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Posttranscriptional Regulation of HLA-A Protein Expression By Posttranscriptional Regulation of HLA-A Protein Expression by Alternative Polyadenylation Signals Involving the RNA-Binding Protein Syncrip This information is current as of September 27, 2021. Smita Kulkarni, Veron Ramsuran, Marijana Rucevic, Sukhvinder Singh, Alexandra Lied, Viraj Kulkarni, Colm O'hUigin, Sylvie Le Gall and Mary Carrington J Immunol 2017; 199:3892-3899; Prepublished online 20 October 2017; Downloaded from doi: 10.4049/jimmunol.1700697 http://www.jimmunol.org/content/199/11/3892 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2017/10/20/jimmunol.170069 Material 7.DCSupplemental References This article cites 66 articles, 30 of which you can access for free at: http://www.jimmunol.org/content/199/11/3892.full#ref-list-1 Why The JI? Submit online. by guest on September 27, 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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Posttranscriptional Regulation of HLA-A Protein Expression by Alternative Polyadenylation Signals Involving the RNA-Binding Protein Syncrip Smita Kulkarni,*,† Veron Ramsuran,*,‡,x,{ Marijana Rucevic,* Sukhvinder Singh,† Alexandra Lied,* Viraj Kulkarni,*,‖ Colm O’hUigin,‡ Sylvie Le Gall,* and Mary Carrington*,‡ Genomic variation in the untranslated region (UTR) has been shown to influence HLA class I expression level and associate with disease outcomes. Sequencing of the 39UTR of common HLA-A alleles indicated the presence of two polyadenylation signals (PAS). The proximal PAS is conserved, whereas the distal PAS is disrupted within certain alleles by sequence variants. Using 39RACE, we confirmed expression of two distinct forms of the HLA-A 39UTR based on use of either the proximal or the distal PAS, which differ Downloaded from in length by 100 bp. Specific HLA-A alleles varied in the usage of the proximal versus distal PAS, with some alleles using only the proximal PAS, and others using both the proximal and distal PAS to differing degrees. We show that the short and the long 39UTR produced similar mRNA expression levels. However, the long 39UTR conferred lower luciferase activity as compared with the short form, indicating translation inhibition of the long 39UTR. RNA affinity pull-down followed by mass spectrometry analysis as well as RNA coimmunoprecipitation indicated differential binding of Syncrip to the long versus short 39UTR. Depletion of Syncrip by small interfering RNA increased surface expression of an HLA-A allotype that uses primarily the long 39UTR, whereas an http://www.jimmunol.org/ allotype expressing only the short form was unaffected. Furthermore, specific blocking of the proximal 39UTR reduced surface expression without decreasing mRNA expression. These data demonstrate HLA-A allele-specific variation in PAS usage, which modulates their cell surface expression posttranscriptionally. The Journal of Immunology, 2017, 199: 3892–3899. he HLA class I molecules are expressed on virtually all recently, a variant in an Oct1-binding site 800 bp upstream of the nucleated cells and present peptides to CTL, initiating an HLA-C coding region was also shown to regulate HLA-C ex- T adaptive immune response. They also serve as ligands for pression levels (2), and this variant along with the polymorphic the killer cell Ig-like receptors expressed on NK cells, thereby miRNA-binding site account for 40% of the differential cell sur- by guest on September 27, 2021 regulating NK cell responses. HLA class I genes are the most face expression levels of HLA-C. Thus, variation in regulatory polymorphic loci in the human genome, and along with the HLA regions of the HLA class I loci that affect their expression levels class II genes, they associate with more human diseases than any may influence the immune response and disease susceptibility. other locus genome-wide (GWAS catalog; http://www.ebi.ac.uk/ The mRNA expression levels of HLA-A alleles were recently gwas). Disease associations with HLA class I variation have shown to vary in an allele-dependent manner as a function of the largely been attributed to variants encoding polymorphic amino degree of methylation in the promoter region of each allele (3). acid positions in the peptide-binding groove of the class I mole- Thus, epigenetic mechanisms account for a portion of the differ- cules. We have shown previously that a polymorphic microRNA ential mRNA expression patterns across HLA-A alleles. In this (miRNA)-binding site in the 39 untranslated region (UTR) of study, we describe, to our knowledge, a novel mechanism, the use HLA-C contributes to allele-specific variation in expression levels of alternative polyadenylation (APA) signals in the 39UTR of and associates with both HIV viral control and risk of Crohn HLA-A, which modulates expression levels of HLA-A protein disease independently of individual HLA allelic effects (1). More through regulation of translation. These data underscore the *Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139; †Department of cation does not necessarily reflect the views or policies of the Department of Health Genetics, Texas Biomedical Research Institute, San Antonio, TX 78227; ‡Cancer and and Human Services, nor does mention of trade names, commercial products, or Inflammation Program, Leidos Biomedical Research Inc., Frederick National Laboratory organizations imply endorsement by the U.S. Government. This research was sup- for Cancer Research, Frederick, MD 21702; xKwaZulu-Natal Research Innovation and ported in part by the Intramural Research Program of the National Institutes of Sequencing Platform, School of Laboratory Medicine and Medical Sciences, University Health, Frederick National Laboratory, and Center for Cancer Research. of KwaZulu-Natal, Durban 4001, South Africa; {Centre for the AIDS Programme of Address correspondence and reprint requests to Smita Kulkarni, Department of Ge- Research in South Africa, Nelson R. Mandela School of Medicine, University of ‖ netics, Texas Biomedical Research Institute, Building 12, Room 12.126, 7620 NW KwaZulu-Natal, Durban 4001, South Africa; and Department of Virology and Immu- Loop 410, San Antonio, TX 78227-5301. E-mail address: [email protected] nology, Texas Biomedical Research Institute, San Antonio, TX 78227 The online version of this article contains supplemental material. ORCIDs: 0000-0003-1590-9893 (V.R.); 0000-0002-2692-2180 (M.C.). Abbreviations used in this article: APA, alternative polyadenylation; miRNA, micro- Received for publication May 11, 2017. Accepted for publication September 25, RNA; MS, mass spectrometry; ORF, open reading frame; PAS, polyadenylation 2017. signal; PASD, distal PAS; PASP, proximal PAS; PASPD, proximal and distal PAS; This work was supported by institutional funds from the Texas Biomedical Research qPCR, quantitative PCR; RBP, RNA-binding protein; RIP, RNA immunoprecipita- Institute and the Ragon Institute of MGH, MIT and Harvard. V.R. is supported by tion; RNP, ribonucleoprotein; siRNA, small interfering RNA; UTR, untranslated South African Medical Research Council Grant MRC-RFA-UFSP-01-2013/UKZN- region. HIVEPI. S.S. is supported by the Cowles Postdoctoral Fellowship. This work has been supported in part with federal funds from the Frederick National Laboratory for Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 Cancer Research under Contract HHSN261200800001E. The content of this publi- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700697 The Journal of Immunology 3893 complex regulatory mechanisms that dictate HLA class I expression Construction of the HLA-A open reading frame–c-Flag vector levels. HLA-A open reading frame (ORF) with a C-terminal DYKDDK (Flag) tag was amplified using PCR (clone OHu21196; NM_002116; GenScript). The Materials and Methods Renilla luciferase sequence was replaced by Flag-tagged HLA-A ORF in Samples the PASP, proximal and distal PAS (PASPD), and PASD vectors described earlier. Oligonucleotide sequences for the cloning primers used are listed Healthy European American donors recruited at the National Cancer In- in Supplemental Table I. stitute (Frederick, MD) were used for determination of HLA-A genotypes and the length of the HLA-A 39UTR. The respective Institutional Review Cell lines, cell transfection, luciferase reporter assays, and Boards approved the study, and all subjects gave written informed consent. Western blots HLA genotyping Transformed B cell lines from three individuals homozygous for HLA-A*11, A*03,orHLA-A*26, and the human T cell line Jurkat were grown in RPMI DNA samples were genotyped for HLA-A, -B,and-C genes by sequence- 1640 medium (Life Technologies) with 10% heat-inactivated FBS (Atlanta based typing of exons 2 and 3 and/or the PCR sequence-specific oligonu- Biologicals). PBLs were plated at a
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