DOCSLIB.ORG

Long Noncoding RNA MALAT1 Promotes Hepatocellular

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Long Noncoding RNA MALAT1 Promotes Hepatocellular Published OnlineFirst December 19, 2016; DOI: 10.1158/0008-5472.CAN-16-1508 Cancer Molecular and Cellular Pathobiology Research Long Noncoding RNA MALAT1 Promotes Hepatocellular Carcinoma Development by SRSF1 Upregulation and mTOR Activation Pushkar Malakar1,AsafShilo1, Adi Mogilevsky1,IlanStein2,EliPikarsky2, Yuval Nevo3, Hadar Benyamini3, Sharona Elgavish3, Xinying Zong4, Kannanganattu V. Prasanth4, and Rotem Karni1 Abstract Several long noncoding RNAs (lncRNA) are abrogated in way by modulating the alternative splicing of S6K1. Inhibition cancer but their precise contributions to oncogenesis are still of SRSF1 expression or mTOR activity abolishes the oncogenic emerging. Here we report that the lncRNA MALAT1 is upregu- properties of MALAT1, suggesting that SRSF1 induction and lated in hepatocellular carcinoma and acts as a proto-oncogene mTOR activation are essential for MALAT1-induced transfor- through Wnt pathway activation and induction of the onco- mation. Our results reveal a mechanism by which lncRNA genic splicing factor SRSF1. Induction of SRSF1 by MALAT1 MALAT1 acts as a proto-oncogene in hepatocellular carcinoma, modulates SRSF1 splicing targets, enhancing the production of modulating oncogenic alternative splicing through SRSF1 upre- antiapoptotic splicing isoforms and activating the mTOR path- gulation. Cancer Res; 77(5); 1–13. Ó2016 AACR. Introduction sequence is highly conserved among mammals (7). MALAT1 is assumed to play an important role in regulation of gene Recent estimations from transcriptome studies suggest that expression due to its residence in nuclear speckles (8). Local- more than 75% of the human genome is transcribed, generating ization of MALAT1 in nuclear speckles is dependent on active thousands of noncoding RNAs (ncRNA), which are not trans- transcription by RNA-polymerase II (9). lated into proteins (1). This large group of noncoding tran- MALAT1 was shown to modulate the positioning of a member scripts contains many small RNAs (2). However, the majority of of the SR family of pre-mRNA splicing factors to the transcription ncRNAs is longer than 200 nucleotides and is designated as site of an inducible transgene array (9). SR proteins are a family of long ncRNA (lncRNA; ref. 2). lncRNAs are involved in the RNA-binding proteins that regulate both general and alternative regulation of almost every step of gene expression, ranging splicing. SRSF1, a classic example of a SR protein family member, from chromatin remodeling, transcriptional control, regulation has been shown to regulate the alternative splicing of various of splicing, mRNA stability, mRNA translation, miRNA proces- oncogenes and tumor suppressor genes important for tumor sing, and protein stability (3). Expression and function of progression and maintenance (10). SRSF1 was shown to act as lncRNAs are deregulated in several human diseases, including an oncogene by activating the mTORC1 pathway (11). MALAT1 cancer (4). The first lncRNA discovered with an established role was shown to bind active chromatin sites of many genes and to in cancer is metastasis-associated lung adenocarcinoma tran- bind several splicing factors, among them SRSF1. This binding script 1 (MALAT1), later referred to as nuclear-enriched abun- affects both its localization and phosphorylation by the kinase dant transcript 2 (NEAT2; ref. 5). The MALAT1 transcript is SRPK1, leading to changes in alternative splicing of its splicing greater than 6 kb in length and is highly abundant (6). MALAT1 targets (12, 13). The process of alternative splicing is widely deregulated in various cancers and many tumors express can- fi 1Department of Biochemistry and Molecular Biology, Hebrew University-Hadas- cer-speci c splicing isoforms that are absent or are expressed at sah Medical School, Ein Karem, Jerusalem, Israel. 2Department of Immunology different levels in the corresponding normal tissues (14, 15). and Cancer Research, Hebrew University-Hadassah Medical School, Ein Karem, Many of these transcripts encode for oncogenes and tumor Jerusalem, Israel. 3Bioinformatics unit, the Institute for Medical Research Israel- suppressor genes (16–18). Canada, Hebrew University-Hadassah Medical School, Ein Karem, Jerusalem, In spite of its abundance, MALAT1 is dispensable for viabil- 4 Israel. Department of Cell and Developmental Biology, University of Illinois at ity,andMALAT1knockoutmicedonotpresentanyobvious Urbana-Champaign, Urbana, Illinois. abnormal phenotype (19, 20). One report using MALAT1 Note: Supplementary data for this article are available at Cancer Research knockout mice suggested that MALAT1 is not necessary for Online (http://cancerres.aacrjournals.org/). development but has the potential to regulate the expression of Corresponding Author: Rotem Karni, Hebrew University-Hadassah Medical nearby genes (21). In the past few years, studies have found that School, Ein Karem, Jerusalem 91120, Israel. Phone: 972-2675-8289; Fax: 972- MALAT1 is upregulated in several cancers, and its knockdown 2675-7379; E-mail: [email protected] inhibited tumorigenesis (22, 23). Several reports showed that doi: 10.1158/0008-5472.CAN-16-1508 MALAT1 regulates the Wnt-b-catenin pathway by enhancing Ó2016 American Association for Cancer Research. nuclear b-catenin levels and elevating c-Myc expression www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst December 19, 2016; DOI: 10.1158/0008-5472.CAN-16-1508 Malakar et al. (24, 25). It was shown recently that MALAT1 regulates the RT-PCR differentiation and metastasis of mammary tumors (26). How- Total RNA was extracted with TRI Reagent (Sigma) and 1 mgof ever, neither a direct causative role for MALAT1 in early steps of total RNA was reverse transcribed using M-MLV reverse transcrip- transformation and tumorigenesis, nor the mechanism by tase (Promega). PCR was performed on 1/10th volume (2 mL) of which MALAT1 causes cellular transformation has been shown the cDNA using PCR Mix (Kapa Biosystems). to date. In this study, we show that MALAT1 is upregulated in hepa- tocellular carcinoma, and acts as a proto-oncogene to induce qRT-PCR m transformation and tumorigenesis of liver progenitor cells by Total RNA was extracted with TRI Reagent (Sigma), and 1 gof Wnt pathway activation, SRSF1 upregulation, and mTORC1 total RNA was reverse transcribed using M-MLV reverse transcrip- activation. tase (Promega) after DNASE treatment (Promega). qPCR was performed on the cDNA using SYBR Green Mix (Roche) and CFX96 (Bio-Rad) real-time PCR machine. Primer list is supplied Materials and Methods in Supplementary Table S1. Animal care All animal experiments were performed in accordance with the Immunoblotting guidelines of the Hebrew University committee for the use of Cells were lysed in Laemmli buffer and analyzed for total animals for research and under the approval of the Hebrew protein concentration as described (10). Twenty micrograms of University Ethics committee. Veterinary care was provided to all total protein from each cell lysate was separated by SDS-PAGE animals by the Hebrew University animal care facility staff in and transferred on to a polyvinylidene difluoride (PVDF) mem- accord with AAALAC standard procedures and as approved by the brane. Primary antibodies used were TCF7L2 EP20334 Hebrew University Ethics committee. (1:10,000; Abcam), c-MYC Sc-40 (1:1,000; Santa Cruz Biotech- nology), SRSF1 (AK96 culture supernatant 1:300; ref. 29), Cell culture GAPDH (1:500; Santa Cruz Biotechnology), a-tubulin Liver progenitor cells from embryonic day 18 fetal livers from (1:1,000; Santa Cruz Biotechnology), b-catenin (1:2,000; À À TP53 / mice were isolated and immortalized with MSCV-based Sigma), b-actin (1:2,000; Santa Cruz Biotechnology), total retroviruses expressing MYC-IRES-GFP as previously described to S6K1-anti-p70 (1:1,000; BD Transduction Laboratories), phos- À À generate TP53 / hepatocytes overexpressing c-MYC (PHM-1) pho-4E-BP1 Thr70 (1:1,000; Cell Signaling Technology), 4E- cells (27, 28). PHM-1, FLC4, and BWTG-3 cells were grown in BP1 (1:1,000; Cell Signaling Technology). Secondary antibodies DMEM supplemented with 10% FCS, 0.1 mg/mL penicillin, and used were HRP-conjugated goat anti-mouse, goat anti-rabbit, 0.1 mg/mL streptomycin. All cell lines have been tested and donkey anti-goat IgG (HþL; 1:10,000; Jackson Laboratories). authenticated using STR loci (TH01, TPOX, vWA, CSF1PO, D16S539, D7S820, D13S317, and D5S818) plus Amelogenin for gender identification for human cell line authentication by the Colony formation assay biosynthesis DNA Identity Testing Centre. Cells were seeded in 6-well plates (1,000 cells/well) and grown for 10 days. After fixation with 2.5% glutaraldehyde, the plates were washed three times. Fixed cells were then stained with Stable cell lines methylene blue solution (1% methylene blue in 0.1 mol/L borate pCD513B1 empty (System Biosciences) and pCD513B1-hMA- buffer, pH 8.5) for 60 minutes at room temperature. Plates were LAT1 lentiviruses were prepared using the manufacturer's instruc- photographed after extensive washing and air drying. tions. These viruses were used to infect PHM-1 cells. Cells were selected by the addition of puromycin (2 mg/mL) for 72–96 hours. In the case of infection with MLP-puro-shRNA viruses, cells were Anisomycin-mediated cell death assay selected with puromycin (2 mg/mL) for 96 hours. Following transduction and selection, cells were seeded in 6-well plates (2 Â 105 cells/well). Twenty-four hours later, cells Growth curve were incubated with 1 mmol/L anisomycin in DMEM containing PHM-1, FLC4, and BWTG-3 cells were either infected with 0.1% serum for 24 hours. Medium and PBS washes were collected the indicated lentiviruses or treated with siRNAs. After selection together with trypsinized cells from each well into 15-mL tubes or siRNA treatment, 500 (PHM-1) or 2,000 cells (FLC4 and and centrifuged at 1,500 rpm for 5 minutes. Cells were washed BWTG-3) were seeded in 96-well plates. Cells were stained with with PBS and resuspended in 50 mL of HEPES buffer. Ten micro- 1% methylene blue in 0.1 mol/L borate buffer and fixed.
Load more

Recommended publications
  • Serine Arginine-Rich Protein-Dependent Suppression Of
    Serine͞arginine-rich protein-dependent suppression of exon skipping by exonic splicing enhancers El Che´ rif Ibrahim*, Thomas D. Schaal†, Klemens J. Hertel‡, Robin Reed*, and Tom Maniatis†§ *Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115; †Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138; and ‡Department of Microbiology and Molecular Genetics, University of California, Irvine, CA 92697-4025 Contributed by Tom Maniatis, January 28, 2005 The 5؅ and 3؅ splice sites within an intron can, in principle, be joined mechanisms by which exon skipping is prevented. Our data to those within any other intron during pre-mRNA splicing. How- reveal that splicing to the distal 3Ј splice site is suppressed by ever, exons are joined in a strict 5؅ to 3؅ linear order in constitu- proximal exonic sequences and that SR proteins are required for tively spliced pre-mRNAs. Thus, specific mechanisms must exist to this suppression. Thus, SR protein͞exonic enhancer complexes prevent the random joining of exons. Here we report that insertion not only function in exon and splice-site recognition but also play of exon sequences into an intron can inhibit splicing to the a role in ensuring that 5Ј and 3Ј splice sites within the same intron .downstream 3؅ splice site and that this inhibition is independent of are used, thus suppressing exon skipping intron size. The exon sequences required for splicing inhibition were found to be exonic enhancer elements, and their inhibitory Materials and Methods activity requires the binding of serine͞arginine-rich splicing fac- Construction of Plasmids.
    [Show full text]
  • Sorting out the Complexity of SR Protein Functions
    Downloaded from rnajournal.cshlp.org on February 6, 2009 - Published by Cold Spring Harbor Laboratory Press RNA (2000), 6:1197–1211+ Cambridge University Press+ Printed in the USA+ Copyright © 2000 RNA Society+ REVIEW Sorting out the complexity of SR protein functions BRENTON R. GRAVELEY Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA INTRODUCTION been clear whether all of these activities occur during the removal of each intron+ Recent studies now sug- Members of the serine/arginine-rich (SR) protein fam- gest that all of the proposed SR protein functions are ily have multiple functions in the pre-mRNA splicing carried out during each round of splicing, and at least reaction+ In addition to being required for the removal some of these functions are performed by independent of constitutively spliced introns, SR proteins can func- SR protein molecules+ This review discusses recent tion to regulate alternative splicing both in vitro and in advances in understanding the diverse functions of SR vivo (Ge & Manley, 1990; Krainer et al+, 1990a; Fu proteins in metazoan pre-mRNA splicing and presents et al+, 1992; Zahler et al+, 1993a; Caceres et al+, 1994; a model that takes these new findings into account+ Wang & Manley, 1995)+ In the cell, SR proteins migrate Although the reader should keep in mind that the ac- from speckles—subnuclear domains that may function tivity of SR proteins in vivo can be influenced by mod- as storage sites for certain splicing factors—to
    [Show full text]
  • Functional Control of HIV-1 Post-Transcriptional Gene Expression by Host Cell Factors
    Functional control of HIV-1 post-transcriptional gene expression by host cell factors DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Amit Sharma, B.Tech. Graduate Program in Molecular Genetics The Ohio State University 2012 Dissertation Committee Dr. Kathleen Boris-Lawrie, Advisor Dr. Anita Hopper Dr. Karin Musier-Forsyth Dr. Stephen Osmani Copyright by Amit Sharma 2012 Abstract Retroviruses are etiological agents of several human and animal immunosuppressive disorders. They are associated with certain types of cancer and are useful tools for gene transfer applications. All retroviruses encode a single primary transcript that encodes a complex proteome. The RNA genome is reverse transcribed into DNA, integrated into the host genome, and uses host cell factors to transcribe, process and traffic transcripts that encode viral proteins and act as virion precursor RNA, which is packaged into the progeny virions. The functionality of retroviral RNA is governed by ribonucleoprotein (RNP) complexes formed by host RNA helicases and other RNA- binding proteins. The 5’ leader of retroviral RNA undergoes alternative inter- and intra- molecular RNA-RNA and RNA-protein interactions to complete multiple steps of the viral life cycle. Retroviruses do not encode any RNA helicases and are dependent on host enzymes and RNA chaperones. Several members of the host RNA helicase superfamily are necessary for progressive steps during the retroviral replication. RNA helicase A (RHA) interacts with the redundant structural elements in the 5’ untranslated region (UTR) of retroviral and selected cellular mRNAs and this interaction is necessary to facilitate polyribosome formation and productive protein synthesis.
    [Show full text]
  • Srsf10 and the Minor Spliceosome Control Tissue-Specific and Dynamic
    SHORT REPORT Srsf10 and the minor spliceosome control tissue-specific and dynamic SR protein expression Stefan Meinke1, Gesine Goldammer1, A Ioana Weber1,2, Victor Tarabykin2, Alexander Neumann1†, Marco Preussner1*, Florian Heyd1* 1Freie Universita¨ t Berlin, Institute of Chemistry and Biochemistry, Laboratory of RNA Biochemistry, Berlin, Germany; 2Institute of Cell Biology and Neurobiology, Charite´-Universita¨ tsmedizin Berlin, corporate member of Freie Universita¨ t Berlin, Humboldt-Universita¨ t zu Berlin, and Berlin Institute of Health, Berlin, Germany Abstract Minor and major spliceosomes control splicing of distinct intron types and are thought to act largely independent of one another. SR proteins are essential splicing regulators mostly connected to the major spliceosome. Here, we show that Srsf10 expression is controlled through an autoregulated minor intron, tightly correlating Srsf10 with minor spliceosome abundance across different tissues and differentiation stages in mammals. Surprisingly, all other SR proteins also correlate with the minor spliceosome and Srsf10, and abolishing Srsf10 autoregulation by Crispr/ Cas9-mediated deletion of the autoregulatory exon induces expression of all SR proteins in a human cell line. Our data thus reveal extensive crosstalk and a global impact of the minor spliceosome on major intron splicing. *For correspondence: [email protected] (MP); [email protected] (FH) Introduction Present address: †Omiqa Alternative splicing (AS) is a major mechanism that controls gene expression (GE) and expands the Corporation, c/o Freie proteome diversity generated from a limited number of primary transcripts (Nilsen and Graveley, Universita¨ t Berlin, Altensteinstraße, Germany 2010). Splicing is carried out by a multi-megadalton molecular machinery called the spliceosome of which two distinct complexes exist.
    [Show full text]
  • SR Proteins: a Conserved Family of Pre-Mrna Splicing Factors
    Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press SR proteins: a conserved family of pre-mRNA splicing factors Alan M. Zahler, William S. Lane/ John A. Stolk, and Mark B. Roth^ Fred Hutchinson Cancer Research Center, Division of Basic Sciences, Seattle, Washington 98104 USA; ^Harvard Microchemistry Facility, Cambridge, Massachusetts 02138 USA We demonstrate that four different proteins from calf thymus are able to restore splicing in the same splicing-deficient extract using several different pre-mRNA substrates. These proteins are members of a conserved family of proteins recognized by a monoclonal antibody that binds to active sites of RNA polymerase II transcription. We purified this family of nuclear phosphoproteins to apparent homogeneity by two salt precipitations. The family, called SR proteins for their serine- and arginine-rich carboxy-terminal domains, consists of at least five different proteins with molecular masses of 20, 30, 40, 55, and 75 kD. Microsequencing revealed that they are related but not identical. In four of the family members a repeated protein sequence that encompasses an RNA recognition motif was observed. We discuss the potential role of this highly conserved, functionally related set of proteins in pre-mRNA splicing. [Key Words: SR proteins; alternative splicing; RNA splicing; splicing factors] Received lanuary 21, 1992; revised version accepted March 2, 1992. Studies of mRNAs from many different tissues and de­ high levels of spliceosomal components, including sites velopmental stages show that regulation of RNA pro­ of RNA polymerase II transcription on lampbrush chro­ cessing can lead to the expression of multiple proteins mosomes and B "snurposomes" in oocyte nuclei, and from single genes (Smith et al.
    [Show full text]
  • Protection Against Retrovirus Pathogenesis by SR Protein Inhibitors
    Protection against retrovirus pathogenesis by SR protein inhibitors. Anne Keriel, Florence Mahuteau-Betzer, Chantal Jacquet, Marc Plays, David Grierson, Marc Sitbon, Jamal Tazi To cite this version: Anne Keriel, Florence Mahuteau-Betzer, Chantal Jacquet, Marc Plays, David Grierson, et al.. Protec- tion against retrovirus pathogenesis by SR protein inhibitors.. PLoS ONE, Public Library of Science, 2009, 4 (2), pp.e4533. 10.1371/journal.pone.0004533. hal-00368669 HAL Id: hal-00368669 https://hal.archives-ouvertes.fr/hal-00368669 Submitted on 25 May 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License Protection against Retrovirus Pathogenesis by SR Protein Inhibitors Anne Keriel1, Florence Mahuteau-Betzer2, Chantal Jacquet1, Marc Plays1, David Grierson3,Marc Sitbon1*, Jamal Tazi1* 1 Universite´ Montpellier 2 Universite´ Montpellier 1 CNRS, Institut de Ge´ne´tique Mole´culaire de Montpellier (IGMM), UMR5535, IFR122, Montpellier, France, 2 Laboratoire de Pharmaco-chimie, CNRS-Institut Curie, UMR 176 Bat 110 Centre Universitaire, Orsay, France, 3 Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia, Canada Abstract Indole derivatives compounds (IDC) are a new class of splicing inhibitors that have a selective action on exonic splicing enhancers (ESE)-dependent activity of individual serine-arginine-rich (SR) proteins.
    [Show full text]
  • HIV-1: to Splice Or Not to Splice, That Is the Question
    viruses Review HIV-1: To Splice or Not to Splice, That Is the Question Ann Emery 1 and Ronald Swanstrom 1,2,3,* 1 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA; [email protected] 2 Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA 3 Center for AIDS Research, University of North Carolina, Chapel Hill, NC 27599, USA * Correspondence: [email protected] Abstract: The transcription of the HIV-1 provirus results in only one type of transcript—full length genomic RNA. To make the mRNA transcripts for the accessory proteins Tat and Rev, the genomic RNA must completely splice. The mRNA transcripts for Vif, Vpr, and Env must undergo splicing but not completely. Genomic RNA (which also functions as mRNA for the Gag and Gag/Pro/Pol precursor polyproteins) must not splice at all. HIV-1 can tolerate a surprising range in the relative abundance of individual transcript types, and a surprising amount of aberrant and even odd splicing; however, it must not over-splice, which results in the loss of full-length genomic RNA and has a dramatic fitness cost. Cells typically do not tolerate unspliced/incompletely spliced transcripts, so HIV-1 must circumvent this cell policing mechanism to allow some splicing while suppressing most. Splicing is controlled by RNA secondary structure, cis-acting regulatory sequences which bind splicing factors, and the viral protein Rev. There is still much work to be done to clarify the combinatorial effects of these splicing regulators. These control mechanisms represent attractive targets to induce over-splicing as an antiviral strategy.
    [Show full text]
  • The Transcriptional and Epigenetic Role of Brd4 in the Regulation of the Cellular Stress Response
    THE TRANSCRIPTIONAL AND EPIGENETIC ROLE OF BRD4 IN THE REGULATION OF THE CELLULAR STRESS RESPONSE INAUGURAL-DISSERTATION to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by Michelle Hussong from Zweibrücken 2015 Die vorliegende Arbeit wurde im Zeitraum von Juli 2012 bis September 2015 am Max- Planck-Institut für Molekulare Genetik in Berlin sowie an der Universität zu Köln unter der Leitung von Frau Prof. Dr. Dr. Michal-Ruth Schweiger angefertigt. 1. Gutachter: Prof. Dr. Dr. Michal-Ruth Schweiger 2. Gutachter: Prof. Dr. Rupert Mutzel Disputation am 07.12.2015 ACKNOWLEDGMENT ACKNOWLEDGMENT This dissertation would not have been possible without the guidance and the help of many people who in one way or another contributed to the preparation and completion of this study. Firstly, I would like to express my sincere gratitude to my advisor Prof. Dr. Dr. Michal-Ruth Schweiger, for her continuous support throughout my PhD study, for her patience, motivation, and immense knowledge. I am eminently thankful for the multiple possibilities she gave me to work on this interesting and challenging field of research. I also want to thank Professor Dr. Rupert Mutzel for taking the time of being my second supervisor. My sincere thanks also goes to Prof. Dr. Hans Lehrach for having given me the opportunity to do my PhD thesis in the extraordinary and inspiring environment at the Max-Planck- Institute for Molecular Genetics in Berlin. Especially, the multitude of technologies and knowledge in his department made my work successful.
    [Show full text]
  • Untersuchung Zur Rolle Des La-Verwandten Proteins LARP4B Im Mrna- Metabolismus
    Untersuchung zur Rolle des La-verwandten Proteins LARP4B im mRNA-Metabolismus Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Julius-Maximilians-Universität Würzburg vorgelegt von Maritta Küspert aus Schwäbisch Hall Würzburg 2014 Eingereicht bei der Fakultät für Chemie und Pharmazie am: ....................................... Gutachter der schriftlichen Arbeit: 1. Gutachter: Prof. Dr. Utz Fischer 2. Gutachter: Prof. Dr. Alexander Buchberger Prüfer des öffentlichen Promotionskolloquiums: 1. Prüfer: Prof. Dr. Utz Fischer 2. Prüfer: Prof. Dr. Alexander Buchberger 3. Prüfer: Prof. Dr. Stefan Gaubatz Datum des öffentlichen Promotionskolloquiums: ......................................................... Doktorurkunde ausgehändigt am: ................................................................................ Zusammenfassung Eukaryotische messenger-RNAs (mRNAs) müssen diverse Prozessierungsreaktionen durchlaufen, bevor sie der Translationsmaschinerie als Template für die Proteinbiosynthese dienen können. Diese Reaktionen beginnen bereits kotranskriptionell und schließen das Capping, das Spleißen und die Polyadenylierung ein. Erst nach dem die Prozessierung abschlossen ist, kann die reife mRNA ins Zytoplasma transportiert und translatiert werden. mRNAs interagieren in jeder Phase ihres Metabolismus mit verschiedenen trans-agierenden Faktoren und bilden mRNA-Ribonukleoproteinkomplexe (mRNPs) aus. Dieser „mRNP-Code“ bestimmt das Schicksal jeder mRNA und reguliert dadurch die Genexpression auf posttranskriptioneller
    [Show full text]
  • The Splicing-Factor Oncoprotein SF2/ASF Activates Mtorc1
    The splicing-factor oncoprotein SF2/ASF activates mTORC1 Rotem Karni*†‡, Yoshitaka Hippo*, Scott W. Lowe*§, and Adrian R. Krainer*‡ *Cold Spring Harbor Laboratory and §Howard Hughes Medical Institute, P.O. Box 100, Cold Spring Harbor, NY 11724 Edited by Thomas Maniatis, Harvard University, Cambridge, MA, and approved August 22, 2008 (received for review February 11, 2008) The splicing factor SF2/ASF is an oncoprotein that is up-regulated to measure the status of this pathway in cells transformed by in many cancers and can transform immortal rodent fibroblasts SF2/ASF. when slightly overexpressed. The mTOR signaling pathway is Akt, a major effector of the PI3K pathway, is both an upstream activated in many cancers, and pharmacological blockers of this activator of mTOR and a substrate of mTORC2 (2, 3, 13). We pathway are in clinical trials as anticancer drugs. We examined the examined the activity of this pathway in cells transformed by activity of the mTOR pathway in cells transformed by SF2/ASF and SF2/ASF overexpression. We observed no activation of Akt, but found that this splicing factor activates the mTORC1 branch of the phosphorylation of substrates of the mTORC1 complex, S6K pathway, as measured by S6K and eIF4EBP1 phosphorylation. This and 4E-BP1, was elevated in these cells and was reduced upon activation is specific to mTORC1 because no activation of Akt, an SF2/ASF knockdown. To examine whether the activation of this mTORC2 substrate, was detected. mTORC1 activation by SF2/ASF pathway is important for SF2/ASF-mediated transformation, we bypasses upstream PI3K/Akt signaling and is essential for SF2/ASF- blocked mTOR pharmacologically using rapamycin and genet- mediated transformation, as inhibition of mTOR by rapamycin ically using shRNAs to mTOR, Raptor, or Rictor and found that blocked transformation by SF2/ASF in vitro and in vivo.
    [Show full text]
  • The SR Protein Family Peter J Shepard and Klemens J Hertel
    Protein family review The SR protein family Peter J Shepard and Klemens J Hertel Address: Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697-4025, USA. Correspondence: Klemens J Hertel. Email: [email protected] More recent genome-wide studies have identified several Summary other RS-domain-containing proteins, most of which are The processing of pre-mRNAs is a fundamental step required conserved in higher eukaryotes and function in pre-mRNA for the expression of most metazoan genes. Members of the family of serine/arginine (SR)-rich proteins are critical compo- splicing or RNA metabolism [10]. Because of differences in nents of the machineries carrying out these essential processing domain structure, lack of mAb104 recognition, or lack of a events, highlighting their importance in maintaining efficient prototypical RRM, these proteins are referred to as SR-like gene expression. SR proteins are characterized by their ability or SR-related proteins. An extensive list of SR-related to interact simultaneously with RNA and other protein compo- proteins and their functional roles in RNA metabolism was nents via an RNA recognition motif (RRM) and through a recently discussed [11]. domain rich in arginine and serine residues, the RS domain. Their functional roles in gene expression are surprisingly diverse, ranging from their classical involvement in constitutive and While introns are common to all eukaryotes, the complex- alternative pre-mRNA splicing to various post-splicing activities, ity of alternative splicing varies among species. SR proteins including mRNA nuclear export, nonsense-mediated decay, and exist in all metazoan species [8] as well as in some lower mRNA translation.
    [Show full text]
  • Emerging Functions of SRSF1, Splicing Factor and Oncoprotein, in RNA Metabolism and Cancer
    Published OnlineFirst May 7, 2014; DOI: 10.1158/1541-7786.MCR-14-0131 Molecular Cancer Review Research Emerging Functions of SRSF1, Splicing Factor and Oncoprotein, in RNA Metabolism and Cancer Shipra Das and Adrian R. Krainer Abstract Serine/Arginine Splicing Factor 1 (SRSF1) is the archetype member of the SR protein family of splicing regulators. Since its discovery over two decades ago, SRSF1 has been repeatedly surprising and intriguing investigators by the plethora of complex biologic pathways it regulates. These include several key aspects of mRNA metabolism, such as mRNA splicing, stability, and translation, as well as other mRNA-independent processes, such as miRNA processing, protein sumoylation, and the nucleolar stress response. In this review, the structural features of SRSF1 are discussed as they relate to the intricate mechanism of splicing and the multiplicity of functions it performs. Similarly, a list of relevant alternatively spliced transcripts and SRSF1 interacting proteins is provided. Finally, emphasis is given to the deleterious consequences of overexpression of the SRSF1 proto-oncogene in human cancers, and the complex mechanisms and pathways underlying SRSF1-mediated transformation. The accumulated knowledge about SRSF1 provides critical insight into the integral role it plays in maintaining cellular homeostasis and suggests new targets for anticancer therapy. Mol Cancer Res; 12(9); 1195–204. Ó2014 AACR. Introduction splicing, the process through which approximately 95% of Eukaryotic gene expression is a complex process, com- human genes produce multiple mRNA transcripts by the prising several intermediary steps between transcription of differential inclusion of exons or exon segments. Although the pre-mRNA in the nucleus and translation in the cyto- different SR proteins can interchangeably restore constitu- plasm.
    [Show full text]