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Bcalm (Ac099524.1) BCALM (AC099524.1) Is a Human B Lymphocyte-Specific Long Noncoding RNA That Modulates B Cell Receptor−Mediated Calcium Signaling This information is current as of October 4, 2021. Sarah C. Pyfrom, Chaz C. Quinn, Hannah K. Dorando, Hong Luo and Jacqueline E. Payton J Immunol published online 22 June 2020 http://www.jimmunol.org/content/early/2020/06/19/jimmun ol.2000088 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2020/06/19/jimmunol.200008 Material 8.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • 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 by guest on October 4, 2021 *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 © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 22, 2020, doi:10.4049/jimmunol.2000088 The Journal of Immunology BCALM (AC099524.1) Is a Human B Lymphocyte-Specific Long Noncoding RNA That Modulates B Cell Receptor–Mediated Calcium Signaling Sarah C. Pyfrom,1 Chaz C. Quinn, Hannah K. Dorando, Hong Luo,2 and Jacqueline E. Payton Of the thousands of long noncoding RNAs (lncRNA) identified in lymphocytes, very few have defined functions. In this study, we report the discovery and functional elucidation of a human B cell–specific lncRNA with high levels of expression in three types of B cell cancer and normal B cells. The AC099524.1 gene is upstream of the gene encoding the B cell–specific phospholipase C g 2 (PLCG2), a B cell–specific enzyme that stimulates intracellular Ca2+ signaling in response to BCR activation. AC099524.1 (B cell– associated lncRNA modulator of BCR-mediated Ca+ signaling [BCALM]) transcripts are localized in the cytoplasm and, as expected, CRISPR/Cas9 knockout of AC099524.1 did not affect PLCG2 mRNA or protein expression. lncRNA interactome, Downloaded from RNA immunoprecipitation, and coimmunoprecipitation studies identified BCALM-interacting proteins in B cells, including phospholipase D 1 (PLD1), and kinase adaptor proteins AKAP9 (AKAP450) and AKAP13 (AKAP-Lbc). These two AKAP proteins form signaling complexes containing protein kinases A and C, which phosphorylate and activate PLD1 to produce phosphatidic acid (PA). BCR stimulation of BCALM-deficient B cells resulted in decreased PLD1 phosphorylation and increased intracellular Ca+ flux relative to wild-type cells. These results suggest that BCALM promotes negative feedback that down- modulates BCR-mediated Ca+ signaling by promoting phosphorylation of PLD1 by AKAP-associated kinases, enhancing pro- http://www.jimmunol.org/ duction of PA. PA activates SHP-1, which negatively regulates BCR signaling. We propose the name BCALM for B-Cell Associated LncRNA Modulator of BCR-mediated Ca+ signaling. Our findings suggest a new, to our knowledge, paradigm for lncRNA-mediated modulation of lymphocyte activation and signaling, with implications for B cell immune response and BCR- dependent cancers. The Journal of Immunology, 2020, 205: 000–000. ppropriate response to environmental cues is critical for B lymphocyte activation pathways and consequent gene expression normal differentiation and cellular homeostasis and also changes promote survival, proliferation, and differentiation, but A serve as an essential firewall to prevent disease. This is when deregulated can drive lymphomagenesis (1–3). However, the by guest on October 4, 2021 true of innate and adaptive immune cells, which must differentiate mechanisms that drive and sustain these changes remain incom- self-antigen versus nonself-antigens and respond accordingly. pletely defined. Binding of Ag to the BCR in B lymphocytes triggers assembly Long noncoding RNAs (lncRNAs) are involved in the regulation of the BCR signalosome and a cascade of cellular activity, in- of gene transcription and signal transduction in normal and dis- cluding calcium flux, activation of transcription factors, and eased cells, including cancer (4–8). The lncRNA’s functional changes in gene expression. We and others have demonstrated that versatility includes epigenetic modification, nuclear domain Department of Pathology and Immunology, Washington University School of Med- agency. The corresponding author (J.E.P.) had full access to all of the data in the icine, St. Louis, MO 63110 study and had final responsibility for the decision to submit for publication. 1Current address: University of Pennsylvania School of Veterinary Medicine, Phila- The sequences presented in this article have been submitted to the Gene Expression delphia, PA. Omnibus under accession numbers GSE62246 and GSE132053. 2Current address: MilliporeSigma Inc., St. Louis, MO. Address correspondence and reprint requests to Dr. Jacqueline E. Payton, Washington University School of Medicine, 660 South Euclid Avenue, Box 8118, St. Louis, MO ORCIDs: 0000-0002-3781-6463 (S.C.P.); 0000-0002-9600-588X (H.K.D.); 0000- 63110. E-mail address: [email protected] 0001-8832-3661 (J.E.P.). The online version of this article contains supplemental material. Received for publication January 24, 2020. Accepted for publication May 27, 2020. Abbreviations used in this article: BCALM, B cell–associated lncRNA modulator of This work was supported by National Institutes of Health (NIH), National Cancer BCR-mediated Ca+ signaling; ChIP, chromatin immunoprecipitation; CLL, chronic Institute (NCI) Grants CA156690 and CA188286, Washington University Institute of lymphocytic lymphoma/leukemia; Co-IP, coimmunoprecipitation; DAG, diacylgly- Clinical and Translational Sciences (ICTS) Grant UL1 TR000448 from the National cerol; DLBCL, diffuse large B cell lymphoma; eCLIP, enhanced cross-linking im- Center for Advancing Translational Sciences (NCATS), and the Alvin J. Siteman munoprecipitation; FL, follicular lymphoma; gDNA, genomic DNA; gRNA, guide Cancer Center (CA091842). Sequencing was provided by the Genome Technology RNA; IP , inositol-1,4,5,-triphosphate; KO, knockout; lncRNA, long noncoding Access Center, which is partially supported by NCI Cancer Center Support Grant P30 3 RNA; MS, mass spectrometry; PA, phosphatidic acid; PIP , phosphatidylinositol- CA91842 to the Alvin J. Siteman Cancer Center and by ICTS/Clinical and Transla- 2 4,5-bisphosphate; PKA, protein kinase A; PKC, protein kinase C; PLAIDOH, Pre- tional Science Award (CTSA) Grant UL1 TR000448 from the National Center for dicting lncRNA Activity through Integrative Data-Driven ’Omics and Heuristics; Research Resources (NCRR), a component of the NIH, and the NIH Roadmap for PLD1, phospholipase D 1; PLDG2, phospholipase C g 2; qRT-PCR, real-time quan- Medical Research. The ICTS is funded by the NIH NCATS CTSA program Grant titative RT-PCR; RBP, RNA binding protein; RCI, relative concentration index; RIP, UL1 TR002345. RNA immunoprecipitation; RNA-seq, RNA sequencing; shRNA, short hairpin RNA; This publication is solely the responsibility of the authors and does not necessarily UCSC, University of California, Santa Cruz; WT, wild-type; WU, Washington represent the official view of NCRR or NIH. None of these sources had any role in University. data collection, analysis, interpretation, trial design, patient recruitment, writing the manuscript, the decision to submit the manuscript, or any other aspect pertinent to the Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 study. None of the authors were paid to write the article by a company or any other www.jimmunol.org/cgi/doi/10.4049/jimmunol.2000088 2 BCALM: A B CELL–SPECIFIC lncRNA THAT MODULATES BCR SIGNALING organization, transcriptional control, regulation of RNA splicing PLD1 enhances SHP-1 phosphatase activity on BCR-associated and translation, and modulation of protein signaling activity (9–12). kinases, attenuating BCR signaling. Based on these findings, we Although lncRNAs have emerged as key players in immune re- suggest the name BCALM for B cell–associated lncRNA modulator sponse and homeostasis, most reports concern innate or T cells, of BCR-mediated Ca+ signaling. Taken together, these studies and little has been reported on the function of lncRNAs in human demonstrate a novel mechanism for negative feedback modulation B lymphocytes (5, 11, 13, 14). To address this gap, we previously of BCR signaling via an lncRNA. developed a bioinformatic tool called Predicting lncRNA Activity through Integrative Data-Driven ’Omics and Heuristics (PLAI- Materials and Methods DOH), which constructs a statistical model using chromatin, gene Lymphoma samples and normal B cell RNA-seq analysis expression, subcellular localization, and genome architecture data Lymph node biopsies, bone marrow, and peripheral blood were collected coupled to biologically based logic rules to predict lncRNA from patients with FL, DLBCL, or CLL seen at the Washington Uni- function. We validated its accuracy by comparison with published versity (WU) Oncology Clinic. Patient characteristics are summarized in Table I. Tonsils were collected from
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