DOCSLIB.ORG

Broad Susceptibility of Nucleolar Proteins and Autoantigens to Complement C1 Protease Degradation

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Broad Susceptibility of Nucleolar Proteins and Autoantigens to Complement C1 Protease Degradation Broad Susceptibility of Nucleolar Proteins and Autoantigens to Complement C1 Protease Degradation This information is current as Yitian Cai, Seng Yin Kelly Wee, Junjie Chen, Boon Heng of September 25, 2021. Dennis Teo, Yee Leng Carol Ng, Khai Pang Leong and Jinhua Lu J Immunol published online 25 October 2017 http://www.jimmunol.org/content/early/2017/10/25/jimmun ol.1700728 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2017/10/25/jimmunol.170072 Material 8.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 25, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 25, 2017, doi:10.4049/jimmunol.1700728 The Journal of Immunology Broad Susceptibility of Nucleolar Proteins and Autoantigens to Complement C1 Protease Degradation Yitian Cai,*,1 Seng Yin Kelly Wee,*,1 Junjie Chen,* Boon Heng Dennis Teo,* Yee Leng Carol Ng,† Khai Pang Leong,† and Jinhua Lu* Anti-nuclear autoantibodies, which frequently target the nucleoli, are pathogenic hallmarks of systemic lupus erythematosus (SLE). Although the causes of these Abs remain broad and ill-defined, a genetic deficiency in C1 complex (C1qC1r2C1s2) or C4 is able to induce these Abs. Considering a recent finding that, in dead cells, nucleoli were targeted by C1q and two nucleolar autoantigens were degraded by C1r/C1s proteases, we considered that C1 could help protect against antinuclear autoimmunity by broadly degrading nucleolar proteins or autoantigens. Nucleoli were isolated to homogeneity and structurally defined. After C1 treatment, cleaved nucleolar proteins were identified by proteomic two-dimensional fluorescence difference gel electrophoresis and mass spectrometry, and further verified by Western blotting using specific Abs. The extent of nucleolar autoantigen degradation upon Downloaded from C1 treatment was estimated using SLE patient autoantibodies. The isolated nucleoli were broadly reactive with SLE patient autoantibodies. These nucleoli lacked significant autoproteolysis, but many nucleolar proteins and autoantigens were degraded by C1 proteases; >20 nucleolar proteins were identified as C1 cleavable. These were further validated by Western blotting using specific Abs. The broad autoantigenicity of the nucleoli may attribute to their poor autoproteolysis, causing autologous immune stimulation upon necrotic exposure. However, C1q targets at these nucleoli to cause C1 protease activation and the cleavage of many nucleolar proteins or autoantigens. This may represent one important surveillance mechanism against antinuclear auto- http://www.jimmunol.org/ immunity because C1 genetic deficiency causes anti-nuclear autoantibodies and SLE disease. The Journal of Immunology, 2017, 199: 000–000. ystemic lupus erythematosus (SLE) is a complex auto- of anti-nuclear autoantibodies remain unclear, and understanding immune disease with limited treatment options (1, 2). their origins can greatly expand therapeutic options. S Although progress has been made in understanding the In live cells, nuclear Ags are segregated from autologous im- underlying pathogenesis of this disease, including the hallmark mune cell recognition and responses. Early apoptotic cells similarly contributions of anti-nuclear autoantibodies (3–5), chronic eleva- conceal these Ags, and also actively suppress proinflammatory by guest on September 25, 2021 tion of IFN-a (6–10), and accumulated apoptotic bodies (11–13), responses from phagocytes and other immune cells (16). However, how these modular mechanisms are activated and rally toward exposure of these intracellular materials can occur when cell death SLE disease remains poorly understood. Anti-nuclear autoanti- takes the necrotic pathway (17, 18). The naive B cell repertoire bodies are early pathogenic factors that can manifest long before contains significant autoreactive B cells (19). The autoimmunogenicity SLE disease onset, whereas IFN-a and apoptotic cellular Ags of nuclear materials has been demonstrated when anti-nuclear au- appear to surge at disease flare (1, 3, 14, 15). The primary causes toantibodies were found induced following the injection of dead cells into mice (11, 20). This was especially prominent when ad- juvant was also injected (20). This reaction inevitably involves in- *Department of Microbiology and Immunology, Yong Loo Lin School of Medicine tracellular autoantigens and potentially intracellular adjuvants such and Immunology Programme, National University of Singapore, Singapore 117597, as IL-1a, IL-33, S100 proteins or high mobility group box 1 Singapore; and †Department of Rheumatology, Allergy and Immunology, Tan Tock Seng Hospital, Singapore 308433, Singapore (HMGB1), which are also known as danger-associated molecular 1Y.C. and S.Y.K.W. contributed equally to this work. patterns (DAMPs) or alarmins (18, 21, 22). Incidentally, HMGB1 ORCIDs: 0000-0002-5462-2118 (Y.C.); 0000-0001-9403-505X (S.Y.K.W.); 0000- is released in complex with chromatin fragments from necrotic 0003-4696-9813 (B.H.D.T.). cells, which induces lupus-like antinuclear responses in mice (21). Received for publication May 19, 2017. Accepted for publication October 5, 2017. Genetic studies have revealed .50 SLE risk genes, broadly This work was supported by a Singapore Ministry of Education Tier 2 grant related to inflammation, immune clearance, and IFN-a–producing (MOE2012-T2-2-122), a Singapore Ministry of Health National Medical Research or signaling pathways (23–25). These risk genes mostly exhibit Council grant (NMRC/OFIRG/0013/2016), and a National University Health System low-to-moderate impacts on SLE disease and are mostly not seed fund (T1-BSRG 2015-07). specific for SLE pathogenesis. More than 20 monogenic SLE risk Address correspondence and reprint requests to Dr. Jinhua Lu, Department of Mi- crobiology and Immunology, Yong Loo Lin School of Medicine, National University genes have also been reported, which also broadly impact on other of Singapore, Blk MD4, 5 Science Drive 2, Singapore 117597, Singapore. E-mail autoimmune diseases (26). However, genetic deficiencies in a address: [email protected] group of complement proteins (i.e., C1q, C1r/C1s, and C4) show The online version of this article contains supplemental material. strong and highly specific association with SLE pathogenesis (23, Abbreviations used in this article: C1-inh, C1 inhibitor; DAMP, danger-associated 27–29). These patients develop anti-nuclear autoantibodies and molecular pattern; DC, dendritic cell; 2D-DIGE, two-dimensional fluorescence dif- ference gel electrophoresis; HMGB1, high mobility group box 1; hnRNPU, hetero- manifest severe SLE disease characterized by early disease onset geneous nuclear ribonucleoprotein U; NCL, nucleolin; NG1, nuclear ghost 1; NPM1, and equal gender susceptibility. These complement proteins are nucleophosmin 1; SLE, systemic lupus erythematosus. intimately related; C1q, C1r, and C1s form the C1 (C1qC1r2C1s2) Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 complex in which C1q is a scaffold, and C1r and C1s are effector www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700728 2 COMPLEMENT DEGRADATION OF NUCLEOLAR Ags serine proteases (30, 31). C1 complex defines the complement In determining C1q binding sites in dead cells, we recently classical pathway: binding of C1q to Ag-bound Abs causes acti- observed that C1q bound to the cell periphery in early apoptosis, vation of C1r followed by C1s, which subsequently cleaves C4 but it bound predominantly to the nucleoli during late apoptosis and C2 to trigger the complement cascade (32). (48). Isolated nucleoli were also recognized by C1q, causing ac- The strong causal association between C1 or C4 deficiency and tivation of the C1s protease and the cleavage of two major nu- SLE pathogenesis cannot be explained in the traditional paradigm cleolar proteins, nucleophosmin-1 (NPM1) and nucleolin (NCL) of the complement system, which consists of nearly 30 plasma (48). Because anti-nucleolus autoantibodies are prevalent in SLE proteins (23, 28). C2 deficiency is relatively common and also patients and are also significant autoantibody specificities in SLE contributes to SLE pathogenesis (27, 33). However, it lacks the mice and some cancer patients (49–52), in this study, we estimated type of strong and specific effect, early disease onset, and equal the extent of nucleolar protein and autoantigen degradation by C1 gender susceptibility found with C1 and C4 deficiencies. In some proteases through a proteomic approach. patients, C1 inhibitor (C1-inh) deficiency can manifest mild SLE- like conditions, and this could be explained by excessive C1r or Materials and Methods C1s activation and consumption of intimately related complement Cell culture and Abs elements
Load more

Recommended publications
  • The DEAD-Box RNA Helicase-Like Utp25 Is an SSU Processome Component
    Downloaded from rnajournal.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press The DEAD-box RNA helicase-like Utp25 is an SSU processome component J. MICHAEL CHARETTE1,2 and SUSAN J. BASERGA1,2,3 1Department of Molecular Biophysics & Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520, USA 2Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA 3Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06520, USA ABSTRACT The SSU processome is a large ribonucleoprotein complex consisting of the U3 snoRNA and at least 43 proteins. A database search, initiated in an effort to discover additional SSU processome components, identified the uncharacterized, conserved and essential yeast nucleolar protein YIL091C/UTP25 as one such candidate. The C-terminal DUF1253 motif, a domain of unknown function, displays limited sequence similarity to DEAD-box RNA helicases. In the absence of the conserved DEAD-box sequence, motif Ia is the only clearly identifiable helicase element. Since the yeast homolog is nucleolar and interacts with components of the SSU processome, we examined its role in pre-rRNA processing. Genetic depletion of Utp25 resulted in slowed growth. Northern analysis of pre-rRNA revealed an 18S rRNA maturation defect at sites A0,A1, and A2. Coimmunoprecipitation confirmed association with U3 snoRNA and with Mpp10, and with components of the t-Utp/UtpA, UtpB, and U3 snoRNP subcomplexes. Mutation of the conserved motif Ia residues resulted in no discernable temperature- sensitive or cold-sensitive growth defects, implying that this motif is dispensable for Utp25 function.
    [Show full text]
  • Fibrillarin from Archaea to Human
    Biol. Cell (2015) 107, 1–16 DOI: 10.1111/boc.201400077 Review Fibrillarin from Archaea to human Ulises Rodriguez-Corona*, Margarita Sobol†, Luis Carlos Rodriguez-Zapata‡, Pavel Hozak† and Enrique Castano*1 *Unidad de Bioquımica´ y Biologıa´ molecular de plantas, Centro de Investigacion´ Cientıfica´ de Yucatan,´ Colonia Chuburna´ de Hidalgo, Merida,´ Yucatan, Mexico, †Department of Biology of the Cell Nucleus, Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic, Prague 14220, Czech Republic, and ‡Unidad de Biotecnologıa,´ Centro de Investigacion´ Cientıfica´ de Yucatan,´ Colonia Chuburna´ de Hidalgo, Merida,´ Yucatan, Mexico Fibrillarin is an essential protein that is well known as a molecular marker of transcriptionally active RNA polyme- rase I. Fibrillarin methyltransferase activity is the primary known source of methylation for more than 100 methylated sites involved in the first steps of preribosomal processing and required for structural ribosome stability. High expression levels of fibrillarin have been observed in several types of cancer cells, particularly when p53 levels are reduced, because p53 is a direct negative regulator of fibrillarin transcription. Here, we show fibrillarin domain conservation, structure and interacting molecules in different cellular processes as well as with several viral proteins during virus infection. Additional supporting information may be found in the online version of this article at the publisher’s web-site Introduction progression, senescence and biogenesis of small nu- The nucleolus is the largest visible structure inside clear RNA and tRNAs proliferation and many forms the cell nucleus. It exists both as a dynamic and sta- of stress response (Andersen et al., 2005; Hinsby ble region depending of the nature and amount of et al., 2006; Boisvert et al., 2007; Shaw and Brown, the molecules that it is made of.
    [Show full text]
  • Analysis of Gene Expression Data for Gene Ontology
    ANALYSIS OF GENE EXPRESSION DATA FOR GENE ONTOLOGY BASED PROTEIN FUNCTION PREDICTION A Thesis Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Master of Science Robert Daniel Macholan May 2011 ANALYSIS OF GENE EXPRESSION DATA FOR GENE ONTOLOGY BASED PROTEIN FUNCTION PREDICTION Robert Daniel Macholan Thesis Approved: Accepted: _______________________________ _______________________________ Advisor Department Chair Dr. Zhong-Hui Duan Dr. Chien-Chung Chan _______________________________ _______________________________ Committee Member Dean of the College Dr. Chien-Chung Chan Dr. Chand K. Midha _______________________________ _______________________________ Committee Member Dean of the Graduate School Dr. Yingcai Xiao Dr. George R. Newkome _______________________________ Date ii ABSTRACT A tremendous increase in genomic data has encouraged biologists to turn to bioinformatics in order to assist in its interpretation and processing. One of the present challenges that need to be overcome in order to understand this data more completely is the development of a reliable method to accurately predict the function of a protein from its genomic information. This study focuses on developing an effective algorithm for protein function prediction. The algorithm is based on proteins that have similar expression patterns. The similarity of the expression data is determined using a novel measure, the slope matrix. The slope matrix introduces a normalized method for the comparison of expression levels throughout a proteome. The algorithm is tested using real microarray gene expression data. Their functions are characterized using gene ontology annotations. The results of the case study indicate the protein function prediction algorithm developed is comparable to the prediction algorithms that are based on the annotations of homologous proteins.
    [Show full text]
  • The Nucleolus – a Gateway to Viral Infection? Brief Review
    Arch Virol (2002) 147: 1077–1089 DOI 10.1007/s00705-001-0792-0 The nucleolus – a gateway to viral infection? Brief Review J. A. Hiscox School of Animal and Microbial Sciences, University of Reading, U.K. Received August 24, 2001; accepted December 26, 2001 Published online March 18, 2002, © Springer-Verlag 2002 Summary. A number of viruses and viral proteins interact with a dynamic sub- nuclear structure called the nucleolus. The nucleolus is present during interphase in mammalian cells and is the site of ribosome biogenesis, and has been implicated in controlling regulatory processes such as the cell cycle. Viruses interact with the nucleolus and its antigens; viral proteins co-localise with factors such as nucleolin, B23 and fibrillarin, and can cause their redistribution during infection. Viruses can use these components as part of their replication process, and also use the nucleolus as a site of replication itself. Many of these properties are not restricted to any particular type of virus or replication mechanism, and examples of these processes can be found in DNA, RNA and retroviruses. Evidence suggests that viruses may target the nucleolus and its components to favour viral transcription, translation and perhaps alter the cell cycle in order to promote virus replication. Autoimmunity to nucleolin and fibrillarin have been associated with a number of diseases, and by targeting the nucleolus and displacing nucleolar antigens, virus infection might play a role in the initiation of these conditions. Introduction The eukaryotic nucleus contains a number of domains or subcompartments, which include nucleoli, nuclear Cajal bodies, nuclear speckles, transcription and replica- tion foci, and chromosome territories [34].
    [Show full text]
  • DDX5 Targets Tissue-Specific Rnas to Promote Intestine Tumorigenesis
    bioRxiv preprint doi: https://doi.org/10.1101/2020.03.25.006668; this version posted March 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. DDX5 targets tissue-specific RNAs to promote intestine tumorigenesis Nazia Abbasi1,4, Tianyun Long1,4, Yuxin Li1, Evelyn Ma1, Brian A. Yee1, Parth R. Patel1, Ibrahim M SayeD2, Nissi Varki2, Soumita Das2, PraDipta Ghosh1, 3, Gene W. Yeo1, WenDy J.M. Huang1,5 1 Department of Cellular anD Molecular MeDicine, University of California San Diego, La Jolla, CA 2 Department of Pathology, University of California San Diego, La Jolla, CA 3 Department of MeDicine, University of California San Diego, La Jolla, CA 4 These authors contributeD equally 5 CorresponDing author email: [email protected] Abstract Tumorigenesis in Different segments of the intestinal tract involves tissue-specific oncogenic Drivers. In the colon, complement component 3 (C3) activation is a major contributor to inflammation anD malignancies. By contrast, tumorigenesis in the small intestine involves fatty aciD-binding protein 1 (FABP1). However, little is known of the upstream mechanisms Driving their expressions in Different segments of the intestinal tract. Here, we report that an RNA binDing protein DDX5 augments C3 and FABP1 expressions post-transcriptionally to promote tumorigenesis in the colon anD small intestine, respectively. Mice with epithelial-specific knockout of DDX5 are protecteD from intestine tumorigenesis. The iDentification of DDX5 as the common upstream regulator of tissue-specific oncogenic molecules proviDes a new therapeutic target for intestine cancers.
    [Show full text]
  • Kras Mutant Genetically Engineered Mouse Models of Human Cancers
    Kras mutant genetically engineered mouse models of PNAS PLUS human cancers are genomically heterogeneous Wei-Jen Chunga,1,2, Anneleen Daemena,1, Jason H. Chengb, Jason E. Longb, Jonathan E. Cooperb, Bu-er Wangb, Christopher Tranb, Mallika Singhb,3, Florian Gnada,4, Zora Modrusanc, Oded Foremand, and Melissa R. Junttilab,5 aBioinformatics & Computational Biology, Genentech, Inc., South San Francisco, CA 94080; bTranslational Oncology, Genentech, Inc., South San Francisco, CA 94080; cMolecular Biology, Genentech, Inc., South San Francisco, CA 94080; and dPathology, Genentech, Inc., South San Francisco, CA 94080 Edited by Anton Berns, The Netherlands Cancer Institute, Amsterdam, The Netherlands, and approved November 6, 2017 (received for review May 23, 2017) KRAS mutant tumors are largely recalcitrant to targeted therapies. treatment susceptibility and tumor evolution under selective pres- Genetically engineered mouse models (GEMMs) of Kras mutant sure of drug therapy. cancer recapitulate critical aspects of this disease and are widely used for preclinical validation of targets and therapies. Through Results comprehensive profiling of exomes and matched transcriptomes Spontaneous Acquisition of Genomic Aberrations in KrasG12D-Initiated of >200 KrasG12D-initiated GEMM tumors from one lung and two GEMM Tumors. To better define the genomic landscape of estab- pancreatic cancer models, we discover that significant intratumoral lished Kras mutant tumor models, we focused on three models of and intertumoral genomic heterogeneity evolves during tumorigen- Kras mutant cancer: a nonsmall cell lung cancer (NSCLC) model esis. Known oncogenes and tumor suppressor genes, beyond those with adenovirus-induced expression of KrasG12D and homozygous engineered, are mutated, amplified, and deleted. Unlike human tu- p53 targeting (KP:KrasLSL.G12D/wt;p53frt/frt) (11, 13), and two models mors, the GEMM genomic landscapes are dominated by copy num- ber alterations, while protein-altering mutations are rare.
    [Show full text]
  • Utpa and Utpb Chaperone Nascent Pre-Ribosomal RNA and U3 Snorna to Initiate Eukaryotic Ribosome Assembly
    ARTICLE Received 6 Apr 2016 | Accepted 27 May 2016 | Published 29 Jun 2016 DOI: 10.1038/ncomms12090 OPEN UtpA and UtpB chaperone nascent pre-ribosomal RNA and U3 snoRNA to initiate eukaryotic ribosome assembly Mirjam Hunziker1,*, Jonas Barandun1,*, Elisabeth Petfalski2, Dongyan Tan3, Cle´mentine Delan-Forino2, Kelly R. Molloy4, Kelly H. Kim5, Hywel Dunn-Davies2, Yi Shi4, Malik Chaker-Margot1,6, Brian T. Chait4, Thomas Walz5, David Tollervey2 & Sebastian Klinge1 Early eukaryotic ribosome biogenesis involves large multi-protein complexes, which co-transcriptionally associate with pre-ribosomal RNA to form the small subunit processome. The precise mechanisms by which two of the largest multi-protein complexes—UtpA and UtpB—interact with nascent pre-ribosomal RNA are poorly understood. Here, we combined biochemical and structural biology approaches with ensembles of RNA–protein cross-linking data to elucidate the essential functions of both complexes. We show that UtpA contains a large composite RNA-binding site and captures the 50 end of pre-ribosomal RNA. UtpB forms an extended structure that binds early pre-ribosomal intermediates in close proximity to architectural sites such as an RNA duplex formed by the 50 ETS and U3 snoRNA as well as the 30 boundary of the 18S rRNA. Both complexes therefore act as vital RNA chaperones to initiate eukaryotic ribosome assembly. 1 Laboratory of Protein and Nucleic Acid Chemistry, The Rockefeller University, New York, New York 10065, USA. 2 Wellcome Trust Centre for Cell Biology, University of Edinburgh, Michael Swann Building, Max Born Crescent, Edinburgh EH9 3BF, UK. 3 Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
    [Show full text]
  • Rrp9 As a Potential Novel Antifungal Target in Candida Albicans: Evidences from in Silico Studies Awad Ali, Archana Wakharde and Sankunny Mohan Karuppayil*
    Research Article iMedPub Journals Medical Mycology: Open Access 2017 http://www.imedpub.com/ Vol.3 No.2:26 ISSN 2471-8521 DOI: 10.4172/2167-7972.100026 Rrp9 as a Potential Novel Antifungal Target in Candida albicans: Evidences from In Silico Studies Awad Ali, Archana Wakharde and Sankunny Mohan Karuppayil* School of Life Sciences (DST-FIST&UGC-SAP Sponsored), S.R.T.M. University (NAAC Accredited with "A" grade), Nanded, 431606, Maharashtra state, India *Corresponding author: Sankunny Mohan Karuppayil, Professor and Director, School of Life Sciences (DST-FIST&UGC-SAP Sponsored), S.R.T.M. University (NAAC Accredited with "A" grade), Nanded, 431606, Maharashtra state, India, Tel: +919764386253; E-mail: [email protected] Received date: November 06, 2017; Accepted date: November 22, 2017; Published date: November 30, 2017 Citation: Ali A, Wakharde A, Karuppayil SM (2017) Rrp9 as a Potential Novel Antifungal Target in Candida albicans: Evidences from In Silico Studies. Med Mycol Open Access Vol.3 No. 2: 26. Copyright: ©2017 Ali A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Human G-protein coupled receptors (GPCRs) are complex compared to C. albicans GPCR. C. albicans has GPCR proteins Abstract such as Gpr1, Ste2, and Ste3. Gpr1 is a glucose sensor that plays important roles in morphogenesis [4]. Ste2 and Ste3 are Dicyclomine is a selective muscarinic M1 receptor pheromone receptors which play roles in mating and antagonist in humans.
    [Show full text]
  • The Complete Structure of the Small-Subunit Processome
    ARTICLES The complete structure of the small-subunit processome Jonas Barandun1,4 , Malik Chaker-Margot1,2,4 , Mirjam Hunziker1,4 , Kelly R Molloy3, Brian T Chait3 & Sebastian Klinge1 The small-subunit processome represents the earliest stable precursor of the eukaryotic small ribosomal subunit. Here we present the cryo-EM structure of the Saccharomyces cerevisiae small-subunit processome at an overall resolution of 3.8 Å, which provides an essentially complete near-atomic model of this assembly. In this nucleolar superstructure, 51 ribosome-assembly factors and two RNAs encapsulate the 18S rRNA precursor and 15 ribosomal proteins in a state that precedes pre-rRNA cleavage at site A1. Extended flexible proteins are employed to connect distant sites in this particle. Molecular mimicry and steric hindrance, as well as protein- and RNA-mediated RNA remodeling, are used in a concerted fashion to prevent the premature formation of the central pseudoknot and its surrounding elements within the small ribosomal subunit. Eukaryotic ribosome assembly is a highly dynamic process involving during which the four rRNA domains of the 18S rRNA (5′, central, in excess of 200 non-ribosomal proteins and RNAs. This process 3′ major and 3′ minor) are bound by a set of specific ribosome-assem- starts in the nucleolus where rRNAs for the small ribosomal subunit bly factors7,8. Biochemical studies indicated that these factors and the (18S rRNA) and the large ribosomal subunit (25S and 5.8S rRNA) 5′-ETS particle probably contribute to the independent maturation are initially transcribed as part of a large 35S pre-rRNA precursor of the domains of the SSU7,8.
    [Show full text]
  • Dual Function of C/D Box Small Nucleolar Rnas in Rrna PNAS PLUS Modification and Alternative Pre-Mrna Splicing
    Dual function of C/D box small nucleolar RNAs in rRNA PNAS PLUS modification and alternative pre-mRNA splicing Marina Falaleevaa, Amadis Pagesb, Zaneta Matuszeka, Sana Hidmic, Lily Agranat-Tamirc, Konstantin Korotkova, Yuval Nevod, Eduardo Eyrasb,e, Ruth Sperlingc, and Stefan Stamma,1 aDepartment of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536; bDepartment of Experimental and Health Sciences, Universitat Pompeu Fabra, E08003 Barcelona, Spain; cDepartment of Genetics, Hebrew University of Jerusalem, 91904 Jerusalem, Israel; dDepartment of Microbiology and Molecular Genetics, Computation Center at the Hebrew University and Hadassah Medical Center, 91904 Jerusalem, Israel; and eCatalan Institution for Research and Advanced Studies, E08010 Barcelona, Spain Edited by James E. Dahlberg, University of Wisconsin Medical School, Madison, WI, and approved February 5, 2016 (received for review October 1, 2015) C/D box small nucleolar RNAs (SNORDs) are small noncoding RNAs, SNORD fragments are not microRNAs, which have an average and their best-understood function is to target the methyltrans- length of 21–22 nt (13–16), and suggests that these fragments may ferase fibrillarin to rRNA (for example, SNORD27 performs 2′-O- have additional functions. methylation of A27 in 18S rRNA). Unexpectedly, we found a subset The association of some SNORDs with specific diseases sug- of SNORDs, including SNORD27, in soluble nuclear extract made gests that they may possess functions in addition to directing the under native conditions, where fibrillarin was not detected, indi- 2′-O-methylation of rRNA. For example, the loss of SNORD116 cating that a fraction of the SNORD27 RNA likely forms a protein expression is a decisive factor in Prader–Willi syndrome, the most complex different from canonical snoRNAs found in the insoluble common genetic cause for hyperphagia and obesity (17, 18).
    [Show full text]
  • Anti-Fibrillarin Antibody (ARG10749)
    Product datasheet [email protected] ARG10749 Package: 50 μl anti-Fibrillarin antibody Store at: -20°C Summary Product Description Rabbit Polyclonal antibody recognizes Fibrillarin Tested Reactivity Hu, Ms, Rat, Mk Tested Application ICC/IF, IHC-Fr, WB Host Rabbit Clonality Polyclonal Isotype IgG Target Name Fibrillarin Antigen Species Human Immunogen Full length Human Fibrillarin expressed in and purified from E. coli. Conjugation Un-conjugated Alternate Names rRNA 2'-O-methyltransferase fibrillarin; RNU3IP1; 34 kDa nucleolar scleroderma antigen; FIB; FLRN; EC 2.1.1.-; Histone-glutamine methyltransferase Application Instructions Application table Application Dilution ICC/IF 1:2000 - 1:5000 IHC-Fr Assay-dependent WB 1:2000 - 1:5000 Application Note * The dilutions indicate recommended starting dilutions and the optimal dilutions or concentrations should be determined by the scientist. Calculated Mw 34 kDa Properties Form Liquid Purification Affinity purification. Buffer PBS and 50% Glycerol. Stabilizer 50% Glycerol Concentration 1 mg/ml Storage instruction For continuous use, store undiluted antibody at 2-8°C for up to a week. For long-term storage, aliquot and store at -20°C. Storage in frost free freezers is not recommended. Avoid repeated freeze/thaw cycles. Suggest spin the vial prior to opening. The antibody solution should be gently mixed before use. Note For laboratory research only, not for drug, diagnostic or other use. www.arigobio.com 1/3 Bioinformation Gene Symbol FBL Gene Full Name fibrillarin Background This gene product is a component of a nucleolar small nuclear ribonucleoprotein (snRNP) particle thought to participate in the first step in processing preribosomal RNA. It is associated with the U3, U8, and U13 small nuclear RNAs and is located in the dense fibrillar component (DFC) of the nucleolus.
    [Show full text]
  • Interdependent Action of KH Domain Proteins Krr1 and Dim2 Drive the 40S Platform Assembly
    ARTICLE DOI: 10.1038/s41467-017-02199-4 OPEN Interdependent action of KH domain proteins Krr1 and Dim2 drive the 40S platform assembly Miriam Sturm1, Jingdong Cheng 2, Jochen Baßler 1, Roland Beckmann2 & Ed Hurt 1 Ribosome biogenesis begins in the nucleolus with the formation of 90S pre-ribosomes, from which pre-40S and pre-60S particles arise that subsequently follow separate maturation pathways. Here, we show how structurally related assembly factors, the KH domain proteins 1234567890 Krr1 and Dim2, participate in ribosome assembly. Initially, Dim2 (Pno1) orchestrates an early step in small subunit biogenesis through its binding to a distinct region of the 90S pre- ribosome. This involves Utp1 of the UTP-B module, and Utp14, an activator of the DEAH-box helicase Dhr1 that catalyzes the removal of U3 snoRNP from the 90S. Following this dis- mantling reaction, the pre-40S subunit emerges, but Dim2 relocates to the pre-40S platform domain, previously occupied in the 90S by the other KH factor Krr1 through its interaction with Rps14 and the UTP-C module. Our findings show how the structurally related Krr1 and Dim2 can control stepwise ribosome assembly during the 90S-to-pre-40S subunit transition. 1 Biochemistry Centre, University of Heidelberg, Heidelberg 69221, Germany. 2 Department of Biochemistry, Gene Center, Center for integrated Protein Science - Munich (CiPS-M), Ludwig-Maximillian University, Munich 81377, Germany. Correspondence and requests for materials should be addressed to E.H. (email: [email protected]) NATURE COMMUNICATIONS | 8: 2213 | DOI: 10.1038/s41467-017-02199-4 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02199-4 he biogenesis of eukaryotic ribosomes is a complex and compacted, processes during which they migrate from the extremely energy-consuming process, during which nucleolus to the nucleoplasm, before export into the cytoplasm, T 2–4 actively growing cells devote most of their RNA poly- where final maturation occurs .
    [Show full text]