The Impact of Hud Protein on the Intestinal Nervous System in the Terminal Rectum of Animal Models of Congenital Anorectal Malformation

Total Page:16

File Type:pdf, Size:1020Kb

The Impact of Hud Protein on the Intestinal Nervous System in the Terminal Rectum of Animal Models of Congenital Anorectal Malformation MOLECULAR MEDICINE REPORTS 16: 4797-4802, 2017 The impact of HuD protein on the intestinal nervous system in the terminal rectum of animal models of congenital anorectal malformation MENG KONG1,2*, YURUI WU1* and YUANMEI LIU2* 1Department of Pediatric Surgery, Qilu Children's Hospital of Shandong University (Jinan Children's Hospital), Jinan, Shandong 250022; 2Department of Pediatric Surgery, Affiliated Hospital of Zunyi Medical College, Zunyi, Guizhou 563003, .R.P China Received January 20, 2016; Accepted January 19, 2017 DOI: 10.3892/mmr.2017.7204 Abstract. Patients with congenital anorectal malformation study may provide a useful theoretical reference for the treat- (ARM) often present with different degrees of defecation ment of postoperative defecation dysfunction in patients with dysfunction severity following corrective operations. Therefore, ARM. studies on how to improve the postoperative defecation func- tion of patients with ARM are of clinical importance. The Introduction present study investigated the expression of the HuD protein in the terminal rectum of ARM embryonic rats and explored the Congenital anorectal malformation (ARM) is one of the effect of HuD expression on the development of the intestinal more common digestive tract abnormalities in children nervous system. Pregnant Sprague Dawley rats were random- with an incidence of ~1/1,500-1/5,000 (1). With advances in ized into a control or ARM (induced by ethylene thiourea) surgical techniques, the ARM recovery rate has improved. group. The terminal rectums of the embryonic rats were However, certain patients continue to suffer from defecation obtained during pregnancy (20 days). The histological changes dysfunctions, including fecal soiling and constipation, with an of the terminal rectum were observed using hematoxylin incidence of ~20-30% due to the multifactorial pathogenesis and eosin staining. The expression of the HuD protein was mechanisms and complex pathological changes associated assessed by immunohistochemistry and western blot analysis. with ARM. However, morphological abnormalities can be In the control group, the histological structure of the terminal corrected following surgery (2), which greatly influence the rectum was well‑defined and a large number of submucosal quality of life of patients (3). Previously, these dysfunctions and intermuscular neurons with a rich cytoplasm and strong were thought to be associated with a postoperative megacolon neuritis were observed. In the ARM group, the histological or megarectum or postoperative rectal weakness. It is suggested layers were ill‑defined and the number of neurons was small. that maldevelopment of features, including the perianal muscle Immunohistochemistry and western blot analysis demon- groups and self-enteric nervous system (self-ENS), causes a strated that the concentration of the HuD protein in the ARM change in enteric dynamics, which is closely associated with group was significantly lower compared with the control group the development of postoperative defecation dysfunction in (312.90±53.40:456.40±57.13; 0.24±0.05:0.45±0.06, P<0.05). patients with ARM (3). Patients with ARM present with no HuD was abnormally expressed in the terminal rectum of the anus however additionally with noticeable enteric nervous ARM embryonic rats and may be involved in the development maldevelopment, which is particularly associated with the and maturation of the enteric nervous system. The present maldevelopment of the ENS in the terminal rectum (4). Originating from neural crest cells, the ENS is a complex, self-created somatic nervous system. Subsequent to entering the intestinal tract and phases of gradual migration, prolif- eration and agglomeration, it develops into gangliocytes and Correspondence to: Professor Yuanmei Liu, Department of colloid cells (5). During this process, the disturbance of any Pediatric Surgery, Affiliated Hospital of Zunyi Medical College, step can affect the development of gangliocytes, resulting in 201 Dalian Road, Zunyi, Guizhou 563003, P.R. China E-mail: [email protected] ENS maldevelopment. The ENS forms different layers of nerve plexuses in the digestive tract, including the myenteric nervous *Contributed equally plexus (MP) and submucosal nervous plexus (SP). The MP is located between the circular muscle and longitudinal muscle Key words: malformation, intermuscular neuron, immuno- layers, while the SP lies between the stratum mucosum and the histochemistry, protein circular layer. The MP and SP are responsible for processing the signals from the enteric cavity and intestinal wall and regulating the contraction, secretion and blood supply of the 4798 KONG et al: HuD IN ENS intestinal tract. Therefore, they are of great significance for the administered physiological saline at the same dose. At day 20, formation of the defecation reflex (6). Maldevelopment of the 10% chloral hydrate (NanJing SenBeiJia Biotechnology Co., ENS in the terminal rectum reaches up to 60% in cases of anal Ltd., Nanjing, China) was given at 0.4 ml/100 g to the animals atresia (7). Kong et al (8) identified that there are no notice- and then the intrauterine embryonic rats were removed. able intestinal glands distributed throughout the rectal cecum; Abnormalities were judged by an experienced chief physician however, proliferated squamous epithelial tissue and thickened using a blinding method. A section of the terminal rectum muscular layers are present in ARM embryonic rats and the was stored at 80˚C and the remaining specimen fixed in 4% number of gangliocytes in the terminal rectum of ARM rats formaldehyde for 12‑36 h. Routine dehydration and paraffin were significantly decreased compared with that in the normal wax embedding were then performed. group. The ENS is a critical system for regulating the functions All the procedures were approved by the Institute of of the intestinal tract. Defecation function is closely associated Animal Ethics of the Affiliated Hospital of Zunyi Medical with the developmental condition of the ENS in the terminal College (Zunyi, China). rectum (4). Therefore, recovery of the normal function of the ENS in the terminal rectum has become a primary focus in the Hematoxylin and eosin (HE) staining. Paraffin wax‑embedded treatment of ARM in recent years. pelvic perineal tissue and terminal rectum tissue were seri- HuD is a member of the highly conserved, neuron‑specific ally sectioned at 4 mm thickness. Routine HE staining was RNA-binding Hu family, which is homologous to the ELAV then performed. The structures of the terminal rectums from protein in Drosophila. Following transcription, HuD regulates the two groups were observed under an optical microscope. the expression of neuron-specific genes and serves impor- A total of 10 sections were then obtained from each group tant roles in the growth, development and differentiation of and 10 fields were randomly selected in each section to neurons (9-11). It is a necessary protein for the formation and calculate the average number of intermuscular and submu- regeneration of nervous processes (12-15). HuD demonstrates cosal neurons. abnormal expression in diseased intestinal canals of patients with a congenital megacolon, which indicates that HuD has a Immunohistochemistry. A total of 10 paraffin-embedded close association with the development of the ENS. The intes- sections were randomly selected from each group. They tinal canal in a congenital megacolon and the terminal rectum were stored at 60˚C for 2 h and then brought to room of ARM children demonstrate gangliocyte maldevelop- temperature. Deparaffinization was performed with xylene. ment (16). ARM tends to be accompanied by a megacolon (17). The specimens were then blocked in 3% H2O2 at room How HuD is expressed in the terminal rectum of the ARM temperature for 15-30 min. A citrate antigen-retrieval embryonic rat model and whether its expression influences solution (pH 6.0) was used for room-temperature cooling the development of ENS in the terminal rectum of the model of the microwave-processed specimens (heated to boiling remains to be elucidated. for 6 min at a power level of 100 W). The specimens were Based on the aforementioned studies, an ARM animal incubated with a rabbit anti-mouse HuD antibody (1:200; cat. model was established in the present study using ethylene- no., 300065; CapitalBio, Beijing, China) at 4˚C overnight and thiourea (ETU) (18). The expression of HuD in the terminal then washed with PBS. A goat anti-rabbit secondary antibody rectum of the embryonic rats with ARM was assessed by (cat. no. 400087; OriGene Technologies, Inc., Beijing, China) immunohistochemistry and western blotting. Its role in the was applied for 20 min at room temperature. Hematoxylin development of the nervous system in the intestinal wall was counterstaining, dehydration, mounting and 3,3'-diami- then explored. nobenzidine coloration were then performed. Cells with cytoplasm and nuclei stained yellow-brown were considered Materials and methods HuD-positive. The expression of HuD was analyzed with a Leica QWin relative optic density analytical system (Thermo ARM model establishment and grouping. A total of 40 healthy, Fisher Scientific, Inc., Waltham, MA, USA) and Image‑pro nulliparous Sprague Dawley rats aged 6-8 weeks (comprising plus version 6.0 image analysis system (Media Cybernetics, 30 females and 10 males, specific pathogen‑free grade) and Inc., Rockville, MD, USA) and an average integral optical weighing 200-250 g were supplied by the animal center of density (IOD) was calculated. the Third Military Medical University (Chongqing, China). The animals were caged at a female-male ratio of 3:1 for Western blot analysis. A total of 10 normal specimens were mating. At 8:00 a.m. on the second day, a vaginal smear test randomly selected from each group. The total protein was was performed. The smears were evaluated using dark field extracted according to the protocol provided with the bicin- microscopy (x100 magnification). An observation of sperm choninic acid (BCA) protein concentration assay kit (P0009; was noted as pregnancy day 0. Each pregnant rat was weighed Beyotime Institute of Biotechnology, Nantong, China). Protein at day 0 and 10.
Recommended publications
  • Distinct Basket Nucleoporins Roles in Nuclear Pore Function and Gene Expression
    bioRxiv preprint doi: https://doi.org/10.1101/685263; this version posted June 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. Distinct Basket Nucleoporins roles in Nuclear Pore Function and Gene Expression: Tpr is an integral component of the TREX-2 mRNA export pathway Vasilisa Aksenova1, Hang Noh Lee1, †, Alexandra Smith1, †, Shane Chen1, †, Prasanna Bhat3, †, James Iben2, Carlos Echeverria1, Beatriz Fontoura3, Alexei Arnaoutov1 and Mary Dasso1, * 1Division of Molecular and Cellular Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA. 2Molecular Genomics Core, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20879 3Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. † These authors contributed equally to this work. *Correspondence: [email protected]. Acronyms: NPC – nuclear pore complex; BSK-NUPs – basket nucleoporins; NG – NeonGreen; AID - Auxin Inducible Degron 1 bioRxiv preprint doi: https://doi.org/10.1101/685263; this version posted June 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. Abstract Nuclear pore complexes (NPCs) are important for many processes beyond nucleocytoplasmic trafficking, including protein modification, chromatin remodeling, transcription, mRNA processing and mRNA export.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • DEAD-Box RNA Helicases in Cell Cycle Control and Clinical Therapy
    cells Review DEAD-Box RNA Helicases in Cell Cycle Control and Clinical Therapy Lu Zhang 1,2 and Xiaogang Li 2,3,* 1 Department of Nephrology, Renmin Hospital of Wuhan University, Wuhan 430060, China; [email protected] 2 Department of Internal Medicine, Mayo Clinic, 200 1st Street, SW, Rochester, MN 55905, USA 3 Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 1st Street, SW, Rochester, MN 55905, USA * Correspondence: [email protected]; Tel.: +1-507-266-0110 Abstract: Cell cycle is regulated through numerous signaling pathways that determine whether cells will proliferate, remain quiescent, arrest, or undergo apoptosis. Abnormal cell cycle regula- tion has been linked to many diseases. Thus, there is an urgent need to understand the diverse molecular mechanisms of how the cell cycle is controlled. RNA helicases constitute a large family of proteins with functions in all aspects of RNA metabolism, including unwinding or annealing of RNA molecules to regulate pre-mRNA, rRNA and miRNA processing, clamping protein complexes on RNA, or remodeling ribonucleoprotein complexes, to regulate gene expression. RNA helicases also regulate the activity of specific proteins through direct interaction. Abnormal expression of RNA helicases has been associated with different diseases, including cancer, neurological disorders, aging, and autosomal dominant polycystic kidney disease (ADPKD) via regulation of a diverse range of cellular processes such as cell proliferation, cell cycle arrest, and apoptosis. Recent studies showed that RNA helicases participate in the regulation of the cell cycle progression at each cell cycle phase, including G1-S transition, S phase, G2-M transition, mitosis, and cytokinesis.
    [Show full text]
  • Metabolic Labeling Enables Selective Photocrosslinking of O-Glcnac-Modified Proteins to Their Binding Partners
    Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners Seok-Ho Yua, Michael Boyceb, Amberlyn M. Wandsa, Michelle R. Bonda, Carolyn R. Bertozzib, and Jennifer J. Kohlera,1 aDepartment of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-9038 and bDepartment of Chemistry, University of California, Berkeley, CA 94720 Edited by Barbara Imperiali, Massachusetts Institute of Technology, Cambridge, MA, and approved January 30, 2012 (received for review August 31, 2011) O-linked β-N-acetylglucosamine (O-GlcNAc) is a reversible post- modified protein, although recent findings suggest that increased translational modification found on hundreds of nuclear and cyto- levels of O-GlcNAc can be associated with acquisition of function plasmic proteins in higher eukaryotes. Despite its ubiquity and (12–14). essentiality in mammals, functional roles for the O-GlcNAc modifi- Selectively observing the cellular behavior of O-GlcNAc- cation remain poorly defined. Here we develop a combined genetic modified proteins remains challenging. Most methods report and chemical approach that enables introduction of the diazirine on the bulk behavior of the protein of interest and do not distin- photocrosslinker onto the O-GlcNAc modification in cells. We engi- guish among the different posttranslationally modified forms. We neered mammalian cells to produce diazirine-modified O-GlcNAc reasoned that appending a small photoactivatable crosslinking by expressing a mutant form of UDP-GlcNAc pyrophosphorylase group to the O-GlcNAc modification would enable selectively and subsequently culturing these cells with a cell-permeable, induced covalent crosslinking between an O-GlcNAc-modified diazirine-modified form of GlcNAc-1-phosphate.
    [Show full text]
  • Structural Requirements for the UBA Domain of the Mrna Export
    Structural requirements for the UBA domain of the mRNA export receptor Mex67 to bind its specific targets, the transcription elongation THO complex component Hpr1 and nucleoporin FXFG repeats. Maria Hobeika, Christoph Brockmann, Florian Gruessing, David Neuhaus, Gilles Divita, Murray Stewart, Catherine Dargemont To cite this version: Maria Hobeika, Christoph Brockmann, Florian Gruessing, David Neuhaus, Gilles Divita, et al.. Struc- tural requirements for the UBA domain of the mRNA export receptor Mex67 to bind its specific targets, the transcription elongation THO complex component Hpr1 and nucleoporin FXFG repeats.. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2009, pp.17575-17583. 10.1074/jbc.M109.004374. hal-00394024 HAL Id: hal-00394024 https://hal.archives-ouvertes.fr/hal-00394024 Submitted on 28 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 THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 26, pp. 17575–17583,
    [Show full text]
  • Assembly and Mobility of Exon–Exon Junction Complexes in Living Cells
    Downloaded from rnajournal.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Assembly and mobility of exon–exon junction complexes in living cells UTE SCHMIDT,1,2,5 KANG-BIN IM,2 CAROLA BENZING,1 SNJEZANA JANJETOVIC,1 KARSTEN RIPPE,3 PETER LICHTER,1 and MALTE WACHSMUTH2,4 1Division of Molecular Genetics, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany 2Cell Biophysics Group, Institut Pasteur Korea, Seoul 136-791, Republic of Korea 3Research Group Genome Organization and Function, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany 4Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany ABSTRACT The exon–exon junction complex (EJC) forms via association of proteins during splicing of mRNA in a defined manner. Its organization provides a link between biogenesis, nuclear export, and translation of the transcripts. The EJC proteins accumulate in nuclear speckles alongside most other splicing-related factors. We followed the establishment of the EJC on mRNA by investigating the mobility and interactions of a representative set of EJC factors in vivo using a complementary analysis with different fluorescence fluctuation microscopy techniques. Our observations are compatible with cotranscriptional binding of the EJC protein UAP56 confirming that it is involved in the initial phase of EJC formation. RNPS1, REF/Aly, Y14/Magoh, and NXF1 showed a reduction in their nuclear mobility when complexed with RNA. They interacted with nuclear speckles, in which both transiently and long-term immobilized factors were identified. The location- and RNA-dependent differences in the mobility between factors of the so-called outer shell and inner core of the EJC suggest a hypothetical model, in which mRNA is retained in speckles when EJC outer-shell factors are missing.
    [Show full text]
  • Cytoplasmic Circular Stable Intronic Sequence
    CYTOPLASMIC CIRCULAR STABLE INTRONIC SEQUENCE RNA by Gaëlle Talhouarne A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland November, 2017 © Gaëlle J. S. Talhouarne 2017 All rights reserved ABSTRACT Introns represent one of the largest fractions of our genomes but also one of the least understood. Soon after transcription, most intronic RNAs are released as lariats and degraded within minutes; therefore, they are seen as discarded byproducts of splicing or junk. However, the Gall laboratory reported the existence of thousands of stable intronic sequence RNAs or sisRNAs in the oocyte nucleus (germinal vesicle) of the frog Xenopus. Also, highlighted in this thesis, I observed sisRNAs in the cytoplasm of these oocytes and eventually in other cells and species. My thesis addresses the characterization of sisRNAs in the cytoplasm of the frog oocyte. I demonstrate that these transcripts are resistant to the exonuclease RNase R, and I confirm that most cytoplasmic sisRNAs accumulate as short lariats. I propose that sisRNAs can accumulate in the cytoplasm because this compartment lacks the lariat degradation machinery. Additionally, I report that circular sisRNAs occur in human, mouse, chicken, and zebrafish as well. Most of these lariats are C-branched (unlike in Xenopus) and many accumulate in the cytoplasm. Surprisingly, hundreds of circular sisRNAs occur in human and mouse red blood cells, which lack nuclei and both transcription and translation machineries. Finally, I catalog the circular sisRNAs which contain small nucleolar (sno)RNA motifs. These stable lariats bearing a snoRNA, or slb-snoRNAs, associate with the canonical snoRNA binding proteins, namely dyskerin and 15.5K, but do not function as canonical modification guide RNAs.
    [Show full text]
  • TREX Exposes the RNA-Binding Domain of Nxf1 to Enable Mrna Export
    ARTICLE Received 22 Feb 2012 | Accepted 11 Jul 2012 | Published 14 Aug 2012 DOI: 10.1038/ncomms2005 TREX exposes the RNA-binding domain of Nxf1 to enable mRNA export Nicolas Viphakone1, Guillaume M. Hautbergue1, Matthew Walsh1, Chung-Te Chang1, Arthur Holland1, Eric G. Folco2, Robin Reed2 & Stuart A. Wilson1 The metazoan TREX complex is recruited to mRNA during nuclear RNA processing and functions in exporting mRNA to the cytoplasm. Nxf1 is an mRNA export receptor, which binds processed mRNA and transports it through the nuclear pore complex. At present, the relationship between TREX and Nxf1 is not understood. Here we show that Nxf1 uses an intramolecular interaction to inhibit its own RNA-binding activity. When the TREX subunits Aly and Thoc5 make contact with Nxf1, Nxf1 is driven into an open conformation, exposing its RNA-binding domain, allowing RNA binding. Moreover, the combined knockdown of Aly and Thoc5 markedly reduces the amount of Nxf1 bound to mRNA in vivo and also causes a severe mRNA export block. Together, our data indicate that TREX provides a license for mRNA export by driving Nxf1 into a conformation capable of binding mRNA. 1 Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.2 Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA. Correspondence and requests for materials should be addressed to S.A.W. (email: [email protected]). NATURE COMMUNICATIONS | 3:1006 | DOI: 10.1038/ncomms2005 | www.nature.com/naturecommunications © 2012 Macmillan Publishers Limited. All rights reserved.
    [Show full text]
  • Identification of RNA-Binding Proteins As Targetable Putative Oncogenes
    International Journal of Molecular Sciences Article Identification of RNA-Binding Proteins as Targetable Putative Oncogenes in Neuroblastoma Jessica L. Bell 1,2,*, Sven Hagemann 1, Jessica K. Holien 3,4 , Tao Liu 2, Zsuzsanna Nagy 2,5, Johannes H. Schulte 6,7, Danny Misiak 1 and Stefan Hüttelmaier 1,* 1 Institute of Molecular Medicine, Sect. Molecular Cell Biology, Martin Luther University Halle-Wittenberg, Charles Tanford Protein Center, 06120 Halle Saale, Germany; [email protected] (S.H.); [email protected] (D.M.) 2 Children’s Cancer Institute Australia, Randwick, NSW 2031, Australia; [email protected] (T.L.); [email protected] (Z.N.) 3 St. Vincent’s Institute of Medical Research, Fitzroy, Victoria 3065, Australia; [email protected] 4 Biosciences and Food Technology, School of Science, College of Science, Engineering and Health, RMIT University, Melbourne, Victoria 3053, Australia 5 School of Women’s & Children’s Health, UNSW Sydney, Randwick, NSW 2031, Australia 6 Department of Pediatric Oncology/Hematology, Charité-Universitätsmedizin Berlin, 10117 Berlin, Germany; [email protected] 7 German Consortium for Translational Cancer Research (DKTK), Partner Site Charité Berlin, 10117 Berlin, Germany * Correspondence: [email protected] (J.L.B.); [email protected] (S.H.) Received: 23 April 2020; Accepted: 14 July 2020; Published: 19 July 2020 Abstract: Neuroblastoma is a common childhood cancer with almost a third of those affected still dying, thus new therapeutic strategies need to be explored. Current experimental therapies focus mostly on inhibiting oncogenic transcription factor signalling. Although LIN28B, DICER and other RNA-binding proteins (RBPs) have reported roles in neuroblastoma development and patient outcome, the role of RBPs in neuroblastoma is relatively unstudied.
    [Show full text]
  • SRSF1-Dependent Nuclear Export Inhibition of C9ORF72 Repeat Transcripts Prevents Neurodegeneration and Associated Motor Deficits
    ARTICLE Received 4 Dec 2016 | Accepted 24 May 2017 | Published 5 Jul 2017 DOI: 10.1038/ncomms16063 OPEN SRSF1-dependent nuclear export inhibition of C9ORF72 repeat transcripts prevents neurodegeneration and associated motor deficits Guillaume M. Hautbergue1,*,**, Lydia M. Castelli1,*, Laura Ferraiuolo1,*, Alvaro Sanchez-Martinez2, Johnathan Cooper-Knock1, Adrian Higginbottom1, Ya-Hui Lin1, Claudia S. Bauer1, Jennifer E. Dodd1, Monika A. Myszczynska1, Sarah M. Alam2, Pierre Garneret1, Jayanth S. Chandran1, Evangelia Karyka1, Matthew J. Stopford1, Emma F. Smith1, Janine Kirby1, Kathrin Meyer3, Brian K. Kaspar3, Adrian M. Isaacs4, Sherif F. El-Khamisy5, Kurt J. De Vos1, Ke Ning1, Mimoun Azzouz1, Alexander J. Whitworth2,** & Pamela J. Shaw1,** Hexanucleotide repeat expansions in the C9ORF72 gene are the commonest known genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Expression of repeat transcripts and dipeptide repeat proteins trigger multiple mechanisms of neurotoxicity. How repeat transcripts get exported from the nucleus is unknown. Here, we show that depletion of the nuclear export adaptor SRSF1 prevents neurodegeneration and locomotor deficits in a Drosophila model of C9ORF72-related disease. This intervention suppresses cell death of patient-derived motor neuron and astrocytic-mediated neurotoxicity in co-culture assays. We further demonstrate that either depleting SRSF1 or preventing its interaction with NXF1 specifically inhibits the nuclear export of pathological C9ORF72 transcripts, the production of dipeptide-repeat proteins and alleviates neurotoxicity in Drosophila, patient-derived neurons and neuronal cell models. Taken together, we show that repeat RNA-sequestration of SRSF1 triggers the NXF1-dependent nuclear export of C9ORF72 transcripts retaining expanded hexanucleotide repeats and reveal a novel promising therapeutic target for neuroprotection.
    [Show full text]
  • The RNA-Binding Protein SBR (Dm NXF1) Is Required for the Constitution of Medulla Boundaries in Drosophila Melanogaster Optic Lobes
    cells Article The RNA-Binding Protein SBR (Dm NXF1) Is Required for the Constitution of Medulla Boundaries in Drosophila melanogaster Optic Lobes Ludmila Mamon 1, Anna Yakimova 2, Daria Kopytova 3 and Elena Golubkova 1,* 1 Department of Genetics and Biotechnology, Saint-Petersburg State University, Universitetskaya Emb. 7/9, 199034 St. Petersburg, Russia; [email protected] or [email protected] 2 A. Tsyb Medical Radiological Research Center—Branch of the National Medical Research Radiological Center of the Ministry of Health of the Russian Federation, Koroleva Str. 4, 249036 Obninsk, Russia; [email protected] 3 Institute of Gene Biology, Russian Academy of Sciences, Vavilov St. 34/5, 119334 Moscow, Russia; [email protected] * Correspondence: [email protected] or [email protected] Abstract: Drosophila melanogaster sbr (small bristles) is an orthologue of the Nxf1 (nuclear export factor 1) genes in different Opisthokonta. The known function of Nxf1 genes is the export of various mRNAs from the nucleus to the cytoplasm. The cytoplasmic localization of the SBR protein indicates that the nuclear export function is not the only function of this gene in Drosophila. RNA-binding protein SBR enriches the nucleus and cytoplasm of specific neurons and glial cells. In sbr12 mutant males, the disturbance of medulla boundaries correlates with the defects of photoreceptor axons pathfinding, axon bundle individualization, and developmental neurodegeneration. RNA-binding protein SBR Citation: Mamon, L.; Yakimova, A.; participates in processes allowing axons to reach and identify their targets. Kopytova, D.; Golubkova, E. The RNA-Binding Protein SBR (Dm Keywords: neurogenesis; optic lobe; Drosophila; NXF; RNA-binding protein NXF1) Is Required for the Constitution of Medulla Boundaries in Drosophila melanogaster Optic Lobes.
    [Show full text]
  • The Principal Mrna Nuclear Export Factor NXF1:NXT1 Forms A
    Published online 27 January 2015 Nucleic Acids Research, 2015, Vol. 43, No. 3 1883–1893 doi: 10.1093/nar/gkv032 The principal mRNA nuclear export factor NXF1:NXT1 forms a symmetric binding platform that facilitates export of retroviral CTE-RNA Shintaro Aibara1, Jun Katahira2,3, Eugene Valkov1 and Murray Stewart1,* 1MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK, 2Biomolecular Networks Laboratories, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadoka, Suita, Osaka 565-0871, Japan and 3Department of Biochemistry, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka Suita, Osaka 565-0871, Japan Received November 21, 2014; Revised January 06, 2015; Accepted January 09, 2015 ABSTRACT tion (reviewed by (1–3)). The movement of transcripts through NPCs is mediated by NXF1:NXT1 (also known as The NXF1:NXT1 complex (also known as TAP:p15) is TAP:p15), which is the metazoan homolog of Mex67:Mtr2, a general mRNA nuclear export factor that is con- the principal mRNA export factor in Saccharomyces cere- served from yeast to humans. NXF1 is a modular visiae (4,5). NXF1:NXT1 is functionally complementary protein constructed from four domains (RRM, LRR, to Mex67:Mtr2 and can rescue, at least partially, an oth- NTF2-like and UBA domains). It is currently unclear erwise inviable Mex67:Mtr2 deletion in S. cerevisiae (6). how NXF1:NXT1 binds transcripts and whether there NXF1 is a modular protein comprised of four domains: is higher organization of the NXF1 domains. We re- an N-terminal RNA recognition motif (RRM), a leucine- port here the 3.4 A˚ resolution crystal structure of rich repeat (LRR), a nuclear transport factor 2-like domain the first three domains of human NXF1 together with (NTF2L) and an ubiquitin-associated (UBA) domain (re- / NXT1 that has two copies of the complex in the asym- viewed by (7)).
    [Show full text]