. We dissect the relationship between protein and mRNA levels for both bulk gene expression and for over ∼6000 genes individually across the cell cycle, revealing complex, gene-specific patterns. This data set, one of the deepest surveys to date of gene expression in human cells, is presented in an online, searchable database, the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/). DOI: http://dx.doi.org/10.7554/eLife.01630.001.
    eLife Sciences 03/2014; 3:e01630. · 8.52 Impact Factor
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    ABSTRACT: Cajal bodies are nuclear structures involved in snRNP and snoRNP biogenesis, telomere maintenance and histone mRNA processing. Recently, the SUMO isopeptidase USPL1 was identified as a Cajal body component essential for cellular growth and Cajal body integrity. However, a cellular function for USPL1 is so far unknown. Here, we use RNAi mediated knockdown in human cells in combination with biochemical and fluorescence microscopy approaches to investigate the function of USPL1 and its relation to Cajal bodies. We demonstrate that the levels of RNAPII-transcribed snRNAs are reduced upon knockdown of USPL1 and that downstream processes such as snRNP assembly and pre-mRNA splicing are compromised. Importantly, we find that USPL1 associates directly with U snRNA loci and that it interacts and colocalizes with components of the Little Elongation Complex, which is involved in RNAPII-mediated snRNA transcription. Thus our data indicate that USPL1 plays a key role in the process of RNAPII-mediated snRNA transcription.
    Journal of Cell Science 01/2014; · 5.33 Impact Factor
  • Yasmeen Ahmad, Angus I Lamond
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    ABSTRACT: During the past 15 years mass spectrometry (MS)-based analyses have become established as the method of choice for direct protein identification and measurement. Owing to the remarkable improvements in the sensitivity and resolution of MS instruments, this technology has revolutionised the opportunities available for the system-wide characterisation of proteins, with wide applications across virtually the whole of cell biology. In this article we provide a perspective on the current state of the art and discuss how the future of cell biology research may benefit from further developments and applications in the field of MS and proteomics, highlighting the major challenges ahead for the community in organising the effective sharing and integration of the resulting data mountain.
    Trends in cell biology 11/2013; · 12.12 Impact Factor
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    ABSTRACT: Messenger RNA translation is regulated by RNA-binding proteins and small non-coding RNAs called microRNAs. Even though we know the majority of RNA-binding proteins and microRNAs that regulate messenger RNA expression, evidence of interactions between the two remain elusive. The role of the RNA-binding protein GLD-1 as a translational repressor is well studied during Caenorhabditis elegans germline development and maintenance. Possible functions of GLD-1 during somatic development and the mechanism of how GLD-1 acts as a translational repressor are not known. Its human homologue, quaking (QKI), is essential for embryonic development. Here, we report that the RNA-binding protein GLD-1 in C. elegans affects multiple microRNA pathways and interacts with proteins required for microRNA function. Using genome-wide RNAi screening, we found that nhl-2 and vig-1, two known modulators of miRNA function, genetically interact with GLD-1. gld-1 mutations enhance multiple phenotypes conferred by mir-35 and let-7 family mutants during somatic development. We used stable isotope labelling with amino acids in cell culture to globally analyse the changes in the proteome conferred by let-7 and gld-1 during animal development. We identified the histone mRNA-binding protein CDL-1 to be, in part, responsible for the phenotypes observed in let-7 and gld-1 mutants. The link between GLD-1 and miRNA-mediated gene regulation is further supported by its biochemical interaction with ALG-1, CGH-1 and PAB-1, proteins implicated in miRNA regulation. Overall, we have uncovered genetic and biochemical interactions between GLD-1 and miRNA pathways.
    Open Biology 11/2013; 3(11):130151. · 4.56 Impact Factor
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    ABSTRACT: Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we have combined native size-exclusion chromatography (SEC) with high throughput proteomic analysis to characterise soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1- MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific GO terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data sharing resource available to the community.
    Molecular &amp Cellular Proteomics 09/2013; · 7.25 Impact Factor
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    ABSTRACT: Stable Isotope Labelling by Amino acids in Cell culture (SILAC) is a powerful technique for comparative quantitative proteomics, which has recently been applied to a number of different eukaryotic organisms. Inefficient incorporation of labelled amino acids in cell cultures of Arabidopsis thaliana has led to very limited use of SILAC in plant systems. We present a method allowing, for the first time, efficient labelling with stable isotope-containing arginine and lysine of whole Arabidopsis seedlings. To illustrate the utility of this method, we have combined the high labelling efficiency (>95%) with quantitative proteomics analyses of seedlings exposed to increased salt concentration. In plants treated for 7 days with 80 mM NaCl, a relatively mild salt stress, 215 proteins were identified whose expression levels changed significantly compared to untreated seedling controls. The 92 up-regulated proteins included proteins involved in abiotic stress responses and photosynthesis, while the 123 down-regulated proteins were enriched in proteins involved in reduction of oxidative stress and other stress responses, respectively. Efficient labelling of whole Arabidopsis seedlings by this modified SILAC method opens new opportunities to exploit the genetic resources of Arabidopsis and analyse the impact of mutations on quantitative protein dynamics in vivo.
    PLoS ONE 08/2013; 8(8):e72207. · 3.53 Impact Factor
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    ABSTRACT: PHD1 belongs to the family of prolyl-4-hydroxylases (PHDs) that is responsible for posttranslational modification of prolines on specific target proteins. Because PHD activity is sensitive to oxygen levels and certain byproducts of the tricarboxylic acid cycle, PHDs act as sensors of the cell's metabolic state. Here, we identify PHD1 as a critical molecular link between oxygen sensing and cell-cycle control. We show that PHD1 function is required for centrosome duplication and maturation through modification of the critical centrosome component Cep192. Importantly, PHD1 is also required for primary cilia formation. Cep192 is hydroxylated by PHD1 on proline residue 1717. This hydroxylation is required for binding of the E3 ubiquitin ligase SCF(Skp2), which ubiquitinates Cep192, targeting it for proteasomal degradation. By modulating Cep192 levels, PHD1 thereby affects the processes of centriole duplication and centrosome maturation and contributes to the regulation of cell-cycle progression.
    Developmental Cell 08/2013; · 10.37 Impact Factor
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    ABSTRACT: We have identified the human FMN2 gene as a novel target regulated by induction of p14ARF and by multiple other stress responses, including DNA damage and hypoxia, which have in common activation of cell cycle arrest. We showed that increased expression of the FMN2 gene following p14ARF induction is caused, at the transcriptional level, by relief of repression by RelA and E2F1, which, under non-induced conditions, bind the FMN2 promoter. Increased FMN2 protein levels promote cell cycle arrest by inhibiting the degradation of p21, and our data show that control of p21 stability is a key part of the mechanism that regulates p21 induction. Consistent with this model, we have shown that transient expression of exogenous FMN2 protein alone is sufficient to increase p21 protein levels in cells, without altering p21 mRNA levels. Here, we provide additional evidence for the role of the N terminus of FMN2 as being the important domain required for p21 stability. In addition, we also investigate the role of RelA's threonine 505 residue in the control of FMN2. Our results identify FMN2 as a crucial protein involved in the control of p21.
    Cell cycle (Georgetown, Tex.) 07/2013; 12(15). · 5.24 Impact Factor
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    ABSTRACT: The organisation of the large volume of mammalian genomic DNA within cell nuclei requires mechanisms to regulate chromatin compaction involving the reversible formation of higher order structures. The compaction state of chromatin varies between interphase and mitosis and is also subject to rapid and reversible change upon ATP depletion/repletion. In this study we have investigated mechanisms that may be involved in promoting the hyper-condensation of chromatin when ATP levels are depleted by treating cells with sodium azide and 2-deoxyglucose. Chromatin conformation was analysed in both live and permeabilised HeLa cells using FLIM-FRET, high resolution fluorescence microscopy and by electron spectroscopic imaging microscopy. We show that chromatin compaction following ATP depletion is not caused by loss of transcription activity and that it can occur at a similar level in both interphase and mitotic cells. Analysis of both live and permeabilised HeLa cells shows that chromatin conformation within nuclei is strongly influenced by the levels of divalent cations, including calcium and magnesium. While ATP depletion results in an increase in the level of unbound calcium, chromatin condensation still occurs even in the presence of a calcium chelator. Chromatin compaction is shown to be strongly affected by small changes in the levels of polyamines, including spermine and spermidine. The data are consistent with a model in which the increased intracellular pool of polyamines and divalent cations, resulting from depletion of ATP, bind to DNA and contribute to the large scale hyper-compaction of chromatin by a charge neutralisation mechanism.
    PLoS ONE 06/2013; 8(6):e67689. · 3.53 Impact Factor
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    ABSTRACT: The study of the function of many human proteins is often hampered by technical limitations, such as cytotoxicity and phenotypes that result from overexpression of the protein of interest together with the endogenous version. Here we present the snoMEN (snoRNA Modulator of gene ExpressioN) vector technology for generating stable cell lines where expression of the endogenous protein can be reduced and replaced by an exogenous protein, such as a fluorescent protein (FP)-tagged version. SnoMEN are snoRNAs engineered to contain complementary sequences that can promote knock-down of targeted RNAs. We have established and characterised two such partial protein replacement human cell lines (snoMEN-PR). Quantitative mass spectrometry was used to analyse the specificity of knock-down and replacement at the protein level and also showed an increased pull-down efficiency of protein complexes containing exogenous, tagged proteins in the protein replacement cell lines, as compared with conventional co-expression strategies. The snoMEN approach facilitates the study of mammalian proteins, particularly those that have so far been difficult to investigate by exogenous expression and has wide applications in basic and applied gene-expression research.
    PLoS ONE 04/2013; 8(4):e62305. · 3.53 Impact Factor
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    Motoharu Ono, Kayo Yamada, Angus I Lamond
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    ABSTRACT: The present invention relates to a method of targeting miRNA and/or pre-miRNA molecules in order to treat diseases that are linked with miRNA expression, such as certain cancers. The present invention also provides modified snoRNA molecules for targeting mi RNA molecules for use in treating diseases that are linked with miRNA expression, such as certain cancers.
    Ref. No: WO 2013/054113 A1, Year: 04/2013
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Publication Stats

15k Citations
1,978.42 Total Impact Points

Institutions

  • 1995–2014
    • University of Dundee
      • • Centre for Gene Regulation and Expression
      • • College of Life Sciences
      • • Wellcome Trust Centre for Gene Regulation and Expression
      Dundee, Scotland, United Kingdom
  • 2013
    • Friedrich Miescher Institute for Biomedical Research
      Bâle, Basel-City, Switzerland
  • 2011–2012
    • University of Natural Resources and Life Science Vienna
      • Department für Biotechnologie
      Vienna, Vienna, Austria
  • 2010
    • University of Western Australia
      • Western Australian Institute for Medical Research (WAIMR)
      Perth, Western Australia, Australia
    • Cold Spring Harbor Laboratory
      Cold Spring Harbor, New York, United States
  • 2008
    • The University of Calgary
      • Department of Biological Sciences
      Calgary, Alberta, Canada
  • 2007
    • Leiden University Medical Centre
      • Department of Molecular Cell Biology
      Leiden, South Holland, Netherlands
  • 2005
    • Cyprus Institute of Neurology and Genetics
      Lefkoşa, Lefkosia, Cyprus
  • 2002
    • University of Southern Denmark
      • Center for Experimental Bioinformatics
      Copenhagen, Capital Region, Denmark
  • 1990–1998
    • European Molecular Biology Laboratory
      Heidelburg, Baden-Württemberg, Germany
    • Universität Basel
      Bâle, Basel-City, Switzerland
  • 1997
    • Uppsala University
      Uppsala, Uppsala, Sweden
    • University of California, San Francisco
      • Department of Microbiology and Immunology
      San Francisco, CA, United States
  • 1996
    • Research Institute of Molecular Pathology
      Wien, Vienna, Austria
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
  • 1988–1996
    • Massachusetts Institute of Technology
      • Department of Biology
      Cambridge, MA, United States
  • 1994
    • MRC National Institute for Medical Research
      Londinium, England, United Kingdom
    • University of Lisbon
      • Faculdade de Medicina
      Lisbon, Lisbon, Portugal
  • 1991
    • University of Wisconsin, Madison
      • Department of Biochemistry
      Madison, MS, United States