DOCSLIB.ORG

Original Article Study on the Clinical Significance of Argonaute2 Expression in Colonic Carcinoma by Tissue Microarray

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Original Article Study on the Clinical Significance of Argonaute2 Expression in Colonic Carcinoma by Tissue Microarray Int J Clin Exp Pathol 2013;6(3):476-484 www.ijcep.com /ISSN:1936-2625/IJCEP1212020 Original Article Study on the clinical significance of Argonaute2 expression in colonic carcinoma by tissue microarray Ying-Xin Wang1, Xiao-Yan Zhang2, Bao-Feng Zhang2, Chang-Qing Yang1, Heng-Jun Gao1,2 1Tongji Institute of Digestive Disease, Department of Gastroenterology, Tongji Hospital, Tongji University, Shanghai 200065,China; 2National Engineering Center for Biochip at Shanghai 201023, China Received December 14, 2012; Accepted January 17, 2013; Epub February 15, 2013; Published March 1, 2013 Abstract: Background: To study the expression levels and clinical significance of Argonaute2 (EIF2C2) on colonic carcinomas and normal tissues. Methods: Colon tissue samples from 90 cases of colonic carcinomas and 90 nor- mal subjects were accumulated and made into a tissue microarray containing 360 dots. Expression of Argonaute2 (EIF2C2) was detected by immunohistochemical staining of the tissue microarray. Results: There was significant dif- ference in the expression levels of Argonaute2 (EIF2C2) between colonic carcinomas and normal tissues (P<0.01). However, the expression of Argonaute2 (EIF2C2) was not related to sex, age, position, differentiation, lymphatic metastasis and clinical stage of the tumor (P>0.05). Conclusion: Abnormal expression of Argonaute2 (EIF2C2) may be correlated with colon tumorigenesis. Keywords: Tissue mircroarray, immunohistochemstry, colonic carcinoma, Argonaute2 Introduction allows the detection of multiple parameters simultaneously, and could be of importance in Colonic carcinoma is one of the most frequent treating colonic carcinoma. cancers in the Western world. At the present time, with the changes in living conditions and There are endogenous non-protein-coding RNA life style, colonic carcinoma has become more molecules in the human genome, which include frequent in China. The prognosis in advanced transfer RNA, ribosomal RNA and various small cases is poor and more than one-third of the non-coding RNAs. MicroRNAs (miRNAs) are patients die from progressive disease and the small, non-coding, single-stranded RNAs ~22 overall survival is about 40% after 5 years [1]. nucleotides in length, which regulate gene Given the high level in incidence rate and mor- expression by pairing with messenger RNA of tality rate of colonic carcinoma, it would be target genes [5-8]. After being first identified in important to better understand the biological C.elegans, miRNAs were subsequently found in basis of tumor development and progression, plants and animals, showing that they are high- to develop markers for assessing onset or pre- ly conserved [5-8]. Regulation of miRNA is a diction of therapy outcome, as well as to iden- complex process and is well orchestrated with tify targets for the development of novel thera- many cellular components [5, 7, 8]. From pies. Colonic carcinoma may be considered the genomic DNA, miRNAs are transcribed to pri- final step of a progressive imbalance between mary miRNAs, which are subsequently pro- mucosal cell proliferation and apoptosis due to cessed to precursor miRNAs by Drosha RNAse the activation of oncogenes and the inactiva- III endonuclease in complex with the double- tion of tumor suppressor genes [2-4]. The eval- stranded RNA-binding domain protein DGCR8 uation of the clinical utility of each of these [9, 10]. After export from nucleus to cytoplasm genes would require multiple experiments with by exportin5, precursor miRNAs are further hundreds of tumor specimens. This would be cleaved by Dicer in a complex with TARBP pro- both time-consuming as well as impractical for teins to generate a short RNA duplex [9, 10]. Of more than a handful of genes. Microarray tech- the duplex, one strand becomes a mature nology provides a new and promising tool that miRNA, while the other strand is rapidly degrad- Argonaute2 in colonic carcinoma ed. The mature miRNA directs a RNA-induced peptide to keyhole limpet hemocyanin (KLH) to silencing complex (RISC) to 3’-untranslated construct Argonaute2-KLH conjugate. The region (UTR) of its complementary target genes Argonaute2-KLH conjugate was injected into and causes inhibition of translation and degra- rabbits subcutaneously to produce polyclonal dation of the messenger RNA [13-15]. The antibodies. Evolution of the antibody titer was major components of the RISC are the Ago pro- controlled by measuring the binding of serial teins. Argonaute2 is a member of a family of dilutions of the antisera to microtiter plates eight proteins in mammals, four of which are coated with peptide antigen. Eight weeks after germ line specific [16]. Ago proteins contain a the first injection, sera were collected and used P-element induced wimpy testis (PIWI) domain to prepare purified antibodies. The lgG solution that can adopt a ribomuclease H fole with was fractionated from the rabbit antisera by potentially innate endonuclease activity [17- precipitation with 40% saturated ammonium 20]. However, Argonaute2 is the only Ago pro- sulphate and then affinity-purified on peptide tein shown to mediate miRNA-dependent cleav- affinity columns. The flowthrough was collected age/degradation of target mRNAs in mammals. and stored at -20°C for use in ELISA, Western Recently, studies have also implicated func- blot, and immunohistochemistry analyses. The tional roles for Argonaute2 independent of its specificity and sensitivity of the antibodies endonuclease activity [21-25]. Argonaute2 has were identified by ELISA and Western blotting been observed to be diffuse within the cyto- after purification using affinity chromatorgra- plasm and localized in both processing bodies phy. The tissue chip array was used to study the (P bodies) and the nucleus [26]. distribution and expression levels of Argonaute2. Several lines of evidence indicate that miRNA is important in development, cell proliferation Patients and specimens and cell death, deregulations of which contrib- Demographic and clinical data were collected ute to the pathogenesis of cancers [27-30]. retrospectively. None of the patients received Some miRNAs act as either a tumor suppressor radiotherapy or chemotherapy before surgery. or a tumor promoter and alterations of miRNA Formalin-fixed and paraffin-embedded tumors have been identified in human cancers [27-30]. and normal tissues（≥5cm away from the Transcriptional and epigenetic alterations, tumor） specimens were obtained from the gene mutation and DNA copy number alteration archives of the Department of Surgical of miRNAs have been reported in human can- Gastroenterology, the Tongji Hospital of Tongji cers [27-30, 31]. In addition, altered expression University and National Engineering Center for and mutation of components in miRNA biogen- Biochip at Shanghai. All specimens were viewed esis have been reported [32-35]. However, by one pathologist (Jing Fang). The specimens data on the expression of Argonaute2 in colon- that were interpretable for IHC included: (1) ic carcinoma is lacking. In the present study we Ninety colon cancer specimens including differ- analyzed the expression level of Argonaute2 in ent grades, such as moderate to high (n=69) colonic carcinoma tissues by immunohisto- and low differentiated (n=21); (2) ninety normal chemistry using a tissue microarray (TMA) colon tissues resected from areas of the colon approach. ≥ 5 cms from the corresponding cancer Materials and methods tissues. Patients were only included in the study if they Preparation and identification of Argonaute2 had provided written consent to participate in We adopted the Ensemble database and the the study after receiving oral and written infor- antibody screening software (Dragonfly, USA) to mation regarding its course and purpose. select the distinctive piece of Argonaute2 pro- Approval for the study was received from the Ethics Committee of the host institution. tein and then add aminothipropionic acid on C-terminal to connect peptide fragment and Construction and sectioning of tissue microar- carrier. The peptide fragment was synthesized ray by C-Strong and the molecular weight by mass spectral analysis corresponded to the theoreti- The colon cancer microarray was constructed cal molecular weight. We coupled Argonaute2 as previously described [36]. Briefly, fresh sec- 477 Int J Clin Exp Pathol 2013;6(3):476-484 Argonaute2 in colonic carcinoma tions were cut from the donor block and stained Scoring of Argonaute2 expression with hematoxylin-eosin (HE) and these slides were used to guide the samplings from morpho- The evaluation of the immunohistochemical logically representative regions of the tissues. staining was performed independently by two A tissue array instrument was used to create authors without knowledge of the clinicpotho- holes in a recipient paraffin block and to acquire logical information. The immunoreactive scores tissue cores from the donor block by a thin- besides Argonaute2 were determined by the walled needle with an inner diameter of 1.0 mm sum of extension and intensity as reported pre- or 1.5 mm, held in an X-Y precision guide. The viously [40]. The intensity of the staining was cylindrical samples were retrieved from the scored using the following scale: 0, no staining selected regions in the donors and extruded of the tumor cells; +, mild staining; ++, moder- directly onto the recipient blocks with defined ate staining and +++, marked staining. The array coordinates. After the construction of the area of staining was evaluated and recorded as array block, multiple
Load more

Recommended publications
  • Not Dicer but Argonaute Is Required for a Microrna Production
    Cell Research (2010) 20:735-737. npg © 2010 IBCB, SIBS, CAS All rights reserved 1001-0602/10 $ 32.00 RESEARCH HIGHLIGHT www.nature.com/cr A new twist in the microRNA pathway: Not Dicer but Argonaute is required for a microRNA production Gabriel D Bossé1, Martin J Simard1 1Laval University Cancer Research Centre, Hôtel-Dieu de Québec (CHUQ), Quebec City, Québec G1R 2J6, Canada Cell Research (2010) 20:735-737. doi:10.1038/cr.2010.83; published online 15 June 2010 Found in all metazoans, microRNAs A Canonical pathway B Ago2-dependent pathway or miRNAs are small non-coding RNA Nucleus Cytoplasm Nucleus Cytoplasm of ~22 nucleotides in length that com- Exp.5 Exp.5 pletely reshaped our understanding of gene regulation. This new class of gene pre-miR-451 regulator is mostly transcribed by the pre-miRNA RNA polymerase II producing a long stem-loop structure, called primary- or Ago2 pri-miRNA, that will first be processed Ago2 Dicer in the cell nucleus by a multiprotein TRBP complex called microprocessor to gen- erate a shorter RNA structure called Ago2 RISC precursor- or pre-miRNA. The precisely Ago2 RISC processed pre-miRNA will next be ex- ported into the cytoplasm by Exportin 5 and loaded onto another processing machine containing the ribonuclease III enzyme Dicer, an Argonaute protein Ago2 Ago2 and other accessory cellular factors mRNA mRNA (Figure 1A; [1]). Dicer will mediate the Translation inhibition Translation inhibition cleavage of the pre-miRNA to form the mature miRNA that will then be bound Figure 1 (A) Canonical microRNA biogenesis. In mammals, the pre-miRNA is by the Argonaute protein to form, most loaded onto a multiprotein complex consisting minimally of Dicer, Tar RNA Bind- likely with other cellular factors, the ef- ing Protein (TRBP) and Ago2.
    [Show full text]
  • EIF2C2 Monoclonal Antibody (M01), Clone 2E12-1C9
    EIF2C2 monoclonal antibody (M01), clone 2E12-1C9 Catalog # : H00027161-M01 規格 : [ 100 ug ] List All Specification Application Image Product Mouse monoclonal antibody raised against a full length recombinant Western Blot (Cell lysate) Description: EIF2C2. Immunogen: EIF2C2 (AAH07633.1, 483 a.a. ~ 859 a.a) full-length recombinant protein with GST tag. MW of the GST tag alone is 26 KDa. Sequence: MPIQGQPCFCKYAQGADSVEPMFRHLKNTYAGLQLVVVILPGKTPVYAE VKRVGDTVLGMATQCVQMKNVQRTTPQTLSNLCLKINVKLGGVNNILLP enlarge QGRPPVFQQPVIFLGADVTHPPAGDGKKPSIAAVVGSMDAHPNRYCATV Western Blot (Transfected RVQQHRQEIIQDLAAMVRELLIQFYKSTRFKPTRIIFYRDGVSEGQFQQV lysate) LHHELLAIREACIKLEKDYQPGITFIVVQKRHHTRLFCTDKNERVGKSGNIP AGTTVDTKITHPTEFDFYLCSHAGIQGTSRPSHYHVLWDDNRFSSDELQI LTYQLCHTYVRCTRSVSIPAPAYYAHLVAFRARYHLVDKEHDSAEGSHTS GQSNGRDHQALAKAVQVHQDTLRTMYFA Host: Mouse enlarge Reactivity: Human Western Blot (Recombinant Isotype: IgG1 Kappa protein) Quality Control Antibody Reactive Against Recombinant Protein. Immunofluorescence Testing: enlarge Immunohistochemistry (Formalin/PFA-fixed paraffin- embedded sections) Western Blot detection against Immunogen (68.03 KDa) . Storage Buffer: In 1x PBS, pH 7.4 enlarge Storage Store at -20°C or lower. Aliquot to avoid repeated freezing and thawing. Instruction: Sandwich ELISA (Recombinant protein) MSDS: Download Interspecies Mouse (100); Rat (99) Antigen Sequence: enlarge Datasheet: Download ELISA Publication Reference RNAi Knockdown (Antibody validated) 1. TDP-43 aggregation induced by oxidative stress causes global mitochondrial imbalance
    [Show full text]
  • Mir-125 in Normal and Malignant Hematopoiesis
    Leukemia (2012) 26, 2011–2018 & 2012 Macmillan Publishers Limited All rights reserved 0887-6924/12 www.nature.com/leu SPOTLIGHT REVIEW MiR-125 in normal and malignant hematopoiesis L Shaham1,2, V Binder3,4,NGefen1,5, A Borkhardt3 and S Izraeli1,5 MiR-125 is a highly conserved microRNA throughout many different species from nematode to humans. In humans, there are three homologs (hsa-miR-125b-1, hsa-miR-125b-2 and hsa-miR-125a). Here we review a recent research on the role of miR-125 in normal and malignant hematopoietic cells. Its high expression in hematopoietic stem cells (HSCs) enhances self-renewal and survival. Its expression in specific subtypes of myeloid and lymphoid leukemias provides resistance to apoptosis and blocks further differentiation. A direct oncogenic role in the hematopoietic system has recently been demonstrated by several mouse models. Targets of miR-125b include key proteins regulating apoptosis, innate immunity, inflammation and hematopoietic differentiation. Leukemia (2012) 26, 2011–2018; doi:10.1038/leu.2012.90 Keywords: microRNA; hematopoiesis; hematological malignancies; acute myeloid leukemia; acute lymphoblastic leukemia MicroRNAs (miRNAs) are 21–23-nucleotide non-coding RNAs that nucleotides with the seed region of miR-125b (ebv-miR-BART21-5p, have crucial roles in fundamental biological processes by ebv-miR-BART8 and rlcv-miR-rL1-25). In humans, as in most of the regulating the levels of multiple proteins. They are transcribed genomes, there are two paralogs (hsa-miR-125b-1 on chromosome as primary miRNAs and processed in the nucleus by the RNase III 11 and hsa-miR-125b-2 on chromosome 21), coding for the same endonuclease DROSHA to liberate 70-nucleotide stem loops, the mature sequence.
    [Show full text]
  • EIF2C2 Monoclonal Antibody (M01), Clone 2E12-1C9
    Produktinformation Diagnostik & molekulare Diagnostik Laborgeräte & Service Zellkultur & Verbrauchsmaterial Forschungsprodukte & Biochemikalien Weitere Information auf den folgenden Seiten! See the following pages for more information! Lieferung & Zahlungsart Lieferung: frei Haus Bestellung auf Rechnung SZABO-SCANDIC Lieferung: € 10,- HandelsgmbH & Co KG Erstbestellung Vorauskassa Quellenstraße 110, A-1100 Wien T. +43(0)1 489 3961-0 Zuschläge F. +43(0)1 489 3961-7 [email protected] • Mindermengenzuschlag www.szabo-scandic.com • Trockeneiszuschlag • Gefahrgutzuschlag linkedin.com/company/szaboscandic • Expressversand facebook.com/szaboscandic EIF2C2 monoclonal antibody (M01), clone 2E12-1C9 Catalog # : H00027161-M01 規格 : [ 100 ug ] List All Specification Application Image Product Mouse monoclonal antibody raised against a full length recombinant Western Blot (Cell lysate) Description: EIF2C2. Immunogen: EIF2C2 (AAH07633.1, 1 a.a. ~ 377 a.a) full-length recombinant protein with GST tag. MW of the GST tag alone is 26 KDa. Sequence: MPIQGQPCFCKYAQGADSVEPMFRHLKNTYAGLQLVVVILPGKTPVYAE VKRVGDTVLGMATQCVQMKNVQRTTPQTLSNLCLKINVKLGGVNNILLP enlarge QGRPPVFQQPVIFLGADVTHPPAGDGKKPSIAAVVGSMDAHPNRYCATV Western Blot (Transfected RVQQHRQEIIQDLAAMVRELLIQFYKSTRFKPTRIIFYRDGVSEGQFQQV lysate) LHHELLAIREACIKLEKDYQPGITFIVVQKRHHTRLFCTDKNERVGKSGNIP AGTTVDTKITHPTEFDFYLCSHAGIQGTSRPSHYHVLWDDNRFSSDELQI LTYQLCHTYVRCTRSVSIPAPAYYAHLVAFRARYHLVDKEHDSAEGSHTS GQSNGRDHQALAKAVQVHQDTLRTMYFA Host: Mouse enlarge Reactivity: Human Western Blot (Recombinant Isotype: IgG1 Kappa
    [Show full text]
  • Relative Contribution of Sequence and Structure Features to the Mrna Binding of Argonaute/ EIF2C–Mirna Complexes and the Degradation of Mirna Targets
    Downloaded from genome.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Letter Relative contribution of sequence and structure features to the mRNA binding of Argonaute/ EIF2C–miRNA complexes and the degradation of miRNA targets Jean Hausser,1,3 Markus Landthaler,2,3,4 Lukasz Jaskiewicz,1 Dimos Gaidatzis,1,5 and Mihaela Zavolan1,6 1Biozentrum, University of Basel and Swiss Institute of Bioinformatics, CH-4056 Basel, Switzerland; 2Howard Hughes Medical Institute, Laboratory for RNA Biology, The Rockefeller University, New York, New York 10021, USA How miRNAs recognize their target sites is a puzzle that many experimental and computational studies aimed to solve. Several features, such as perfect pairing of the miRNA seed, additional pairing in the 39 region of the miRNA, relative position in the 39 UTR, and the A/Ucontent of the environment of the putative site, have been found to be relevant. Here we have used a large number of previously published data sets to assess the power that various sequence and structure features have in distinguishing between putative sites that do and those that do not appear to be functional. We found that although different data sets give widely different answers when it comes to ranking the relative importance of these features, the sites inferred from most transcriptomics experiments, as well as from comparative genomics, appear similar at this level. This suggests that miRNA target sites have been selected in evolution on their ability to trigger mRNA degradation. To understand at what step in the miRNA-induced response individual features play a role, we transfected human HEK293 cells with miRNAs and analyzed the association of Argonaute/EIF2C–miRNA complexes with target mRNAs and the degradation of these messages.
    [Show full text]
  • Dysregulated RNA-Induced Silencing Complex (RISC) Assembly Within CNS Corresponds with Abnormal Mirna Expression During Autoimmune Demyelination
    The Journal of Neuroscience, May 13, 2015 • 35(19):7521–7537 • 7521 Neurobiology of Disease Dysregulated RNA-Induced Silencing Complex (RISC) Assembly within CNS Corresponds with Abnormal miRNA Expression during Autoimmune Demyelination Przemysław Lewkowicz, Hanna Cwiklin´ska, Marcin P. Mycko, Maria Cichalewska, Małgorzata Domowicz, Natalia Lewkowicz, Anna Jurewicz, and Krzysztof W. Selmaj Department of Neurology, Laboratory of Neuroimmunology, Medical University of Lodz, 92-213 Lodz, Poland MicroRNAs (miRNAs) associate with Argonaute (Ago), GW182, and FXR1 proteins to form RNA-induced silencing complexes (RISCs). RISCs represent a critical checkpoint in the regulation and bioavailability of miRNAs. Recent studies have revealed dysregulation of miRNAs in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE); however, the function of RISCs in EAE and MS is largely unknown. Here, we examined the expression of Ago, GW182, and FXR1 in CNS tissue, oligodendrocytes (OLs), brain-infiltrating T lymphocytes, and CD3 ϩsplenocytes (SCs) of EAE mic, and found that global RISC protein levels were signifi- cantly dysregulated. Specifically, Ago2 and FXR1 levels were decreased in OLs and brain-infiltrating T cells in EAE mice. Accordingly, assembly of Ago2/GW182/FXR1 complexes in EAE brain tissues was disrupted, as confirmed by immunoprecipitation experiments. In parallel with alterations in RISC complex content in OLs, we found downregulation of miRNAs essential for differentiation and survival of OLs and myelin synthesis. In brain-infiltrating T lymphocytes, aberrant RISC formation contributed to miRNA-dependent proinflam- matory helper T-cell polarization. In CD3 ϩ SCs, we found increased expression of both Ago2 and FXR1 in EAE compared with nonim- munized mice.
    [Show full text]
  • NIH Public Access Author Manuscript Epigenetics
    NIH Public Access Author Manuscript Epigenetics. Author manuscript; available in PMC 2009 June 23. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Epigenetics. 2007 ; 2(2): 126±134. Epigenetics and Neural Developmental Disorders: Washington DC, September 18 and 19, 2006 Xinyu Zhao1,*, ChangHui Pak2, Richard D. Smrt1, and Peng Jin2,* 1 Department of Neuroscience; University of New Mexico School of Medicine; Albuquerque, New Mexico USA 2 Department of Human Genetics; Emory University School of Medicine; Atlanta, Georgia USA Abstract Neural developmental disorders, such as autism, Rett Syndrome, Fragile X syndrome, and Angelman syndrome manifest during early postnatal neural development. Although the genes responsible for some of these disorders have been identified, how the mutations of these genes affect neural development is currently unclear. Emerging evidence suggest that these disorders share common underlying defects in neuronal morphology, synaptic connectivity and brain plasticity. In particular, alterations in dendritic branching and spine morphology play a central role in the pathophysiology of most mental retardation disorders, suggesting that common pathways regulating neuronal function may be affected. Epigenetic modulations, mediated by DNA methylation, RNA-associated silencing, and histone modification, can serve as an intermediate process that imprints dynamic environmental experiences on the “fixed” genome, resulting in stable alterations in phenotypes. Disturbance in epigenetic regulations can lead to inappropriate expression or silencing of genes, causing an array of multi-system disorders and neoplasias. Rett syndrome, the most common form of mental retardation in young girls, is due to l mutation of MECP2, encoding a methylated DNA binding protein that translates DNA methylation into gene repression.
    [Show full text]
  • T Lymphocytes Mrna, and Protein Expression in Activated Networks
    MicroRNA Regulation of Molecular Networks Mapped by Global MicroRNA, mRNA, and Protein Expression in Activated T Lymphocytes This information is current as of September 23, 2021. Yevgeniy A. Grigoryev, Sunil M. Kurian, Traver Hart, Aleksey A. Nakorchevsky, Caifu Chen, Daniel Campbell, Steven R. Head, John R. Yates III and Daniel R. Salomon J Immunol 2011; 187:2233-2243; Prepublished online 25 July 2011; Downloaded from doi: 10.4049/jimmunol.1101233 http://www.jimmunol.org/content/187/5/2233 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2011/07/25/jimmunol.110123 Material 3.DC1 References This article cites 83 articles, 31 of which you can access for free at: http://www.jimmunol.org/content/187/5/2233.full#ref-list-1 Why The JI? Submit online. by guest on September 23, 2021 • 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 *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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology MicroRNA Regulation of Molecular Networks Mapped by Global MicroRNA, mRNA, and Protein Expression in Activated T Lymphocytes Yevgeniy A.
    [Show full text]
  • A Drosophila Fragile X Protein Interacts with Components of Rnai and Ribosomal Proteins
    Downloaded from genesdev.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins Akira Ishizuka,1,2,3 Mikiko C. Siomi,1,3 and Haruhiko Siomi1,4 1Institute for Genome Research, 2Graduate School of Nutrition University of Tokushima, Tokushima 770-8503, Japan Fragile X syndrome is a common form of inherited mental retardation caused by the loss of FMR1 expression. The FMR1 gene encodes an RNA-binding protein that associates with translating ribosomes and acts as a negative translational regulator. In Drosophila, the fly homolog of the FMR1 protein (dFMR1) binds to and represses the translation of an mRNA encoding of the microtuble-associated protein Futsch. We have isolated a dFMR1-associated complex that includes two ribosomal proteins, L5 and L11, along with 5S RNA. The dFMR1 complex also contains Argonaute2 (AGO2) and a Drosophila homolog of p68 RNA helicase (Dmp68). AGO2 is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNA interference (RNAi) in Drosophila. We show that Dmp68 is also required for efficient RNAi. We further show that dFMR1 is associated with Dicer, another essential component of the RNAi pathway, and microRNAs (miRNAs) in vivo, suggesting that dFMR1 is part of the RNAi-related apparatus. Our findings suggest a model in which the RNAi and dFMR1-mediated translational control pathways intersect in Drosophila. Our findings also raise the possibility that defects in an RNAi-related machinery may cause human disease. [Keywords: fragile X syndrome; FMR1; RNAi; RNA helicase; miRNA] Received July 9, 2002; revised version accepted August 22, 2002.
    [Show full text]
  • High-Risk Myeloma Is Associated with Global Elevation of Mirnas and Overexpression of EIF2C2/AGO2
    High-risk myeloma is associated with global elevation of miRNAs and overexpression of EIF2C2/AGO2 Yiming Zhoua,1, Lijuan Chena,b,1, Bart Barlogiea, Owen Stephensa, Xiaosong Wua, David R. Williamsa, Marie-Astrid Cartrona, Frits van Rheea, Bijay Naira, Sarah Waheeda, Mauricio Pineda-Romana, Yazan Alsayeda, Elias Anaissiea, and John D. Shaughnessy, Jr.a,2 aDonna D. and Donald M. Lambert Laboratory for Myeloma Genetics, Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, Little Rock, AR 72205; and bDepartment of Hematology, First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, 210029,China Edited* by Carlo M. Croce, Ohio State University, Columbus, OH, and approved March 17, 2010 (received for review July 30, 2009) MicroRNAs (miRNAs) are noncoding RNAs that regulate global gene liferation in MM cell lines, and Lionetti et al. (14) identified 16 expression. miRNAs often act synergistically to repress target genes, miRNAs sensitive to DNA copy number. and their dysregulation can contribute to the initiation and pro- To further investigate the potential involvement of miRNA gression of a variety of cancers. The clinical relationship between in MM, we performed integrative analyses of both miRNA global expression of miRNA and mRNA in cancer has not been expression profiles and protein-coding gene expression profiles studied in detail. We used whole-genome microarray analyses of (GEPs) of myeloma cells from newly diagnosed patients. Global CD138-enriched plasma cells from 52 newly diagnosed cases of increases in miRNA expression were seen in cases with high-risk multiple myeloma to correlate miRNA expression profiles with a MM, which sharply contrasts with discriminate expression validated mRNA-based risk stratification score, proliferation index, changes of only selected miRNAs that are seen in other cancers and predefined gene sets.
    [Show full text]
  • Integrative Modeling and Visualization of Exosomes 7
    Integrative Modeling and Visualization of Exosomes 7 Integrative Modeling and Visualization of Exosomes Julia Jiménez, Ludovic Autin, Inmaculada Ibáñez de Cáceres, David S. Goodsell As part of this work, we have used small, well-defined systems to Information from proteomics, microscopy, and structural test and apply new methods. Because the work is highly biology are integrated to create structural models of exosomes, small vesicles released from cells. Three visualization methods integrative, a wide variety of data must be gathered (or are employed and compared: 2D painting of a cross section estimated) in order to generate a convincing model. Early using traditional media, manual creation of a cross section illustrative work focused on well-studied systems such as red using the mesoscale 2.5D digital painting software cellPAINT, blood cells and Escherichia coli (Goodsell 1992). For our 3D and generation of a 3D atomic model using the mesoscale modeling work, we have used simpler systems, including HIV modeling program cellPACK. (Johnson et al. 2014) and mycoplasma bacteria (Goodsell, Autin, et al. 2018). Exosomes provide an excellent test case for current mesoscale modeling methods. Sufficient proteomic, structural, and ultrastructural data are available to define a convincing model, Keywords however, there are still grey areas in this knowledge that may Exosome, integrative structural biology, molecular benefit from mesoscale modeling. Given their growing role in visualization, non-photorealistic rendering, protein structure many disease states, many illustrations of exosomes have been created, ranging from simple schematics to highly rendered editorial and advertising imagery (an internet search of Introduction “exosome” will yield hundreds of examples). However, to our Modeling of the molecular structure of cells is rapidly moving knowledge, no detailed integrative models of exosomes have from an art into a science.
    [Show full text]
  • Recombinant AGO2 Protein Catalog No: 31486, 31886 Quantity: 20, 1000 Μg Lot No: 07315001 Concentration: 0.4 Expressed In: Baculovirus Μg/Μl Source: Human
    Recombinant AGO2 protein Catalog No: 31486, 31886 Quantity: 20, 1000 µg Lot No: 07315001 Concentration: 0.4 Expressed In: Baculovirus µg/µl Source: Human Buffer Contents: Full length recombinant AGO2 protein expressed in Sf9 cells and is provided in 25 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 0.04% Triton X-100, 0.2 mM TCEP and 0.2 mg/ml 3X FLAG peptide. Background: AGO2 (Argonaute RISC Catalytic Component 2, also known as Q10; EIF2C2) is a member of the Argonaute family of proteins which play a role in RNA interference. This process is important both in gene regulation by microRNAs and in defense against viral infections. The RNA-induced silencing complex, or RISC, is a multiprotein complex that includes argonaute proteins and one strand of a small interfering RNA (siRNA) or microRNA (miRNA). RISC uses the siRNA or miRNA as a template for recognizing complementary mRNA. When it recognizes a complementary strand, it activates AGO2 to cleave the mRNA. The precise mechanism of gene silencing depends on the degree of complementarity between the miRNA or siRNA and its target. Binding of RISC to a perfectly complementary mRNA generally results in silencing due to endonucleolytic cleavage of the mRNA specifically by AGO2. Binding of RISC to a partially complementary mRNA results in silencing through inhibition of translation, and this is independent of endonuclease activity. In some cases, RISC-mediated translational repression is also observed for miRNAs that perfectly match the 3’ untranslated region (3’UTR). In addition, AGO2 can also up-regulate the posttranscriptional upregulation of specific mRNAs under certain growth conditions.
    [Show full text]