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

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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 patients undergoing tonsillectomy lncRNA functions, primarily in epithelial and myeloid tumors and at Barnes-Jewish and St. Louis Children’s Hospitals. Peripheral blood cell lines (15). from healthy donors was collected by the WU Volunteer for Health To address the function of lncRNAs in B cells, we performed a program. From these samples, CD19+ B cells were purified, and RNA global analysis of gene regulation in primary human B cell extracted as described by Koues et al. (1) and Pyfrom et al. (15). RNA- cancers and normal B cells from tonsil (chromatin immuno- seq was performed and analyzed as by Koues et al. (1), Pyfrom et al. (15), and Andrews et al. (35). Briefly, RNA was isolated from 1 to 2 3 precipitation [ChIP] and RNA sequencing [RNA-seq]). Our 106 cells from each sample with a Qiagen RNeasy Kit (catalog no. discovery analyses identified lncRNAs with high expression in 74104; Qiagen). rRNA-depleted (Epicentre, discontinued; Ribo-Zero) chronic lymphocytic lymphoma/leukemia (CLL), diffuse large libraries were prepared using TruSeq RNA Sample Kits with indexed Downloaded from B cell lymphoma (DLBCL), and follicular lymphoma (FL). We adaptors (Illumina) and subjected to 100-bp paired-end or 50-bp single- end sequencing on an Illumina Hi-Seq 2000 by the WU Genome used PLAIDOH to predict the function of these lncRNAs and Technology Access Center. Sequencing reads were aligned to hg38 then ranked them based on their potential role in B cell acti- using STAR (v2·5·3a) (36). Reads per kb of transcript per million vation, survival, or signaling, based on B cell specificity, ex- mapped reads–normalized genome browser tracks were created with pression level, and the function of the genes or pathways likely deepTools’ (v3·1·0) bamCoverage utility and visualized on the Uni- targeted (15). These analyses identified AC099524.1 (RP11- versity of California, Santa Cruz (UCSC) Genome Browser. Read http://www.jimmunol.org/ quantification was performed by Salmon (v0·11·0) using UCSC hg19 960L18.1, ENSG00000261218), an intergenic multiexon-spliced known gene annotations (37), and differential gene expression analyses lncRNA that is highly and specifically expressed in B cell lym- were performed with the DESeq2 R package (v1·20·0) (38). Genotify phomas and normal B cells. The AC099524.1 gene is located (v1·2·1) was used for manual gene curation (39). Coding Poten- upstream of the gene for the B cell–specific phospholipase C g 2 tial Assessment Tool was used to exclude transcripts with coding potential (40). (PLCG2), which is recruited to the BCR signalosome upon Ag binding, resulting in its phosphorylation and activation. Activated PLAIDOH analyses PLCG2 hydrolyzes the membrane lipid phosphatidylinositol-4,5- The lncRNA analyses with PLAIDOH were performed as in (15) with the bisphosphate (PIP2) to generate second messengers diacylglycerol following changes. Subcellular localization of lncRNAs with expression by guest on October 4, 2021 (DAG) and inositol-1,4,5,-triphosphate (IP3) that then stimulate detectable by RNA-seq in our datasets ($100 average normalized counts intracellular calcium flux and activate multiple downstream sig- in at least one B cell cancer or normal group) was determined using the naling pathways (16–18). Constitutional (germline) mutations lncATLAS database (http://lncatlas.crg.eu/) (41). Seventy-two percent of lncRNAs detected (1270/1765) had localization data in lncATLAS. Lo- in PLCG2 cause the immune dysregulation syndrome PLAID, calization relative concentration index (RCI) scores were downloaded for whereas acquired mutations confer resistance to Btk inhibitor all lncRNAs in the database, then the mean was calculated over all cell therapy in patients with leukemia or lymphoma (19, 20). Because lines for each lncRNA. An lncRNA was determined to be nuclear if its PLCG2 plays an essential role in B cell immune response, we mean RCI was ,20.5, both nuclear and cytoplasmic if its mean RCI was between 20.5 and 0.5, and cytoplasmic if its mean RCI was .0.5. RNA sought to determine the molecular function of AC099524.1 in binding protein (RBP) subcellular localization was determined by lo- B lymphocytes. cation information in the Human Protein Atlas (http://www.proteinatlas. We used lncRNA interactome (pull-down) assays, RNA immu- org) (42). An RBP was determined to be nuclear if it was annotated only noprecipitation (RIP), coimmunoprecipitation (Co-IP), CRISPR as nuclear in the cell atlas or tissue atlas, cytoplasmic if annotated only knockout (KO) of AC099524.1, and B cell activation assays to as cytoplasmic in the cell atlas or tissue atlas, or both nuclear and cy- toplasmic if shown to be located in the nucleus and cytoplasm by either elucidate the function of BCALM. These studies revealed that, the cell atlas or tissue atlas. If an RBP in the Encode enhanced cross- although the AC099524.1 gene coincides with enhancer regulatory linking immunoprecipitation (eCLIP) assay did not have localization data elements, the transcript does not regulate the expression level of in the Human Protein Atlas, subcellular localization was determined using neighboring genes PLCG2 and CMIP. Instead, BCALM is local- immunofluorescence as in (43). An RBP was considered to bind a site on an lncRNA if the same peak from the Encode eCLIP assay (defined as ized to the cytoplasm and interacts with phospholipase D 1 overlapping by at least 50% of its length) was present in at least two (PLD1) and A-kinase proteins AKAP9 (AKAP450) and AKAP13 replicates (https://www.encodeproject.org/eclip/) (44, 45). AC099524.1 (AKAP-Lbc). AKAPs assemble kinase signaling complexes to gene annotations from different genome databases are listed in Table II. ensure correct subcellular targeting, including protein kinase A Subcellular fractionation and real-time quantitative RT-PCR (PKA) and protein kinase C (PKC), which phosphorylate and activate PLD1 (21–30). PLD1 hydrolyzes lipid membrane com- Subcellular fractionation of cytoplasm, nuclear total, nucleoplasm, and ponent phosphatidylcholine to phosphatidic acid (PA), which ac- chromatin for RNA isolation was performed as described in (46). cDNA was generated from whole-cell lysate and cell fractions using the High- tivates the SHP-1 phosphatase (26, 31, 32), a negative regulator of Capacity RNA-to-cDNA Kit from Thermo Fisher Scientific (4387406). BCR signaling (33, 34). KO of AC099524.1 (BCALM) abrogated Real-time quantitative RT-PCR (qRT-PCR) was performed using primers the association of PLD1 and AKAP9, attenuated phosphorylation specific for PLCG2, AC099524.1, 18S, U1 snRNA, GAPDH (Table III), of PLD1 and enhanced calcium flux after BCR stimulation. These and the SYBR Green Real-Time PCR Master Mix. Expression was cal- culated relative to GAPDH using standard methods (15), except in sub- results support a role for BCALM in promoting negative feedback cellular fractionation experiments where inverse log2 cycle threshold modulation of BCR signaling by facilitating phosphorylation and values were calculated relative to the inverse log2 cycle threshold values of activation of PLD1. Increased production of PA by activated the cytoplasmic fraction. The Journal of Immunology 3

Luciferase reporter assays magnet, the supernatant was removed, and 250 ml of RNA lysis buffer was added. One milliliter of TRIzol was added to each tube and RNA purified Putative regulatory regions were PCR amplified from GM12878 B cell line according to manufacturer’s instructions. RNA was DNase-treated using genomic DNA (gDNA) (Table III). Cloning into the luciferase reporter ArticZymes Heat and Run gDNA Removal Kit (80200-250) according to plasmid was performed and luciferase reporter assays performed as in kit instructions. cDNA was made using the Applied Biosystems High- Andrews et al. (35) except that assays were performed in GM12878 Capacity cDNA Reverse Transcription Kit (4368814; Thermo Fisher B cells. All experiments were assayed in triplicates and performed at least Scientific). twice. The average ratios between the firefly luciferase reporter plasmid containing a putative regulatory region and control (NanoLuc) luciferase Co-IP readings were compared with the average ratio for the empty luciferase vector to determine relative luciferase activity. Ten million cells per Ab were lysed by rotation at 4˚C for 30 min in 0.025 M Tris, 0.15 M NaCl, 1% IGEPAL CA-630, and 5% glycerol (pH 7.4) with Cell culture, genome editing, and RNAi knockdown protease and phosphatase inhibitors, spun at 13,000 3 g for 10 min at 4˚C, 10% of lysate supernatant was saved for input samples. Supernatants were OCI-Ly7 and U2932 cells were cultured in RPMI 1640 + 10% FBS or incubated overnight at 4˚C with 5 ml of AKAP9 Ab (A301-662A; Bethyl IMDM + 20% FBS. Knockdown with short hairpin RNA (shRNA) was Laboratories) or 3 ml of PLD1 Ab (A305-241A; Bethyl Laboratories). performed as in (15). CRISPR KO of AC099524.1 in U2932 and OCI-Ly7 Forty microliters of Dynabeads Protein A (10002D; Thermo Fisher Sci- cells was performed as in (3) using target sequences in Table III. Confir- entific) per condition were washed three times with lysis buffer and in- mation of genome editing was performed with PCR of gDNA and qRT- cubated by rotation with Ab + supernatant for 3 h at 4˚C. Beads were PCR using primers in Table III. washed three times with lysis buffer and collected using a Dynamag-2 RNA pull-down Magnet (12321D; Thermo Fisher Scientific). Proteins were eluted with Laemmli sample buffer. In vitro transcription from plasmids containing the sense (59–39) or anti- Western blot sense (39–59) AC099524.1 (BCALM) cDNA (exons only) was performed Downloaded from using the T7 AmpliScribe Kit (AS3107; Lucigen) per manufacturer’s in- Whole cells were lysed in RIPA buffer supplemented with protease struction with 10% of UTP replaced with biotinylated UTP. Sense and inhibitors, an equal volume of loading dye was added, and lysates were antisense RNA were heated to 65˚C for 10 min, then allowed to cool slowly boiled for 10 min at 95˚C. After transfer to nitrocellulose paper, blots ∼ to 4˚C over 1 h to allow secondary structure formation. Ten million cells were incubated in primary Abs for AKAP9 (A301-662A; Bethyl were harvested per pull-down and washed twice in cold PBS, spinning at Laboratories), PLCG2 (sc-407; Santa Cruz Biotechnology), phospho- 3 500 g in a 4˚C tabletop centrifuge for 5 min between washes. The final PLCG2 (3874, Tyr759; Cell Signaling Technologies), PLD1 (A305- pellet was then resuspended in 1 ml of supplemented RNA lysis buffer 241A) PLD1 (28314; Santa Cruz), phospho-PLD1 (PA537688, http://www.jimmunol.org/ (0.025 M Tris, 0.28 M NaCl, 1% NP-40, 10% glycerol [pH 7.4], plus Thr147; Thermo Fisher Scientific), DICER (3363S; Cell Signaling protease inhibitors, phosphatase inhibitors, PMSF [all 1:100], 1:200 RN- Technologies), DHX9 (A300-855A; Bethyl Laboratories), or GAPDH Ase inhibitor, and 0.5 mM DTT) and allowed to lyse on ice for 30 min. (ab9485; Abcam). Lysates were then spun for 10 min at 13,000 3 g at 4˚C. The supernatant was moved to a fresh tube and supplemented with yeast tRNA. The lysate Calcium flux assays was diluted with supplemented RNA lysis buffer until the equivalent of 10 million cells per 750 ml was achieved. Seven hundred and fifty mi- Triplicate aliquots of 200,000 cells per condition were washed and croliters of lysate was then aliquoted into separate Eppendorf tubes, one resuspended in appropriate media plus 2% FBS. Cells were resuspended in per condition. Five micrograms of cooled RNA was added per pull-down Indo-1 working solution (1 mM in media + 2% FBS) and incubated at 37˚C and lysates were rotated for 4 h at 4˚C. Fifty microliters of Dynabeads in the dark for 30–60 min. Cells were washed twice with media + 2% FBS. Calcium flux was measured by flow cytometry on a BD LSRFORTESSA

MyOne Streptavidin C1 (65001) per condition were washed three times by guest on October 4, 2021 with unsupplemented RNA lysis buffer. Beads were then resuspended in X-20 according to manufacturer’s instructions. The emission of Indo-1 ∼ 2+ ∼ 2+ their original volume of lysis buffer and 50 ml were added to each pull- shifts from 475 nm without Ca (unbound) to 400 nm with Ca 2+ ∼ down. Tubes were rotated an additional 45 min at 4˚C. Beads were col- (Ca -bound) when excited at 350 nm. Baseline signal was acquired for lected on a Dynamag-2 Magnet (12321D) and supernatant aspirated. The 60 s, then 1 mg/ml Anti-IgM Ab (clone MHM-88, 314502; BioLegend) was added to stimulate BCR signaling and calcium flux, then signal was beads were then washed five times with RNA lysis buffer supplemented 2+ with PMSF and protease inhibitors, with 5 min rotations at 4˚C in between acquired for four additional minutes. The ratio of Ca -bound/-unbound washes. Finally, beads were resuspended in 50 ml of loading dye, boiled Indo-1 signal was calculated and plotted over time, and the confidence for 10 min at 95˚C, separated on an SDS-PAGE gel, silver stain was interval was calculated from triplicates for each experiment using R performed using the Pierce Silver Stain Kit (24612), and bands were ex- (version 3.6.1, R packages dplyr, smooth, TTR, Mcomp, and ggplot2). cised from the gel and sent for mass spectrometry (MS) (Taplin Biological Statistical analyses MS Facility at Harvard Medical School, Cambridge, MA or Danforth Center Proteomics and MS Facility, St. Louis, MO). Greater than nine All statistical tests were performed with GraphPad Prism (version 8.1.1). unique peptides were required for further analysis. List of proteins mapped to peptides detected by MS in Supplemental Table I. Data were analyzed Data accession using R packages dplyr and ggplot2. Sequencing data were deposited in Gene Expression Omnibus under ac- RIP analysis cession numbers GSE132053 and GSE62246. Ten million cells were harvested and washed twice in cold PBS, spun at Results 500 3 g in a 4˚C tabletop centrifuge for 5 min between washes. The final pellet was then resuspended in 1 ml of RNA lysis buffer (0.025 M Tris, BCALM (AC099524.1) is an lncRNA upstream of PLCG2 that 0.28 M NaCl, 1% NP-40, 10% glycerol (pH 7.4), plus protease inhibitors, is specifically and highly expressed in malignant and normal phosphatase inhibitors, PMSF [all 1:100], 1:200 RNAse inhibitor, and B cells 0.5 mM DTT) and allowed to lyse on ice for 30 min. Lysates were then spun for 10 min at 13,000 3 g at 4˚C. The supernatant was moved to a To identify lncRNAs that may play roles in B cell lymphoma, we fresh tube, and 5 ml were reserved for input. Ab was added to the super- collected lymph node biopsies, bone marrow, and peripheral blood natant at recommended concentrations per manufacturer’s instructions for from patients with FL, DLBCL, and CLL. For nonmalignant immunoprecipitation (Abs used as in Western blot) and rotated overnight B cells, tonsils were collected from patients undergoing tonsil- at 4˚C. Then 50 ml of PureProteome Protein A/G Mix Magnetic Beads (MilliporeSigma) per condition were washed three times with RNA lysis lectomy and peripheral blood was collected from healthy donors. buffer and added to each condition. After rotating for 4 h beads were Patient and sample characteristics are summarized in Table I. From collected on a Dynamag-2 Magnet (12321D) and supernatant aspirated. these samples, CD19+ B cells were purified, subjected to RNA The beads were then washed six times with RNA lysis buffer supple- extraction, and RNA-seq was performed as previously described mented with PMSF and protease inhibitors, with 5 min rotations at 4˚C in between washes. Beads and input tube were then resuspended in Proteinase (1, 15, 35). The lncRNAs have a range of cellular functions that are K Buffer (10% SDS, 10 mg/ml proteinase K in RIPA buffer) and incubated often initially categorized by subcellular localization. Consistent at 55˚C for 30 min with shaking. Tubes were then placed back on the with this, lncRNAs expressed in B cells from CLL, DLBCL, FL, 4 BCALM: A B CELL–SPECIFIC lncRNA THAT MODULATES BCR SIGNALING and tonsil are localized primarily in the nucleus, with smaller We specifically probed for H3K27ac, a chromatin mark associated proportions in the cytoplasm or both nucleus and cytoplasm with active enhancers, and H3K9/14ac (H3ac), a histone mark (Supplemental Fig. 1A). Overall, lncRNA expression levels are associated with active enhancers and promoters. Fig. 2A shows similar in cytoplasmic and nuclear fractions across the three types enrichment of H3K27ac and H3ac in several regions that overlap of B cell cancer and normal B cells (Supplemental Fig. 1B). In and flank AC099524.1 and its neighboring genes, CMIP and addition, a subset of lncRNAs exhibits significant expression dif- PLCG2. Coinciding with many of these regions are DNase hy- ferences across these B cell cancer groups (Supplemental Fig. 1C). persensitivity and binding sites for CTCF and EP300 (47) in In our previous study, we identified an intergenic multiexon-spliced B cells, which mark sites of accessible chromatin, chromatin lncRNA highly expressed in FL, AC099524.1 (ENSG00000261218, looping, and acetylation of chromatin, respectively. These epige- Table II), that is located 13 kb upstream of PLCG2, which encodes netic studies identified several putative enhancers in the region, as the PLCG2 protein, a critical effector of downstream BCR sig- well as the promoter of AC099524.1 (Fig. 2A). To test the tran- naling (15). In this study, we found that AC099524.1 (BCALM) is scriptional regulatory activity of these elements, we cloned them also expressed highly in DLBCL and CLL, as well as in normal into luciferase reporter vectors and assayed them in a B cell line. B cells (Fig. 1A, 1B, Table III). Expression of BCALM is quite We assayed the promoter of AC099524.1 because lncRNA pro- specific for B lymphocytes, as demonstrated by high expression moters often coincide with enhancer elements (5, 7, 48, 49), but in B lymphocytes and lymphocyte-rich tissues (tonsil, B cell not the promoter region of PLCG2 as it is a known gene promoter. lymphoma/leukemia, Fig. 1A, 1B; spleen, small intestine, Nearly all of the regions tested demonstrate transcriptional regu- Fig. 1C), but has no detectable expression in T lymphocytes latory activity that is significantly higher than the empty vector (Fig. 1A). We previously demonstrated that BCALM transcript control (Fig. 2B). The elements with the highest activity are lo- is localized to the cytoplasm (15) and confirmed this in additional cated between CMIP and AC099524.1, in the first intron of Downloaded from B cell lines (OCI-Ly7 and GM12878, Fig. 1D, Supplemental Fig. 1D). AC099524.1 and in the second intron of PLCG2. These results The functions of most lncRNAs have not been fully established, confirm that genomic elements in this locus exhibit cis-regulatory and prior to this study, the function of AC099524.1 (BCALM) was activity and suggest that they may regulate the transcription of unknown. To address this knowledge gap, we previously developed PLCG2, CMIP, and/or AC099524.1. a tool, PLAIDOH, to predict lncRNA function and to prioritize lncRNAs for experimental studies (15). PLAIDOH is a set of Deletion of BCALM (AC099524.1) has no effect on PLCG2 http://www.jimmunol.org/ bioinformatic modules that analyzes and integrates expression, protein expression or phosphorylation state epigenome, proteome, and cellular localization data with biolog- To generate a model with complete loss of BCALM, we designed ically informed rules to predict the function of lncRNAs. BCALM CRISPR/Cas9 gene–editing guides to delete the promoter and first transcript likely does not act as a transcriptional regulator because exon or promoter and first two exons in two additional B cell lines it is localized in the cytoplasm and its expression level does not (OCI-Ly7 and U2932, Fig. 3A, Supplemental Fig. 2A–C). Het- significantly correlate with neighboring genes CMIP and PLCG2 erozygous KO reduced BCALM expression 25–50%, and homo- [Fig. 1A and (15)]. To identify possible nontranscriptional func- zygous KO completely abrogated BCALM expression (Fig. 3B, tions, we previously used PLAIDOH to plot the interactions of 3D). As expected, there was no change in PLCG2 protein levels by guest on October 4, 2021 lncRNAs with RBPs identified by eCLIP sequencing (44) by ex- measured by Western blot (Fig. 3C, 3E). PLCG2 is phosphory- pression level, subcellular localization (41), and the number of lated and activated by BCR-associated kinases upon binding of Ag RBP binding sites (15). However, this approach was inherently to the BCR (16). KO of BCALM (AC099524.1) did not alter limited by the following: 1) the lncRNA-RBP interactions were phospho-PLCG2 levels at multiple timepoints after BCR stimu- identified by RIP of known RBPs, and thus no novel interacting lation with anti-IgM treatment (Fig. 3C, 3E). These findings proteins could be identified; and 2) no B cell lines were used (43, provide further evidence that BCALM transcript does not regulate 45). Therefore, we designed an experimental approach to elucidate RNA or protein levels of neighboring gene PLCG2 in B cells. the function of AC099524.1. BCALM interacts with signal transduction proteins in B cells Genomic regulatory elements in the BCALM locus exhibit Some lncRNAs act as scaffolds to regulate protein/protein inter- enhancer activity actions or to directly inhibit or activate their protein targets in the BCALM (AC099524.1) transcript is localized primarily to the cytoplasm (4, 5, 50–53). To identify BCALM-interacting proteins, cytoplasm and shRNA knockdown of AC099524.1 does not alter we performed an RNA pull-down using in vitro–transcribed, the expression of neighboring genes CMIP or PLCG2 [Fig. 1D, biotinylated BCALM sense (experimental) and antisense (control) Supplemental Fig. 1E and (15)], suggesting that cis-regulatory transcripts (Fig. 4A). Biotinylated sense and antisense transcripts elements (e.g., enhancers), rather than the BCALM transcript, were incubated with whole-cell lysates from B lymphoma cell may control the expression of the genes in this locus. To address lines and streptavidin beads, washed, and the bound proteins were this question, we performed ChIP sequencing in CD19+ B cells eluted, separated by SDS-PAGE, and visualized by silver stain. isolated from FL, DLBCL, and CLL patient samples and tonsils. Fig. 4B shows a representative silver-stained gel comparing

Table I. Patient characteristics

Disease Number Male/Female Age (Median/Range) CLL 19 9/10 60.5 (38–86) DLBCL 12 6/6 64.5 (50–77) Activated B cell (ABC) type 6 Germinal center B cell type 5 Gray zone/double hit type 1 FL 18 7/11 53 (35–78) Stage 1–2 15 The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on October 4, 2021

FIGURE 1. The lncRNA BCALM (AC099524.1) is highly and specifically expressed in B lymphocytes. (A) UCSC Genome Browser screenshot shows the genomic locus containing AC099524.1 and flanking genes. Tracks below show gene expression in representative samples of primary B cell cancers from WUSM patients (CLL412, DL135, FL313); nonmalignant CD19+ B, CD4+, and CD8+ T cells; and a lung adenocarcinoma cell line (A549) (RNA-seq, normalized transcripts per million). (B) Expression of BCALM in purified B cells from WUSM B cell cancer primary samples and normal B cells (RNA- seq, reads per kb of transcript per million mapped reads [RPKM], log10). Unpaired two-tailed t test with Welch correction; ***p , 0.001 (C) Violin plots show expression of BCALM and nearby coding gene PLCG2 across 27 different cell and tissue types (RNA-seq, log10, gtexportal.org). (D) Subcellular localization of RNA transcripts measured by qRT-PCR following fractionation of OCI-Ly7 lymphoma cells. (Fold change relative to same transcript in cytoplasm, mean 6 SD). Data are representative of at least three independent experiments (D). n.s., not significant for any comparison between B cell cancer groups; TPM, transcripts per million; WUSM, WU School of Medicine. 6 BCALM: A B CELL–SPECIFIC lncRNA THAT MODULATES BCR SIGNALING

Table II. AC099524.1 genome annotations signal intensity (Fig. 4C, 4D, Supplemental Table I). Some of these were previously reported to interact with BCALM by RBP Genome Database or Assembly Annotation for BCALM immunoprecipitation and sequencing (eCLIP sequencing) in my- HGNC gene symbol RP11-960L18.1 eloid and hepatic cell lines (15, 43–45). However, none of these Clone-based (Ensembl) AC099524.1 coincided with the most enriched proteins in the RNA pull-down gene name in B cells, suggesting that lncRNA-protein interactions may vary Ensembl gene ENSG00000261218 in different cell types. HAVANA manual ID gene OTTHUMG00000176531.2_5 Human (GRCh38.p13) Chr16: 81, 738, 248–81, 767, 868, Plotting by number of peptides and signal intensity demonstrates forward strand that several proteins were substantially enriched in sense BCALM GRCh37 location Chr16:81, 771, 853–81, 801, 473 pull-down compared with the antisense control (Fig. 4C). Several HGNC, HUGO Gene Nomenclature Committee; ID, identification. of those with the highest enrichment in sense BCALM pull-down have molecular weights that are consistent with bands in the sil- ver stain gel, including DHX9 and PLD1 (Fig. 4C, Supplemental proteins bound by biotinylated sense or antisense BCALM tran- Table I). DHX9, a DEAH-containing RNA helicase, and DICER1, script to input or streptavidin beads alone. Asterisks mark protein a DEXH-containing RNA helicase, both are known to bind RNA bands that are present in sense but not antisense lanes. We sub- molecules, including lncRNA (54–58). PLD1, a phospholipase mitted sense and antisense lanes from a similar gel and from a that hydrolyzes phosphatidylcholine to produce PA, is a compo- high m.w. gel for MS, which identified peptides and quantified nent of multiple signal transduction pathways, including AgR Downloaded from

Table III. qRT-PCR and gDNA PCR primers

Gene mRNA Target Primer Sequence PLCG2 Forward 59-TCAATCCGTCCATGCCTCAG-39 PLCG2 Reverse 59-CCTCGACGTAGTTGGATGGG-39 AC099524.1 Forward 59-GTCACACAGCCAACTTGCG-39 http://www.jimmunol.org/ AC099524.1 Reverse 59-AGCCTCTATCTGCTTACGTGC-39 U1 Forward 59-ATACTTACCTGGCAGGGGAGA-39 U1 Reverse 59-CAGGGGGAAAGCGCGAACGCA-39 18S Forward 59-GTGTGTGGGTTGACTTCGGA-39 18S Reverse 59-AAGGCTTTTCTCACCGAGGG-39 GAPDH Forward 59-ACCCACTCCTCCACCTTTGAC-39 GAPDH Reverse 59-TGTTGCTGTAGCCAAATTCGTT-39 Regulatory element region (luciferase) A Forward 59-GCAGGACTGTCCACAACTGTC-39 Regulatory element region (luciferase) A Reverse 59-GTGGTTACATACAAGAAGCCGTACA-39

Regulatory element region (luciferase) B Forward 59-CGCAAACTGGGTGGCCTAAA-39 by guest on October 4, 2021 Regulatory element region (luciferase) B Reverse 59-AAGTCCAGTGGCATGTGTCC-39 Regulatory element region (luciferase) C Forward 59-AACGTTGTCATGGAGACCCG-39 Regulatory element region (luciferase) C Reverse 59-CCATCCGCTACCCCAGGA-39 Regulatory element region (luciferase) D Forward 59-GGCTGAGGCACAAGAATTGC-39 Regulatory element region (luciferase) D Reverse 59-TATCCCAGCTCAGGGATGCT-39 Regulatory element region (luciferase) E Forward 59-GCAGGCCCAGGAGTGATAAG-39 Regulatory element region (luciferase) E Reverse 59-CAAGTTGGCTGTGTGACTGC-39 Regulatory element region (luciferase) F Forward 59-GGAGCCCCCAGTATTTTCC-39 Regulatory element region (luciferase) F Reverse 59-AACACCCTGTGCCTCCATT-39 Regulatory element region (luciferase) G Forward 59-GGCAGGTACGCAGGTAGTA-39 Regulatory element region (luciferase) G Reverse 59-TGTGGTCAGCATCCCTAGC-39 Regulatory element region (luciferase) H Forward 59-CCTTCCTCCATAGACCAGCG-39 Regulatory element region (luciferase) H Reverse 59-CAAAGCCCCAGCACAATACC-39 Regulatory element region (luciferase) I Forward 59-CTCTCTGTAGGGGCGAGACT-39 Regulatory element region (luciferase) I Reverse 59-GGGCGAAAACTCAAGCACTC-39 Regulatory element region (luciferase) J Forward 59-CTGGCAGAGCTGGGATTGAA-39 Regulatory element region (luciferase) J Reverse 59-CGGCATCCTAGACTGCTGTT-39 Regulatory element region (luciferase) K Forward 59-AGAGGTGGTCCCCTTAGCTC-39 Regulatory element region (luciferase) K Reverse 59-TACCGTGGTTGGTCTCCACT-39 Regulatory element region (luciferase) L Forward 59-GGAGCCCCCAGTATTTTCC-39 Regulatory element region (luciferase) L Reverse 59-AACACCCTGTGCCTCCATT-39 Regulatory element region (luciferase) M Forward 59-GGCAGGTACGCAGGTAGTA-39 Regulatory element region (luciferase) M Reverse 59-TGTGGTCAGCATCCCTAGC-39 CRISPR/Cas9 target upstream gRNA 1 59-CCGCGGGGAGGTTTCGTACC-39 CRISPR/Cas9 target downstream 1 gRNA 3 59-TGCAATCACAGTTACTCGGG-39 CRISPR/Cas9 target downstream 2 gRNA 4 59-GAGCGCGTTCCTCCTACCTA-39 Genome editing intact site Forward 59-CCCTGAAGGCTCTTGTCTGAC-39 Genome editing intact site Reverse 59-CTCAGGAAGGGTTTCCAGATTTAGG-39 gRNA 1/3 deletion Forward 59-CCCTGAAGGCTCTTGTCTGAC-39 gRNA 1/3 deletion Reverse 59-GGTATGCACTGGCGCTG-39 gRNA 1/4 deletion Forward 59-CCCTGAAGGCTCTTGTCTGAC-39 gRNA 1/4 deletion Reverse 59-CCCACAATGGTAACTTTTGTGTGC-39 Control site Forward 59-ACCCACTCCTCCACCTTTGA-39 Control site Reverse 59-GTGGTCCAGGGGTCTTACTC-39 Intact site Forward 59-GCTGCCCCTAAAAGGACAGATT-39 Intact site Reverse 59-GGTATGCACTGGCGCTG-39 The Journal of Immunology 7

FIGURE 2. Active regulatory el- ements are distributed throughout the CMIP/AC099524.1/PLCG2 lo- cus. (A) UCSC Genome Browser screenshot shows the genomic locus containing AC099524.1 and flanking genes. Putative regulatory regions assayed by luciferase reporter in (B) are labeled A–M, and blue or green bars correspond to their size and location (blue: putative enhancer, green: promoter region of AC099524.1). Purple bars show peaks of DNase I hypersensitivity and tracks be- lowshowCTCFandEP300peaks [GM12878 B cells, ENCODE (47)]. Lower tracks show histone acetyla- tion peaks (H3K27ac, H3ac) in representative WU lymphoma and nonmalignant B cell samples (ChIP sequencing, normalized reads per Downloaded from million). (B) Bar graphs show lucif- erase reporter activity normalized to empty vector for each of the indicated regions in (A). Mean 6 SD for two to three experiments. Unpaired two- tailed t test with Welch correction. *p , 0.05, **p , 0.01, ***p , 0.001, http://www.jimmunol.org/ ****p , 0.0001. EV, empty vector.

activation, and is a key player in tumorigenesis but has not been phosphorylation increases 2- to 3-fold over the time course in previously reported to interact with RNA (24, 31, 32, 59–61). The WT cells, whereas in KO cells, phosphorylation levels show high m.w. bands were determined to be AKAP9 (AKAP450) and minimal change at all timepoints. IgM stimulation did not alter the AKAP13 (AKAP-Lbc), scaffold proteins that assemble kinase levels of BCALM transcript in WT or KO cells over the course of by guest on October 4, 2021 complexes, including PKA and PKC (21–23, 29, 30), both of the experiment (Supplemental Fig. 2D). which phosphorylate and activate PLD1 (27, 28, 31, 32, 62) (Fig. BCALM associates with PLD1 and AKAP9, a scaffold protein 4D). We confirmed the MS findings by Western blot, demon- that associates with PKA and PKC, which phosphorylates PLD1. strating that both PLD1 and DHX9 were enriched in sense com- Because loss of BCALM resulted in reduced levels of phospho- pared with antisense AC099524.1 pull-down. DICER1 showed PLD1, we hypothesized that BCALM may bring together PLD1 modest enrichment in sense AC099524.1 and as expected PLCG2 and an AKAP kinase complex, thereby promoting PLD1 phos- showed no enrichment (Fig. 4E). To validate the BCALM-protein phorylation. We tested this by immunoprecipitation of PLD1 in WT interactions identified by RNA pull-down, we performed an or- and KO B cell lines (OCI-Ly7). As shown in Fig. 5C, PLD1 thogonal assay, RIP, in which Abs against PLD1, DHX9, and coprecipitates AKAP9 in WT, but not in BCALM KO lymphoma DICER1 were used to assay interacting RNA transcripts. Results cell lines, suggesting that BCALM is necessary for the association from the RIP assays show that BCALM transcript was signifi- of PLD1 with the PKA/PKC kinase scaffold protein AKAP9. cantly enriched compared with IgG control for each of the three We next assessed the effect of BCALM deficiency on calcium proteins, with PLD1 and DICER1 having 2-fold or greater en- flux downstream of BCR stimulation. In these assays, anti-IgM richment and DXH9 with 1.4-fold enrichment (Fig. 4F). Western stimulation of the BCR causes assembly of the BCR signal- blot confirmed immunoprecipitation of the target proteins (Fig. osome, in which BCR-associated kinases activate PLCG2, which 4G). Taken together, these results identify interactions between hydrolyzes PIP2 to DAG and IP3. IP3 stimulates IP3 receptor BCALM and cytosolic signal transduction proteins, suggesting channels on endoplasmic reticulum to release calcium into the that BCALM may play a role in signaling pathways in B cells. cytoplasm (33, 34). Indo-1 is a fluorescent calcium indicator with emission at 475 nm when unbound that shifts to 400 nm when BCALM (AC099524.1) deficiency leads to increased calcium bound to Ca+, enabling accurate measurement of intracellu- flux in response to BCR stimulation lar calcium concentrations with flow cytometry (68). For these Given its specificity for B cells, interaction with signaling pathway studies, we preincubated WT, heterozygous or homozygous proteins, and proximity to PLCG2, we next assessed the effect of AC099524.1 (BCALM)–deficient cells with Indo-1 prior to BCR BCALM KO on signaling downstream of BCR activation. Al- stimulation with anti-IgM. The resulting calcium release into the though PLD1’s role in BCR signaling has not been previously cytoplasm caused a shift in the ratio of bound/unbound Indo-1, examined, PLD1 is involved in TCR activation and chemotaxis in which was detectable by flow cytometry. Compared with WT, T cells and in Ag stimulation of mast cells (27, 28, 61, 63–67). We heterozygous and homozygous AC099524.1 (BCALM)–deficient treated wild-type (WT) and KO B cell lines (OCI-Ly7) with anti- OCI-Ly7 lymphoma cells generated from two different guide IgM to stimulate the BCR and collected at 0, 2, 5, 10, 30, 60, RNA (gRNA) pairs exhibited higher levels of calcium flux after and 240 min poststimulation. Fig. 5A and 5B show that PLD1 BCR stimulation as shown by higher ratios of Ca+–bound/unbound 8 BCALM: A B CELL–SPECIFIC lncRNA THAT MODULATES BCR SIGNALING

FIGURE 3. KO of BCALM (AC099524.1) does not affect PLCG2 expression or phosphorylation. (A) Diagram depicts the locations of CRISPR/Cas9 targeting gRNAs for gene editing of AC099524.1 (red and blue triangles). (B) Expression of AC099524.1 in U2932 lymphoma cell line subclones post–gene editing. Measured by qRT-PCR, normalized to GAPDH, and shown relative to WT. gRNA pairs 1/3 and 1/4 were used for gene editing and are indi- cated. Mean 6 SD; homozygous (KO); genomic PCR in Supplemental Fig. 2A–C. (C) Western blots show levels of phosphorylated and total PLCG2 protein after BCR stimula- Downloaded from tion with anti-IgM in U2932 WT and KO cells. GAPDH is a loading con- trol. (D) Expression of AC099524.1 in OCI-Ly7 lymphoma cell line sub- clones post–gene editing, as in (B). Heterozygous (Het); genomic PCR in E Supplemental Fig. 2A–C. ( )West- http://www.jimmunol.org/ ern blots of OCI-Ly7 cells treated as in (C). Representative of at least two independent experiments (B–E). by guest on October 4, 2021

Indo-1 (Fig. 5D). A similar increase in calcium flux was observed for functional experimental studies. AC099524.1 (BCALM) had in two AC099524.1 (BCALM) KO U2932 lymphoma cell lines high potential for activity in B cells because of its specific and compared with WT (Supplemental Fig. 2E) and in a nonmalig- high expression in B cell cancers and normal B cells and its nant B cell line treated with BCALM shRNAs (GM12878, proximity to the gene that encodes PLCG2, a critical regulator of Supplemental Fig. 2F). Taken together, these data support a role downstream BCR signaling. In addition, BCALM was intriguing for BCALM in calcium flux downstream of BCR stimulation, because of its cytoplasmic localization, which was less common perhaps through modulation of PLD1 phosphorylation (summa- for lncRNAs but in agreement with PLAIDOH’s prediction (15). rized in Fig. 6; see Discussion). Also in agreement with its functional prediction, BCALM tran- script itself does not regulate the expression of neighboring genes Discussion CMIP or PLCG2, but enhancers in this locus are active and may In mature B lymphocytes, binding of Ag to the BCR activates regulate the expression of AC099524.1 (BCALM), CMIP, and/or signaling pathways and transcription factors that change gene PLCG2. These results are consistent with reports that, for some expression, driving differentiation, survival, and proliferation enhancer lncRNAs, the transcripts themselves are dispensable for (69, 70). Perturbation of these pathways is the hallmark of “cis”-regulation of neighboring gene transcription and instead B cell cancers, but the underlying mechanisms remain in- function in “trans” in other pathways. Indeed, we identified completely defined (71–75). In normal hematopoietic cells and BCALM-interacting proteins that are involved in nontranscrip- non–B cell cancers, lncRNAs have emerged as key players in tional cell processes, including signal transduction proteins PLD1 modulating gene expression, protein translation, and signaling and AKAP9. Our initial RNA pull-down studies used an in vitro– pathways (4–6, 9, 11, 51, 53, 76–86); however, little is known transcribed BCALM that might lack posttranscriptional modifi- regarding their functional roles in B cell activation or lym- cations present on the endogenous lncRNA, and thus could have phoma (6, 13, 87–90). To address this critical knowledge gap, missed protein interactions that require such modifications. we performed global epigenome and transcriptome studies in Nonetheless, subsequent RIP, Co-IP, B cell activation, and calcium human B cell cancers and normal B lymphocytes and identi- flux assays were performed in B cells and demonstrate that en- fied lncRNAs as potential actors in lymphocyte activation dogenous BCALM interacts with PLD1 and AKAP9 and modu- pathways. lates their activity. We used an integrative bioinformatic tool we developed Other cytosolic lncRNAs have been shown to modulate signaling [PLAIDOH (15)] to select one of these lncRNAs, AC099524.1, pathways. For example, lncRNA AK023948 positively regulates The Journal of Immunology 9

FIGURE 4. BCALM (AC099524.1) interacts with RNA binding and sig- naling transduction proteins. (A)Dia- gram of sense and antisense BCALM oligonucleotides used in (B). (B) Silver-stained SDS-PAGE gel shows proteins in input whole-cell lysate, sense and antisense BCALM pull- down, and beads alone control. As- terisks mark bands that are enriched in sense BCALM pull-down. (C) Proteins identified by MS after pull-down as in (B) and plotted by log2 fold change of sense/antisense BCALM for peptide number and signal intensity. (D) (Left panel)

Silver stain of high m.w. gel showing Downloaded from bands enriched in sense AC099524.1 pull-down compared with antisense (asterisks). (Right panel) Table of peptides identified by MS from the highlighted gel bands. (E) Western blots confirm BCALM pull-down of proteins highlighted in (C). (F) http://www.jimmunol.org/ Bar graph shows relative amount of BCALM transcript pulled down by RIP of the indicated proteins. qRT- PCR, unpaired two-tailed t test. *p , 0.05, ****p , 0.0001. (G) Western blots confirm immunopre- cipitation of indicated proteins from RIP in (F). Representative of at least two independent experiments, (B–G). by guest on October 4, 2021

AKTactivity in breast cancer by stabilizing PI3K subunit p85 and is interacts with PLD1, AKAP9 (AKAP450), and AKAP13 (AKAP- associated with poorer prognosis (52). In triple-negative breast Lbc). The AKAP proteins form signaling complexes with PKA cancer, lncRNA LINK-A associates with BRK and promotes and PKC (21–23, 29, 30), which phosphorylate and activate PLD1 the phosphorylation and stabilization of HIFa, leading to ac- (27, 28, 31, 32). PLD1 produces PA, which activates SHP-1 tivation of HIF1a transcriptional programs under normoxic (26, 97), a phosphatase that inhibits BCR-associated kinases and conditions (53). In macrophages, LPS-induced lncRNA Mirt2 downregulates BCR signaling (71, 72, 75, 98, 99) (Fig. 6A). inhibits K63-linked ubiquitination of TRAF6, thereby attenu- Without BCALM, interaction of PLD1 with the AKAP kinase ating NF-kB and MAPK pathways and providing negative complex is abrogated, resulting in decreased PLD1 phosphoryla- feedback regulation of inflammation (77). Many other studies tion and increased intracellular Ca+ concentrations in response in cancer, immune cells, and other organ systems also dem- to BCR stimulation, in a manner commensurate with BCALM onstrate diverse roles for cytoplasmic lncRNAs in metabolic, transcript level (i.e., homozygous loss increased calcium flux more signal transduction, and immune response pathways (4–7, 51, than heterozygous loss). These data suggest that BCALM acts as a 53, 80, 85, 91–93). scaffold and brings together PLD1 and AKAP-associated kinases Similar to the lncRNAs in the above studies, BCALM appears to to promote PLD1 phosphorylation and activation, resulting in act as a modulator of downstream BCR-mediated signaling and increased SHP-1 activity and downregulation of BCR signaling calcium flux. Fig. 6 depicts a model for how this modulation may and calcium flux in B cells. In the absence of BCALM, the in- occur. Ag binding to the BCR activates BCR-associated kinases teraction of PLD1 and AKAP9 is reduced and PLD1 phosphory- and PLCG2, which hydrolyzes PIP2 to second messengers DAG lation is decreased, which reduces the PLD1 activity and PA and IP3. IP3 stimulates IP3 receptor Ca+ channels on the endo- production. Lower levels of PA would result in decreased SHP-1 plasmic reticulum to release Ca+ into the cytoplasm, which, along activity and less inhibition of BCR signaling, and thus higher with DAG, stimulates downstream signaling proteins, including levels of intracellular calcium (Fig. 6B). Together, these data PKC, which promotes cell growth and survival in normal and suggest that BCALM transcript acts to promote negative feedback lymphoma B cells (33, 34, 69, 70, 72–75, 94–96). PKC also of BCR-mediated signaling and calcium flux in response to BCR phosphorylates and activates PLD1 (27, 28, 31, 32). BCALM stimulation. 10 BCALM: A B CELL–SPECIFIC lncRNA THAT MODULATES BCR SIGNALING

FIGURE 5. Loss of BCALM (AC099524.1) decreased PLD1 phos- phorylation and increased calcium flux after BCR stimulation. (A)Western blots show levels of phosphorylated and total PLD1 protein after BCR stimulation with anti-IgM in OCI-Ly7 WT and KO cells. GAPDH is a load- ing control. (B)Linegraphshowsthe ratio of phospho-PLD1 to total PLD1 from (A). (C) Western blots show Co- IP of AKAP9 with anti-PLD1 Ab in

WT but not in BCALM KO OCI-Ly7 Downloaded from cells (arrowheads: IB target protein bands; asterisks: nonspecific bands). (D) Indo-1 ratio measures the calcium flux in OCI-Ly7 WT, heterozygous (Het), and KO cells after addition of anti-IgM (vertical dashed line). Solid or dashed lines with intervening gray http://www.jimmunol.org/ shading indicate the mean, 10th, and 90th percentiles of three replicates. Representative of at least two inde- pendent experiments (A–D). by guest on October 4, 2021

AgR stimulation and downstream PLD and PKC signaling play synapse formation during Ag-dependent T cell activation (110). essential roles in diverse immune response pathways. Phospholipase In addition, AKAP9-deficient T cells exhibit decreased TCR D is a critical component of lipid second messenger signaling, recycling to the cell surface, resulting in reduced TCR reac- which is required for movement of cells (chemotaxis) and tivation and T cell retention in peripheral inflamed tissues cellular components (e.g., secretory vesicles, lysosomes). In (111). AKAP13 (AKAP-Lbc) forms a complex with the IkB T lymphocytes, PLD1 deficiency impairs TCR-mediated sig- to control the production of proinflammatory cytokines in naling, proliferation, cell adhesion, and chemotaxis in auto- cardiac myocytes in response to adrenergic stimulation (23, immune and infectious models (61, 64–66, 100). In mast cells, 112). Taken together, these published studies and the work PLD1 is activated by Ag stimulation and is required for exo- presented in this study suggest that BCR stimulation leads to cytosis of secretory granules (27, 28, 60, 101). In neutrophils phosphorylation and activation of PLD1 via AKAP-signaling and phagocytes, PLD1 is involved in chemotaxis, cell adhesion, kinase complexes, which are modulated by interaction with and migration, and its activation promotes antimicrobial de- the lncRNA BCALM (AC099524.1). Further work is needed fense by facilitating phagolysosomal maturation (66, 67, 102, to determine whether loss of PLD1, AKAP9, or AKAP13 103). There has been less study of PLD enzymes in B cells; in B cells similarly impacts BCR-mediated calcium flux or however, reports in human CLL and FL highlight the impor- functionally impacts BCR dynamics, immune synapse forma- tanceofPLD1inCXCL12-mediatedadhesionandinmTOR tion, or mTOR/NF-kB growth and survival pathways (95, activation downstream of the BCR-associated Syk kinase (104, 113, 114). 105). During BCR activation, PKC is activated by both DAG In summary, the results presented in this study suggest that the and Ca+ and, in turn, activates CARD11/MALT1/BCL-10 B cell–specific lncRNA BCALM (AC099524.1) may play an and NF-kB prosurvival and progrowth pathways (75, 95, 96, important negative regulatory role in modulating calcium sig- 106–108). AKAP proteins assemble kinase-containing com- naling downstream of BCR stimulation. BCALM thus repre- plexes, including PKA and PKC, and are involved in multiple sents a new, to our knowledge, paradigm for lncRNAs in signaling cascades in normal and malignant cells across organ B lymphocyte activation and signaling pathways, with impli- systems (21, 23, 30, 109). Relevant to these studies, AKAP9 cations for B cell development, immune response, and lym- (AKAP450) plays a key role in T cells in immunological phoma pathogenesis. The Journal of Immunology 11

FIGURE 6. Model for BCALM transcript modulation of calcium flux after BCR stimulation. (A) After IgM stimulation, BCR-associated kinases are activated and phosphorylate and activate PLCG2, which hydrolyzes PIP2 to DAG and IP3. IP3 stimulates Ca+ release from endoplasmic reticulum stores. DAG stimulates PKC, which associates with kinase-anchoring (AKAP) proteins. BCALM associates with AKAP proteins 9 and 13 and with PLD1, and thus may facilitate phosphorylation and activation of PLD1 by PKC and/or PKA. PA produced by activated PLD1 activates SHP-1, which dephosphorylates

BCR-associated kinases, resulting in a downregulation of BCR signaling. (B) Loss of BCALM decreased the association of PLD1 with AKAP9 and Downloaded from decreased PLD1 phosphorylation, the latter of which activates PLD1. Reduced PLD1 activity decreases PA production and results in less activation of SHP-1, decreasing the inhibition of BCR signaling. In sum, loss of BCALM could result in increased calcium flux via decreased feedback inhibition after BCR stimulation. Green arrows signify activation; red blunt-ended lines signify inhibition; dark gray arrows signify hydrolysis; pink arrows signify passage through an ion channel.

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Nuclear Cytoplasmic 62.8% 15.1% 2

0 lncRNA median expression (log10)

C CLL DLBCL FL Normal B 25 CLL vs. FL and DLBCL DLBCL vs. CLL and FL FL vs. CLL and DLBCL Non-significant E 1.5 20 BCALM (AC099254.1) CMIP PLCG2 15

10 1.0

Adjusted P-value (-log10) 5

0 0.5 -10 -5 0 5 10 Fold change (log2)

D 1.25 BCALM Expression relative to control shRNA

1.0

shControlshRNA_1shRNA_2 shControlshRNA_1shRNA_2 shControlshRNA_1shRNA_2 0.75

0.5

Expression relative to cytoplasm 0.25

0.0

Nuclear Cytoplasmic Figure S1. LncRNAs are distributed in nucleus, cytoplasm, or both subcellular fractions and differentially regulated in B cell cancers; shRNA targeted to BCALM reduces BCALM levels, but not CMIP or PLCG2. A) Pie graph shows that the majority of lncRNAs in B cells are localized in the nucleus, but a substantial proportion are in the cytoplasm or both nucleus and cytoplasm. B) Violin plots show that lncRNA expression is consistent across subcellular fractions and across B cell cancers and normal B cells. C) Volcano plot shows that 231 lncRNAs are differentially expressed in CLL, DLBCL, and FL B cell cancers (RNA-seq, DESeq2, significant threshold FDR < 0.05, absolute log2 fold change >1). D) Subcellular local- ization of BCALM transcript measured by qRT-PCR following fractionation of GM12878 B cells. (Fold change relative to same transcript in cytoplasm, mean ± SD). E) Expression of BCALM, CMIP, and PLCG2 relative to control shRNA in B lymphoblastoid cells (GM12878) treated with control or BCALM-targeted shRNAs (qRT-PCR, mean +/- standard deviation). Data are representative of at least two independent experiments (D-E). A 2kb Figure S2 gRNA 1 AC099524.1 gRNA 3 gRNA 4 exon 1 2 B

3000 1000 500 100 Het Het WT Het Het WT Het Het WT Het Het WT *Het *WT *KO *WT *Het *Het *WT *KO *WT *Het *Het *WT *KO *WT *Het *Het *WT *KO *WT *Het gRNA pair 1/3 1/4 1/3 1/4 1/3 1/4 1/3 1/4 intact site 1/4 deletion 1/3 deletion control site D 10-1 C WT KO

-2 3000 10 1000 500 10-3 100

Het Het Het Het Het Het Het Het Het -4 *WT *WT *WT 10 *KO_1 *KO_2 *KO_1 *KO_2 *KO_1 *KO_2

intact site 1/4 deletion control site Expression BCALM (log10) 10-5 0 2 5 10 30 60 240 Time (minutes) E F U2932 0.4 0.45 B lymphoblastoid cells WT shRNA control KO_1 shRNA_1 KO_2 shRNA_2 0.40

0.36 0.35

0.30 Indo-1 ratio (calcium bound/calcium free) Indo-1 ratio (calcium bound/calcium free) 0.32 0.25 0 100 200 300 0 100 200 300 400 Time (seconds) Time (seconds)

Figure S2. BCALM (AC099524.1) interacts with kinase scaffold proteins; AC099524.1 CRISPR/Cas9 gene editing scheme and genomic PCR evidence of successful editing; Loss of BCALM (AC099524.1) alters calcium flux after BCR stimulation. A) Diagram showing the locations of CRISPR/Cas9 targeting gRNAs for genome editing of AC099524.1 (red triangles). B) Gel image shows PCR products from OCI-Ly7 cell line subclones post-gene editing (after expressing doxycycline-inducible CRISPR/Cas9 and the indicated gRNA pairs). C) Gel image of PCR products from U2932 cell line subclones as in B. (* subclones used in experiments). D) Bar graph shows expression of BCALM relative to GAPDH after BCR stimulation in OCI-Ly7 WT and KO cells. qRT-PCR, mean +/- standard deviation for at least 2 independent experi- ments. E-F) Calcium flux within U2932 WT and KO subclones (E) or GM12878 cells treated with control or BCALM shRNAs (F) after addition of anti-IgM (vertical dotted line). Solid or dashed lines with intervening grey shading indicate the mean, 10th and 90th percentiles of 3 replicates. Representative of at least 2 independent experiments (D-F). Table S1. BCALM interacting proteins identified by RNA pull-down and mass spectrometry Gene Symbol MW Uniq Total Uniq Tota Sum Sum Log2fc Log2fc Log2fc kD ue Sens ue l AS Intensity Intensity UniquePep Intensity Total Sens e AS Sense AS tides S_AS S_AS Peptides e S_AS PLD1 124.11 19 19 4 4 1.80E+06 2.40E+05 2.24792751 2.9068906 2.24792751 DHX9 140.87 49 151 22 32 1.30E+08 6.00E+06 1.15527823 4.43740531 2.23840474 DICER1 218.54 49 81 20 21 1.20E+07 1.30E+06 1.29278175 3.20645088 1.94753258 DIS3L2 99.22 20 22 4 4 3.10E+06 6.00E+05 2.32192809 2.36923381 2.45943162 MATR3 94.56 23 67 11 19 3.40E+07 2.60E+06 1.06413034 3.70895122 1.81816168 YTHDC2 160.15 14 14 4 4 6.40E+05 1.30E+05 1.80735492 2.29956028 1.80735492 MRPS35 36.82 15 38 10 11 1.40E+07 1.80E+07 0.5849625 -0.3625701 1.78849589 LEO1 75.36 16 103 15 34 3.00E+08 4.90E+07 0.0931094 2.61410885 1.59903769 MPHOSPH10 78.82 10 12 4 4 4.80E+05 5.00E+04 1.32192809 3.26303441 1.5849625 TARBP2 39.01 10 20 7 7 9.80E+06 1.40E+06 0.51457317 2.80735492 1.51457317 TSR1 91.75 20 46 10 17 7.10E+06 1.10E+06 1 2.6903155 1.43609911 SSB 46.81 27 86 19 32 3.60E+07 1.20E+07 0.50695999 1.5849625 1.42626475 PRKRA 34.38 12 33 11 13 6.70E+07 6.20E+06 0.12553088 3.43382097 1.3439544 HTATSF1 85.8 11 15 6 6 8.10E+05 3.00E+05 0.87446912 1.43295941 1.32192809 ADAR 135.98 33 74 23 31 2.20E+07 6.90E+06 0.52083216 1.67283526 1.25525706 RBM19 107.27 14 14 6 6 9.90E+05 3.30E+05 1.22239242 1.5849625 1.22239242 U2SURP 118.22 14 15 6 7 1.60E+06 4.40E+05 1.22239242 1.86249648 1.09953567 IGF2BP3 63.67 24 83 20 39 9.60E+07 1.50E+07 0.26303441 2.67807191 1.08963721 RPL5 34.34 24 107 19 51 4.40E+07 1.30E+07 0.33703499 1.7589919 1.06904164 RRP12 143.61 39 82 26 41 2.70E+07 9.10E+06 0.5849625 1.56902096 1 SF3B1 145.74 41 97 33 49 3.10E+07 7.50E+06 0.31315789 2.04730571 0.985203 IGF2BP1 63.44 10 25 5 13 1.70E+07 5.60E+06 1 1.60203601 0.94341647 NOP14 97.61 23 46 14 24 7.60E+06 2.10E+06 0.71620703 1.85561009 0.93859946 RBM28 85.68 14 23 9 12 3.70E+06 1.60E+06 0.63742992 1.20945337 0.93859946 MRPS9 45.81 19 44 13 23 1.10E+07 3.90E+06 0.5474878 1.49595749 0.93586966 DDX54 98.53 16 19 10 10 2.40E+06 1.80E+06 0.67807191 0.4150375 0.92599942 SDC4 21.63 14 86 11 47 5.00E+07 2.20E+07 0.3479233 1.18442457 0.8716759 NAT10 115.66 19 20 11 11 2.90E+06 1.30E+06 0.78849589 1.15754128 0.86249648 DDB1 126.89 20 20 11 11 1.80E+06 8.70E+05 0.86249648 1.0489096 0.86249648 DHX30 133.85 59 179 39 99 1.70E+08 3.90E+07 0.59724083 2.12398872 0.85445916 RPS6 28.66 20 141 17 78 9.20E+08 2.20E+08 0.23446525 2.06413034 0.85414913 YBX3 40.07 13 66 9 37 1.90E+08 7.80E+07 0.53051472 1.28445339 0.83494075 SF3B3 135.49 38 94 27 53 7.00E+07 2.40E+07 0.49304001 1.54432052 0.8266684 IWS1 91.9 23 90 15 51 1.70E+07 7.60E+06 0.61667136 1.16146342 0.81942775 MYBBP1A 148.76 37 65 22 37 1.20E+07 5.40E+06 0.75002175 1.15200309 0.81291445 RBM34 48.54 14 14 8 8 1.80E+06 1.20E+06 0.80735492 0.5849625 0.80735492 DDX58 106.53 17 17 10 10 1.50E+06 4.20E+05 0.76553475 1.83650127 0.76553475 VPRBP 168.9 10 10 6 6 7.00E+05 2.20E+05 0.73696559 1.6698514 0.73696559 MOV10 113.6 26 28 17 17 4.90E+06 1.60E+06 0.61297688 1.61470984 0.71989208 MRPS26 24.2 11 18 4 11 2.30E+06 1.60E+06 1.45943162 0.52356196 0.71049338 STAU2 62.6 28 44 20 27 8.90E+06 4.60E+06 0.48542683 0.95217147 0.70454412 THRAP3 108.6 11 13 8 8 1.80E+06 8.10E+05 0.45943162 1.15200309 0.70043972 KRI1 82.55 20 37 13 23 5.40E+06 2.50E+06 0.62148838 1.11103131 0.68589141

Table S1. BCALM interacting proteins identified by RNA pull-down and mass spectrometry MRPS22 41.25 23 78 21 49 5.70E+07 3.10E+07 0.13124453 0.8786937 0.67069237 RPL10 24.59 10 51 8 33 9.60E+07 7.90E+06 0.32192809 3.60310985 0.62803122 MVP 99.27 15 17 11 11 1.50E+06 7.60E+05 0.44745898 0.98089118 0.62803122 LARP1 123.43 28 69 24 45 4.70E+07 1.70E+07 0.22239242 1.46712601 0.61667136 MRPS31 45.29 17 35 14 23 2.20E+07 1.30E+07 0.28010792 0.7589919 0.60572106 UPF1 124.27 34 62 26 41 3.00E+07 9.20E+06 0.38702312 1.70525673 0.59664431 DAP3 45.54 14 24 10 16 1.50E+07 3.20E+06 0.48542683 2.22881869 0.5849625 WHSC1 152.16 12 12 8 8 6.00E+05 4.60E+05 0.5849625 0.38332864 0.5849625 ZRANB2 37.38 13 28 7 19 6.20E+06 1.60E+06 0.8930848 1.95419631 0.55942741 XRN2 108.51 18 25 14 17 2.90E+06 1.30E+06 0.36257008 1.15754128 0.55639335 NCL 76.57 46 487 36 334 2.30E+09 4.40E+08 0.35363695 2.38605843 0.54407367 HERC5 116.78 14 16 9 11 2.10E+06 8.40E+05 0.63742992 1.32192809 0.54056838 WDR61 33.56 12 26 10 18 1.90E+07 1.20E+07 0.26303441 0.66296501 0.53051472 STAU1 63.14 24 39 18 27 2.30E+07 1.90E+07 0.4150375 0.27563444 0.53051472 SF3B2 100.16 47 153 36 106 5.40E+07 3.10E+07 0.38466385 0.80069119 0.52946739 POP1 114.64 21 50 18 35 1.40E+07 5.20E+06 0.22239242 1.4288433 0.51457317 MRPS2 33.23 12 20 10 14 7.80E+06 4.60E+06 0.26303441 0.76184026 0.51457317 RPL6 32.71 15 62 11 44 4.90E+07 2.90E+07 0.44745898 0.75672885 0.49476469 RPL8 28.01 12 38 10 27 7.70E+07 2.40E+07 0.26303441 1.68182404 0.49304001 RPS3 26.67 29 287 28 206 2.10E+09 6.40E+08 0.05062607 1.71424552 0.4784064 DDX24 96.27 35 96 29 69 3.90E+07 1.40E+07 0.27130202 1.4780473 0.47643804 ZC3H18 106.32 10 15 8 11 1.90E+06 8.90E+05 0.32192809 1.09412218 0.44745898 SMARCA5 121.83 29 40 22 30 5.70E+06 2.60E+06 0.39854938 1.1324503 0.4150375 CDC5L 92.19 12 12 9 9 5.20E+05 2.90E+05 0.4150375 0.84245872 0.4150375 BMS1 145.72 12 12 7 9 9.50E+05 6.60E+05 0.77760758 0.52546149 0.4150375 CTR9 133.42 34 65 23 49 6.00E+07 1.80E+07 0.56390089 1.73696559 0.40765797 RPS4X 29.58 16 50 14 38 2.70E+07 1.70E+07 0.19264508 0.66742466 0.39592868 NIFK 34.2 11 13 8 10 1.50E+06 9.20E+05 0.45943162 0.70525673 0.37851162 MRPL15 33.4 11 13 10 10 2.40E+06 1.60E+06 0.13750352 0.5849625 0.37851162 GNL2 83.6 19 22 15 17 3.80E+06 1.40E+06 0.34103692 1.44057259 0.37196878 KRR1 43.64 15 22 11 17 6.70E+06 4.70E+06 0.44745898 0.51150034 0.37196878 YBX1 35.9 19 819 18 642 6.00E+09 2.70E+09 0.07800251 1.15200309 0.35129015 HNRNPU 90.53 13 14 9 11 1.80E+06 1.60E+06 0.53051472 0.169925 0.3479233 RPL10A 24.82 12 33 11 26 1.10E+08 4.50E+07 0.12553088 1.28950662 0.3439544 TRIM28 88.49 21 40 19 32 1.10E+07 4.20E+06 0.14438991 1.38904229 0.32192809 RPS3A 29.93 34 441 27 353 2.70E+09 4.10E+08 0.33257534 2.71926359 0.32111047 NOP2 89.25 31 80 28 65 6.70E+07 2.90E+07 0.14684139 1.2081082 0.29956028 PHAX 44.38 10 11 8 9 1.40E+06 9.50E+05 0.32192809 0.55942741 0.28950662 MRPS7 28.12 12 23 10 19 1.30E+07 5.80E+06 0.26303441 1.16438682 0.27563444 RPL7A 29.98 15 60 12 50 1.70E+08 7.00E+07 0.32192809 1.28010792 0.26303441 EIF4A3 46.84 12 12 10 10 1.40E+06 6.80E+05 0.26303441 1.04182018 0.26303441 MRPS34 25.63 11 18 11 15 2.70E+06 1.40E+06 0 0.94753258 0.26303441 RPS2 31.3 23 170 16 142 1.80E+09 1.00E+09 0.52356196 0.84799691 0.25964382 RPL3 46.08 14 86 15 72 8.90E+07 6.80E+07 -0.0995357 0.38827059 0.25633975 BAZ1B 170.8 18 19 15 16 1.90E+06 1.00E+06 0.26303441 0.92599942 0.24792751 MRPS18B 29.38 10 32 9 27 1.70E+07 8.10E+06 0.15200309 1.06954093 0.2451125

Table S1. BCALM interacting proteins identified by RNA pull-down and mass spectrometry 16.76 11 420 10 356 1.30E+10 5.80E+09 0.13750352 1.16438682 0.23851209 RPLP0P6 34.34 15 74 13 63 4.60E+08 5.60E+08 0.20645088 -0.283793 0.23217344 RPL7 29.21 18 55 14 47 8.60E+07 9.70E+07 0.36257008 -0.1736481 0.22677086 UBAP2L 114.47 12 14 12 12 1.80E+06 8.10E+05 0 1.15200309 0.22239242 RPL13 24.25 17 135 15 117 4.70E+08 1.70E+08 0.18057225 1.46712601 0.20645088 EFTUD2 109.37 15 15 13 13 1.50E+06 8.20E+05 0.20645088 0.87126669 0.20645088 LACTB 60.65 15 30 15 26 1.10E+07 6.30E+06 0 0.80407979 0.20645088 CEBPZ 120.9 14 16 12 14 2.30E+06 1.40E+06 0.22239242 0.71620703 0.19264508 CDC73 60.54 29 68 24 61 4.50E+07 3.80E+07 0.27301849 0.24392558 0.1567255 NOC4L 58.43 10 10 9 9 1.30E+06 8.50E+05 0.15200309 0.61297688 0.15200309 HNRNPUL1 95.68 23 42 20 38 9.30E+06 1.10E+07 0.20163386 -0.2422009 0.14438991 DNAJC9 29.89 11 11 9 10 1.70E+07 1.10E+07 0.28950662 0.62803122 0.13750352 UBTF 89.35 11 13 10 12 1.70E+06 2.40E+06 0.13750352 -0.4974997 0.11547722 HP1BP3 61.17 16 35 16 33 8.90E+06 8.40E+06 0 0.08341601 0.0848889 MCCC1 80.42 31 101 27 96 6.10E+07 6.90E+07 0.19930881 -0.1777871 0.07324898 GTPBP4 73.92 17 26 16 25 3.20E+06 3.50E+06 0.08746284 -0.129283 0.05658353 PAF1 59.94 36 103 34 100 8.60E+07 1.20E+08 0.08246216 -0.4806258 0.04264434 MCCC2 61.29 23 88 23 86 6.60E+07 5.50E+07 0 0.26303441 0.03316686 FTSJ3 96.5 10 10 10 10 7.40E+05 4.10E+05 0 0.85190136 0 LYAR 43.59 14 19 13 19 4.50E+06 3.20E+06 0.1069152 0.4918531 0 RPS9 22.58 17 126 20 126 5.60E+08 4.30E+08 -0.2344653 0.38109017 0 BYSL 49.57 19 37 18 37 1.00E+07 8.20E+06 0.07800251 0.28630419 0 UTP14A 87.92 10 11 10 11 5.40E+05 4.60E+05 0 0.23132555 0 GNL3 61.95 16 19 15 19 3.30E+06 2.90E+06 0.0931094 0.18641312 0 LTV1 54.82 17 24 14 24 5.50E+06 5.90E+06 0.28010792 -0.1012833 0 MAP7D1 92.76 13 17 13 17 3.20E+06 4.40E+06 0 -0.4594316 0 PTCD3 78.5 17 38 17 39 1.60E+07 1.50E+07 0 0.0931094 -0.0374747 HSPA5 72.29 25 31 25 32 3.70E+06 5.70E+06 0 -0.6234366 -0.0458037 SKIV2L2 117.73 19 22 17 23 2.50E+06 2.70E+06 0.16046467 -0.1110313 -0.0641303 ZC3HAV1 101.37 14 21 11 22 2.40E+06 1.30E+06 0.3479233 0.88452278 -0.0671142 PCCB 58.18 16 21 14 22 3.60E+06 2.80E+06 0.19264508 0.36257008 -0.0671142 NOP58 59.54 15 17 12 18 1.60E+06 3.00E+06 0.32192809 -0.9068906 -0.0824622 PCCA 80.01 14 14 15 15 1.60E+06 1.80E+06 -0.0995357 -0.169925 -0.0995357 RSL1D1 54.94 19 27 17 29 1.50E+07 9.30E+06 0.16046467 0.68965988 -0.1030935 SRGN 17.64 11 79 12 88 4.40E+08 3.30E+08 -0.1255309 0.4150375 -0.1556509 NAP1L1 45.35 10 17 8 19 8.50E+06 9.50E+06 0.32192809 -0.1604647 -0.1604647 RPL4 47.67 20 55 18 62 3.20E+07 2.50E+07 0.15200309 0.35614381 -0.1728366 FAM120A 121.81 18 22 15 25 1.80E+06 1.50E+06 0.26303441 0.26303441 -0.1844246 MRPS27 47.58 14 35 15 40 1.00E+07 1.50E+07 -0.0995357 -0.5849625 -0.1926451 DDX27 89.78 13 15 16 18 3.20E+06 2.30E+06 -0.2995603 0.47643804 -0.2630344 ACACA 265.38 65 108 68 131 2.80E+07 6.80E+07 -0.065095 -1.2801079 -0.2785355 TUBB2A 49.87 13 52 15 64 9.40E+07 2.10E+08 -0.2064509 -1.1596567 -0.2995603 PABPC4 70.74 13 17 12 21 1.40E+07 1.40E+07 0.11547722 0 -0.3048546 ZCCHC8 78.53 16 22 20 29 4.60E+06 6.80E+06 -0.3219281 -0.5639009 -0.3985494 ZFR 116.94 11 12 12 16 9.80E+05 1.10E+06 -0.1255309 -0.1666499 -0.4150375 TUBA1A 50.1 19 62 18 83 1.40E+08 1.60E+08 0.07800251 -0.1926451 -0.4208431

Table S1. BCALM interacting proteins identified by RNA pull-down and mass spectrometry LARP7 66.86 21 23 16 31 4.60E+06 6.10E+06 0.39231742 -0.4071754 -0.4306344 NKRF 77.62 11 11 14 16 9.10E+06 1.80E+06 -0.3479233 2.33786964 -0.5405684 PABPC1 70.63 30 72 31 105 9.50E+07 2.40E+08 -0.0473057 -1.337035 -0.5443205 ILF2 43.04 14 29 15 43 1.10E+07 2.00E+07 -0.0995357 -0.8624965 -0.5682838 DDX17 80.22 13 13 16 20 1.80E+06 3.50E+06 -0.2995603 -0.959358 -0.6214884 MRPL19 33.51 10 11 12 18 2.00E+06 2.80E+06 -0.2630344 -0.4854268 -0.7104934 NOP56 66.01 15 17 19 28 2.10E+06 4.30E+06 -0.3410369 -1.0339473 -0.7198921 HNRNPUL2 85.05 13 13 17 22 5.30E+05 1.70E+06 -0.3870231 -1.6814705 -0.7589919 HSPA9 73.63 12 12 17 21 1.30E+06 2.70E+06 -0.5025003 -1.0544478 -0.8073549 SART3 109.87 32 52 37 92 9.50E+06 2.60E+07 -0.2094534 -1.4525122 -0.8231222 RUVBL2 51.12 15 15 23 27 1.70E+06 8.90E+06 -0.6166714 -2.3882706 -0.8479969 ILF3 95.28 21 34 20 63 3.70E+06 1.10E+07 0.07038933 -1.5719063 -0.8898171 DDX21 87.29 35 115 40 217 1.20E+08 2.70E+08 -0.1926451 -1.169925 -0.9160612 HSPA8 70.85 17 19 20 36 3.20E+06 1.50E+07 -0.2344653 -2.2288187 -0.9219975 HNRNPM 77.46 37 102 43 200 4.10E+07 1.30E+08 -0.2168114 -1.6648158 -0.9714308 RUVBL1 50.2 12 15 18 30 2.00E+06 9.30E+06 -0.5849625 -2.2172307 -1 DHX36 114.69 17 22 24 47 2.40E+06 5.20E+06 -0.4974997 -1.1154772 -1.0951572 SYNCRIP 69.56 10 16 20 38 1.40E+06 7.70E+06 -1 -2.4594316 -1.2479275 MYH9 226.39 13 13 36 39 5.80E+05 5.00E+06 -1.4694853 -3.1078033 -1.5849625