HNRNPR Regulates the Expression of Classical and Nonclassical MHC Class I Proteins This information is current as Adi Reches, Daphna Nachmani, Orit Berhani, Alexandra of September 29, 2021. Duev-Cohen, Dorin Shreibman, Yael Ophir, Barbara Seliger and Ofer Mandelboim J Immunol 2016; 196:4967-4976; Prepublished online 18 May 2016; doi: 10.4049/jimmunol.1501550 Downloaded from http://www.jimmunol.org/content/196/12/4967 References This article cites 34 articles, 6 of which you can access for free at: http://www.jimmunol.org/content/196/12/4967.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology HNRNPR Regulates the Expression of Classical and Nonclassical MHC Class I Proteins Adi Reches,* Daphna Nachmani,* Orit Berhani,* Alexandra Duev-Cohen,* Dorin Shreibman,* Yael Ophir,* Barbara Seliger,† and Ofer Mandelboim* MHC class I molecules, in addition to their role in specific activation of the CTL of adaptive immune system, function also as the main ligands for NK cell inhibitory receptors, which prevent NK cells from killing normal, healthy cells. MHC class I proteins are divided into classical and nonclassical proteins. The former group consists of hundreds of HLA-A, B, and C alleles, which are universally expressed, whereas several alleles of the latter group, such as HLA-G, manifest a restricted expression pattern. Despite the important role played by these molecules in innate and adaptive immune responses, their complex expression regulation is not fully known. In our study, we investigated the regulation processes controlling the expression of MHC class I molecules, with a particular focus on their 39 untranslated regions. We identified heterogeneous nuclear ribonucleoprotein R (HNRNPR) as an Downloaded from important positive regulator of classical and nonclassical MHC class I molecules. HNRNPR is a RNA-binding protein belonging to the heterogeneous nuclear ribonucleoprotein family, which has a known role in processing of precursor mRNA. We demonstrated that HNRNPR binds MHC class I mRNAs in their 39 untranslated regions and enhances their stability and consequently their expression. Furthermore, regulation by HNRNPR modulates the cytotoxic activity of NK cells. In con- clusion, we show that HNRNPR acts as a general positive regulator of MHC class I expression. The Journal of Immunology, 2016, 196: 4967–4976. http://www.jimmunol.org/ ajor histocompatibility complex class I proteins are one family shows a unique affinity to HLA-C subtypes. Practically of the major immune system modulators. They present all of the HLA-C alleles can be divided into two groups, which M peptides to T cells and activate CTLs of the adaptive are recognized by KIR2DL1 or by KIR2DL2 inhibitory NK re- immune system, and, in contrast, serve as inhibitory ligands for NK ceptors. Generally, the KIR2DL1 receptor recognizes HLA-C sub- cell inhibitory receptors (1). NK cells are innate lymphocytes that, types (C2 group), having lysine at position 80, whereas KIR2DL2 besides supporting developmental processes such as fetal growth receptors recognize HLA-C subtypes (C1 group), having asparagine during pregnancy (2), are best known for their ability to dis- atposition80(5). by guest on September 29, 2021 criminate between self and altered self by killing virally infected, MHC class I proteins are divided into classical and nonclassical. transformed, and damaged cells. NK cell activity is governed by In humans, the classical proteins are named HLA-A, -B, and -C, integrating signals derived from a panel of activating and in- each containing hundreds of different alleles. The nonclassical hibitory receptors. The inhibitory receptors are expressed sto- proteins are significantly less polymorphic and include HLA-E, chastically on NK cells, and their main inhibitory ligands are -F, -G, and HFE (6). MHC class I molecules (3, 4). There are several families of HLA-G is one of the most interesting nonclassical MHC class I MHC class I inhibitory receptors; among them, the KIR2DL proteins. It has a very unique expression pattern, in which it is upregulated on cancer cells, whereas on normal tissues its ex- pression is mainly restricted to the extravillous cytotrophoblasts *Lautenberg Center for General and Tumor Immunology, Institute of Medical Re- of the placenta, which is of fetal origin (7). Although HLA-G search Israel-Canada, Faculty of Medicine, Hebrew University Hadassah Medical School, 9112001 Jerusalem, Israel; and †Institute of Medical Immunology, Martin mRNA can be found in several tissues, HLA-G protein is absent, Luther University Halle-Wittenberg, 06112 Halle (Saale), Germany suggesting for regulation of HLA-G expression at the mRNA ORCID: 0000-0002-8489-9986 (D.N.). level. Received for publication July 9, 2015. Accepted for publication April 20, 2016. The transcription processes regulating MHC gene expression This work was supported by the European Research Council under the European were broadly investigated (8), and a few posttranscription regu- Union’s Seventh Framework Programme (FP/2007-2013)/European Research Council lators were discovered as well (9, 10). However, whether RNA- Grant Agreement 320473-BacNK. Further support came from the Israel Science Foun- dation, the German–Israeli Foundation for Scientific Research and Development (to O.M. binding proteins (RBPs) control the expression of classical and and B.S.), the Lewis Family Foundation, an Israel Cancer Research Fund professorship nonclassical MHC class I proteins is largely unexplored. grant, a Helmholtz Israel grant, and the Rosetrees Trust. This work was also supported RBPs play a part in every aspect of RNA biogenesis, including by Grants GRK1591 and SE-581-22-1 from the Deutscheforschungsgemeinschaft (to B.S.). transcription, pre-mRNA splicing, polyadenylation, RNA modifi- Address correspondence and reprint requests to Prof. Ofer Mandelboim, Lautenberg cation, transport, localization, translation, turnover, and immune ac- Center for General and Tumor Immunology, Hebrew University Hadassah Medical tivities (11–15). One major family of RBPs is the heterogeneous School, P.O. Box 12272, 9112001 Jerusalem, Israel. E-mail address: oferm@ekmd. huji.ac.il nuclear ribonucleoproteins (HNRNPs), which are among the most abundant proteins in the eukaryotic nucleus, taking part in processing Abbreviations used in this article: HNRNP, heterogeneous nuclear ribonucleoprotein; HNRNPR, heterogeneous nuclear ribonucleoprotein R; KD, knockdown; qRT-PCR, of precursor mRNA (16). In this study, we identify a member of this quantitative RT-PCR; RBP, RNA-binding protein; UTR, untranslated region; WB, family, heterogeneous nuclear ribonucleoprotein R (HNRNPR), as Western blot. a general regulator of classical and nonclassical MHC class I Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 expression. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501550 4968 HNRNPR REGULATES THE EXPRESSION OF MHC CLASS I PROTEINS Materials and Methods proteins that bound specifically to the RNAs, a SDS gel analysis was Cell culture performed and specific bands were detected with Coomassie brilliant blue G-250 (Sigma-Aldrich). Specific bands were excised and analyzed by mass JEG-3, U87, and Mel1074 cells were maintained in DMEM. The 721.221, spectrometry. Analysis was performed by the Smoler Proteomics Center Jurkat, Bjab, and U937 cells were maintained in RPMI 1640 medium. All (Technion, Haifa, Israel). media were supplemented with 10% FCS. Generation of lentivirus, knockdown, and overexpression RNA affinity purification and mass spectrometry The various RBP-knockdown (KD) vectors and scrambled vector are in The interactions between RNA and RNA-binding proteins were analyzed by the pLKO.1-puro plasmids and were purchased from Sigma-Aldrich. RNA affinity purification, as previously described (17). In short, the 39 Transduced JEG-3, 721.221, Jurkat, Bjab, and U937 cells were grown untranslated regions (UTRs) of HLA-G (sense and antisense orientation, in the presence of 1 mg/ml, 2 mg/ml, 1 mg/ml, 2 mg/ml, and 7 mg/ml National Center for Biotechnology Information Reference Sequence: puromycin, respectively. Cloning of the HNRNPR and HLA-G 39 UTRs NT_167249.2) and a UTR of a control gene with similar length and GC containing 14-bp deletion and insertion was performed into the content (GPR112) were cloned into the pBSII plasmid using the primers pHAGE-DsRED(2)-eGFP(+) lentiviral vector, which also contains (restriction sites are underlined), as follows: HLA-G sense forward (NotI), GFP. Lentiviruses were generated in 293T cells using a transient three- 59-ACGCGGCCGCATTGAAAGGAGGGAGCTA-39 and HLA-G sense plasmid transfection