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 γ 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, 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 downmodulates BCR-mediated Ca+ signaling by promoting phosphorylation of PLD1 by AKAP-associated kinases, enhancing production 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.

This article is featured in In This Issue, p.553

Appropriate response to environmental cues is critical for normal differentiation and cellular homeostasis and also serve as an essential firewall to prevent disease. This is true of innate and adaptive immune cells, which must differentiate self-antigen versus nonself-antigens and respond accordingly. Binding of Ag to the BCR in B lymphocytes triggers assembly of the BCR signalosome and a cascade of cellular activity, including calcium flux, activation of transcription factors, and changes in gene expression. We and others have demonstrated that B lymphocyte activation pathways and consequent gene expression changes promote survival, proliferation, and differentiation, but when deregulated can drive lymphomagenesis (13). However, the mechanisms that drive and sustain these changes remain incompletely defined.

Long noncoding RNAs (lncRNAs) are involved in the regulation of gene transcription and signal transduction in normal and diseased cells, including cancer (48). The lncRNA’s functional versatility includes epigenetic modification, nuclear domain organization, transcriptional control, regulation of RNA splicing and translation, and modulation of protein signaling activity (912). Although lncRNAs have emerged as key players in immune response and homeostasis, most reports concern innate or T cells, and little has been reported on the function of lncRNAs in human B lymphocytes (5, 11, 13, 14). To address this gap, we previously developed a bioinformatic tool called Predicting lncRNA Activity through Integrative Data-Driven 'Omics and Heuristics (PLAIDOH), which constructs a statistical model using chromatin, gene expression, subcellular localization, and genome architecture data coupled to biologically based logic rules to predict lncRNA function. We validated its accuracy by comparison with published lncRNA functions, primarily in epithelial and myeloid tumors and cell lines (15).

To address the function of lncRNAs in B cells, we performed a global analysis of gene regulation in primary human B cell cancers and normal B cells from tonsil (chromatin immunoprecipitation [ChIP] and RNA sequencing [RNA-seq]). Our discovery analyses identified lncRNAs with high expression in chronic lymphocytic lymphoma/leukemia (CLL), diffuse large B cell lymphoma (DLBCL), and follicular lymphoma (FL). We used PLAIDOH to predict the function of these lncRNAs and then ranked them based on their potential role in B cell activation, survival, or signaling, based on B cell specificity, expression level, and the function of the genes or pathways likely targeted (15). These analyses identified AC099524.1 (RP11-960L18.1, ENSG00000261218), an intergenic multiexon-spliced lncRNA that is highly and specifically expressed in B cell lymphomas and normal B cells. The AC099524.1 gene is located upstream of the gene for the B cell–specific phospholipase C γ 2 (PLCG2), which is recruited to the BCR signalosome upon Ag binding, resulting in its phosphorylation and activation. Activated PLCG2 hydrolyzes the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to generate second messengers diacylglycerol (DAG) and inositol-1,4,5,-triphosphate (IP3) that then stimulate intracellular calcium flux and activate multiple downstream signaling pathways (1618). Constitutional (germline) mutations in PLCG2 cause the immune dysregulation syndrome PLAID, whereas acquired mutations confer resistance to Btk inhibitor therapy in patients with leukemia or lymphoma (19, 20). Because PLCG2 plays an essential role in B cell immune response, we sought to determine the molecular function of AC099524.1 in B lymphocytes.

We used lncRNA interactome (pull-down) assays, RNA immunoprecipitation (RIP), coimmunoprecipitation (Co-IP), CRISPR knockout (KO) of AC099524.1, and B cell activation assays to elucidate the function of BCALM. These studies revealed that, although the AC099524.1 gene coincides with enhancer regulatory elements, the transcript does not regulate the expression level of neighboring genes PLCG2 and CMIP. Instead, BCALM is localized to the cytoplasm and interacts with phospholipase D 1 (PLD1) and A-kinase proteins AKAP9 (AKAP450) and AKAP13 (AKAP-Lbc). AKAPs assemble kinase signaling complexes to ensure correct subcellular targeting, including protein kinase A (PKA) and protein kinase C (PKC), which phosphorylate and activate PLD1 (2130). PLD1 hydrolyzes lipid membrane component phosphatidylcholine to phosphatidic acid (PA), which activates the SHP-1 phosphatase (26, 31, 32), a negative regulator of BCR signaling (33, 34). KO of AC099524.1 (BCALM) abrogated the association of PLD1 and AKAP9, attenuated phosphorylation of PLD1 and enhanced calcium flux after BCR stimulation. These results support a role for BCALM in promoting negative feedback modulation of BCR signaling by facilitating phosphorylation and activation of PLD1. Increased production of PA by activated PLD1 enhances SHP-1 phosphatase activity on BCR-associated kinases, attenuating BCR signaling. Based on these findings, we suggest the name BCALM for B cell–associated lncRNA modulator of BCR-mediated Ca+ signaling. Taken together, these studies demonstrate a novel mechanism for negative feedback modulation of BCR signaling via an lncRNA.

Lymph node biopsies, bone marrow, and peripheral blood were collected from patients with FL, DLBCL, or CLL seen at the Washington University (WU) Oncology Clinic. Patient characteristics are summarized in Table I. Tonsils were collected from patients undergoing tonsillectomy at Barnes-Jewish and St. Louis Children’s Hospitals. Peripheral blood from healthy donors was collected by the WU Volunteer for Health program. From these samples, CD19+ B cells were purified, and RNA extracted as described by Koues et al. (1) and Pyfrom et al. (15). RNA-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 × 106 cells from each sample with a Qiagen RNeasy Kit (catalog no. 74104; Qiagen). rRNA-depleted (Epicentre, discontinued; Ribo-Zero) libraries were prepared using TruSeq RNA Sample Kits with indexed 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 Technology Access Center. Sequencing reads were aligned to hg38 using STAR (v2·5·3a) (36). Reads per kb of transcript per million mapped reads–normalized genome browser tracks were created with deepTools’ (v3·1·0) bamCoverage utility and visualized on the University of California, Santa Cruz (UCSC) Genome Browser. Read quantification was performed by Salmon (v0·11·0) using UCSC hg19 known gene annotations (37), and differential gene expression analyses were performed with the DESeq2 R package (v1·20·0) (38). Genotify (v1·2·1) was used for manual gene curation (39). Coding Potential Assessment Tool was used to exclude transcripts with coding potential (40).

The lncRNA analyses with PLAIDOH were performed as in (15) with the following changes. Subcellular localization of lncRNAs with expression detectable by RNA-seq in our datasets (≥100 average normalized counts in at least one B cell cancer or normal group) was determined using the lncATLAS database (http://lncatlas.crg.eu/) (41). Seventy-two percent of lncRNAs detected (1270/1765) had localization data in lncATLAS. Localization relative concentration index (RCI) scores were downloaded for all lncRNAs in the database, then the mean was calculated over all cell lines for each lncRNA. An lncRNA was determined to be nuclear if its mean RCI was <−0.5, both nuclear and cytoplasmic if its mean RCI was between −0.5 and 0.5, and cytoplasmic if its mean RCI was >0.5. RNA binding protein (RBP) subcellular localization was determined by location information in the Human Protein Atlas (http://www.proteinatlas.org) (42). An RBP was determined to be nuclear if it was annotated only as nuclear in the cell atlas or tissue atlas, cytoplasmic if annotated only as cytoplasmic in the cell atlas or tissue atlas, or both nuclear and cytoplasmic if shown to be located in the nucleus and cytoplasm by either the cell atlas or tissue atlas. If an RBP in the Encode enhanced cross-linking immunoprecipitation (eCLIP) assay did not have localization data in the Human Protein Atlas, subcellular localization was determined using 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 overlapping by at least 50% of its length) was present in at least two replicates (https://www.encodeproject.org/eclip/) (44, 45). AC099524.1 gene annotations from different genome databases are listed in Table II.

Subcellular fractionation of cytoplasm, nuclear total, nucleoplasm, and chromatin for RNA isolation was performed as described in (46). cDNA was generated from whole-cell lysate and cell fractions using the High-Capacity RNA-to-cDNA Kit from Thermo Fisher Scientific (4387406). Real-time quantitative RT-PCR (qRT-PCR) was performed using primers specific for PLCG2, AC099524.1, 18S, U1 snRNA, GAPDH (Table III), and the SYBR Green Real-Time PCR Master Mix. Expression was calculated relative to GAPDH using standard methods (15), except in subcellular fractionation experiments where inverse log2 cycle threshold values were calculated relative to the inverse log2 cycle threshold values of the cytoplasmic fraction.

Putative regulatory regions were PCR amplified from GM12878 B cell line genomic DNA (gDNA) (Table III). Cloning into the luciferase reporter plasmid was performed and luciferase reporter assays performed as in Andrews et al. (35) except that assays were performed in GM12878 B cells. All experiments were assayed in triplicates and performed at least twice. The average ratios between the firefly luciferase reporter plasmid containing a putative regulatory region and control (NanoLuc) luciferase readings were compared with the average ratio for the empty luciferase vector to determine relative luciferase activity.

OCI-Ly7 and U2932 cells were cultured in RPMI 1640 + 10% FBS or IMDM + 20% FBS. Knockdown with short hairpin RNA (shRNA) was performed as in (15). CRISPR KO of AC099524.1 in U2932 and OCI-Ly7 cells was performed as in (3) using target sequences in Table III. Confirmation of genome editing was performed with PCR of gDNA and qRT-PCR using primers in Table III.

In vitro transcription from plasmids containing the sense (5′–3′) or antisense (3′–5′) AC099524.1 (BCALM) cDNA (exons only) was performed using the T7 AmpliScribe Kit (AS3107; Lucigen) per manufacturer’s instruction with 10% of UTP replaced with biotinylated UTP. Sense and antisense RNA were heated to 65°C for 10 min, then allowed to cool slowly to 4°C over∼1 h to allow secondary structure formation. Ten million cells were harvested per pull-down and washed twice in cold PBS, spinning at 500 × g in a 4°C tabletop centrifuge for 5 min between washes. The final pellet was then resuspended in 1 ml of supplemented RNA lysis buffer (0.025 M Tris, 0.28 M NaCl, 1% NP-40, 10% glycerol [pH 7.4], plus protease inhibitors, phosphatase inhibitors, PMSF [all 1:100], 1:200 RNAse inhibitor, and 0.5 mM DTT) and allowed to lyse on ice for 30 min. Lysates were then spun for 10 min at 13,000 × g at 4°C. The supernatant was moved to a fresh tube and supplemented with yeast tRNA. The lysate was diluted with supplemented RNA lysis buffer until the equivalent of 10 million cells per 750 μl was achieved. Seven hundred and fifty microliters of lysate was then aliquoted into separate Eppendorf tubes, one per condition. Five micrograms of cooled RNA was added per pull-down and lysates were rotated for 4 h at 4°C. Fifty microliters of Dynabeads MyOne Streptavidin C1 (65001) per condition were washed three times with unsupplemented RNA lysis buffer. Beads were then resuspended in their original volume of lysis buffer and 50 μl were added to each pull-down. Tubes were rotated an additional 45 min at 4°C. Beads were collected on a Dynamag-2 Magnet (12321D) and supernatant aspirated. The beads were then washed five times with RNA lysis buffer supplemented with PMSF and protease inhibitors, with 5 min rotations at 4°C in between washes. Finally, beads were resuspended in 50 μl of loading dye, boiled for 10 min at 95°C, separated on an SDS-PAGE gel, silver stain was performed using the Pierce Silver Stain Kit (24612), and bands were excised from the gel and sent for mass spectrometry (MS) (Taplin Biological MS Facility at Harvard Medical School, Cambridge, MA or Danforth Center Proteomics and MS Facility, St. Louis, MO). Greater than nine unique peptides were required for further analysis. List of proteins mapped to peptides detected by MS in Supplemental Table I. Data were analyzed using R packages dplyr and ggplot2.

Ten million cells were harvested and washed twice in cold PBS, spun at 500 × 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, 0.28 M NaCl, 1% NP-40, 10% glycerol (pH 7.4), plus protease inhibitors, phosphatase inhibitors, PMSF [all 1:100], 1:200 RNAse inhibitor, and 0.5 mM DTT) and allowed to lyse on ice for 30 min. Lysates were then spun for 10 min at 13,000 × g at 4°C. The supernatant was moved to a fresh tube, and 5 μl were reserved for input. Ab was added to the supernatant at recommended concentrations per manufacturer’s instructions for immunoprecipitation (Abs used as in Western blot) and rotated overnight at 4°C. Then 50 μl of PureProteome Protein A/G Mix Magnetic Beads (MilliporeSigma) per condition were washed three times with RNA lysis buffer and added to each condition. After rotating for 4 h beads were collected on a Dynamag-2 Magnet (12321D) and supernatant aspirated. The beads were then washed six times with RNA lysis buffer supplemented with PMSF and protease inhibitors, with 5 min rotations at 4°C in between washes. Beads and input tube were then resuspended in Proteinase K Buffer (10% SDS, 10 mg/ml proteinase K in RIPA buffer) and incubated at 55°C for 30 min with shaking. Tubes were then placed back on the magnet, the supernatant was removed, and 250 μl of RNA lysis buffer was added. One milliliter of TRIzol was added to each tube and RNA purified according to manufacturer’s instructions. RNA was DNase-treated using ArticZymes Heat and Run gDNA Removal Kit (80200-250) according to kit instructions. cDNA was made using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (4368814; Thermo Fisher Scientific).

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 protease and phosphatase inhibitors, spun at 13,000 × g for 10 min at 4°C, 10% of lysate supernatant was saved for input samples. Supernatants were incubated overnight at 4°C with 5 μl of AKAP9 Ab (A301-662A; Bethyl Laboratories) or 3 μl of PLD1 Ab (A305-241A; Bethyl Laboratories). Forty microliters of Dynabeads Protein A (10002D; Thermo Fisher Scientific) per condition were washed three times with lysis buffer and incubated by rotation with Ab + supernatant for 3 h at 4°C. Beads were washed three times with lysis buffer and collected using a Dynamag-2 Magnet (12321D; Thermo Fisher Scientific). Proteins were eluted with Laemmli sample buffer.

Whole cells were lysed in RIPA buffer supplemented with protease inhibitors, an equal volume of loading dye was added, and lysates were boiled for 10 min at 95°C. After transfer to nitrocellulose paper, blots were incubated in primary Abs for AKAP9 (A301-662A; Bethyl Laboratories), PLCG2 (sc-407; Santa Cruz Biotechnology), phospho-PLCG2 (3874, Tyr759; Cell Signaling Technologies), PLD1 (A305-241A) PLD1 (28314; Santa Cruz), phospho-PLD1 (PA537688, Thr147; Thermo Fisher Scientific), DICER (3363S; Cell Signaling Technologies), DHX9 (A300-855A; Bethyl Laboratories), or GAPDH (ab9485; Abcam).

Triplicate aliquots of 200,000 cells per condition were washed and resuspended in appropriate media plus 2% FBS. Cells were resuspended in Indo-1 working solution (1 μM in media + 2% FBS) and incubated at 37°C 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 X-20 according to manufacturer’s instructions. The emission of Indo-1 shifts from∼475 nm without Ca2+ (unbound) to∼400 nm with Ca2+ (Ca2+-bound) when excited at∼350 nm. Baseline signal was acquired for 60 s, then 1 μg/ml Anti-IgM Ab (clone MHM-88, 314502; BioLegend) was added to stimulate BCR signaling and calcium flux, then signal was acquired for four additional minutes. The ratio of Ca2+-bound/-unbound Indo-1 signal was calculated and plotted over time, and the confidence interval was calculated from triplicates for each experiment using R (version 3.6.1, R packages dplyr, smooth, TTR, Mcomp, and ggplot2).

All statistical tests were performed with GraphPad Prism (version 8.1.1).

Sequencing data were deposited in Gene Expression Omnibus under accession numbers GSE132053 and GSE62246.

To identify lncRNAs that may play roles in B cell lymphoma, we collected lymph node biopsies, bone marrow, and peripheral blood from patients with FL, DLBCL, and CLL. For nonmalignant B cells, tonsils were collected from patients undergoing tonsillectomy and peripheral blood was collected from healthy donors. Patient and sample characteristics are summarized in Table I. From these samples, CD19+ B cells were purified, subjected to RNA extraction, and RNA-seq was performed as previously described (1, 15, 35). The lncRNAs have a range of cellular functions that are often initially categorized by subcellular localization. Consistent with this, lncRNAs expressed in B cells from CLL, DLBCL, FL, and tonsil are localized primarily in the nucleus, with smaller proportions in the cytoplasm or both nucleus and cytoplasm (Supplemental Fig. 1A). Overall, lncRNA expression levels are similar in cytoplasmic and nuclear fractions across the three types of B cell cancer and normal B cells (Supplemental Fig. 1B). In addition, a subset of lncRNAs exhibits significant expression differences across these B cell cancer groups (Supplemental Fig. 1C).

Table I.
Patient characteristics
DiseaseNumberMale/FemaleAge (Median/Range)
CLL 19 9/10 60.5 (38–86) 
DLBCL 12 6/6 64.5 (50–77) 
 Activated B cell (ABC) type   
 Germinal center B cell type   
 Gray zone/double hit type   
FL 18 7/11 53 (35–78) 
 Stage 1–2 15   
DiseaseNumberMale/FemaleAge (Median/Range)
CLL 19 9/10 60.5 (38–86) 
DLBCL 12 6/6 64.5 (50–77) 
 Activated B cell (ABC) type   
 Germinal center B cell type   
 Gray zone/double hit type   
FL 18 7/11 53 (35–78) 
 Stage 1–2 15   

In our previous study, we identified an intergenic multiexon-spliced lncRNA highly expressed in FL, AC099524.1 (ENSG00000261218, Table II), that is located 13 kb upstream of PLCG2, which encodes the PLCG2 protein, a critical effector of downstream BCR signaling (15). In this study, we found that AC099524.1 (BCALM) is also expressed highly in DLBCL and CLL, as well as in normal B cells (Fig. 1A, 1B, Table III). Expression of BCALM is quite specific for B lymphocytes, as demonstrated by high expression in B lymphocytes and lymphocyte-rich tissues (tonsil, B cell lymphoma/leukemia, Fig. 1A, 1B; spleen, small intestine, Fig. 1C), but has no detectable expression in T lymphocytes (Fig. 1A). We previously demonstrated that BCALM transcript is localized to the cytoplasm (15) and confirmed this in additional B cell lines (OCI-Ly7 and GM12878, Fig. 1D, Supplemental Fig. 1D).

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 ± 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.

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 ± 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.

Close modal

The functions of most lncRNAs have not been fully established, and prior to this study, the function of AC099524.1 (BCALM) was unknown. To address this knowledge gap, we previously developed a tool, PLAIDOH, to predict lncRNA function and to prioritize lncRNAs for experimental studies (15). PLAIDOH is a set of bioinformatic modules that analyzes and integrates expression, epigenome, proteome, and cellular localization data with biologically informed rules to predict the function of lncRNAs. BCALM transcript likely does not act as a transcriptional regulator because it is localized in the cytoplasm and its expression level does not significantly correlate with neighboring genes CMIP and PLCG2 [Fig. 1A and (15)]. To identify possible nontranscriptional functions, we previously used PLAIDOH to plot the interactions of lncRNAs with RBPs identified by eCLIP sequencing (44) by expression level, subcellular localization (41), and the number of RBP binding sites (15). However, this approach was inherently limited by the following: 1) the lncRNA-RBP interactions were identified by RIP of known RBPs, and thus no novel interacting proteins could be identified; and 2) no B cell lines were used (43, 45). Therefore, we designed an experimental approach to elucidate the function of AC099524.1.

Table II.
AC099524.1 genome annotations
Genome Database or AssemblyAnnotation for BCALM
HGNC gene symbol RP11-960L18.1 
Clone-based (Ensembl) gene name AC099524.1 
Ensembl gene ENSG00000261218 
HAVANA manual ID gene OTTHUMG00000176531.2_5 
Human (GRCh38.p13) Chr16: 81, 738, 248–81, 767, 868, forward strand 
GRCh37 location Chr16:81, 771, 853–81, 801, 473 
Genome Database or AssemblyAnnotation for BCALM
HGNC gene symbol RP11-960L18.1 
Clone-based (Ensembl) gene name AC099524.1 
Ensembl gene ENSG00000261218 
HAVANA manual ID gene OTTHUMG00000176531.2_5 
Human (GRCh38.p13) Chr16: 81, 738, 248–81, 767, 868, forward strand 
GRCh37 location Chr16:81, 771, 853–81, 801, 473 

HGNC, HUGO Gene Nomenclature Committee; ID, identification.

Table III.
qRT-PCR and gDNA PCR primers
Gene mRNA TargetPrimerSequence
PLCG2 Forward 5′-TCAATCCGTCCATGCCTCAG-3′ 
PLCG2 Reverse 5′-CCTCGACGTAGTTGGATGGG-3′ 
AC099524.1 Forward 5′-GTCACACAGCCAACTTGCG-3′ 
AC099524.1 Reverse 5′-AGCCTCTATCTGCTTACGTGC-3′ 
U1 Forward 5′-ATACTTACCTGGCAGGGGAGA-3′ 
U1 Reverse 5′-CAGGGGGAAAGCGCGAACGCA-3′ 
18S Forward 5′-GTGTGTGGGTTGACTTCGGA-3′ 
18S Reverse 5′-AAGGCTTTTCTCACCGAGGG-3′ 
GAPDH Forward 5′-ACCCACTCCTCCACCTTTGAC-3′ 
GAPDH Reverse 5′-TGTTGCTGTAGCCAAATTCGTT-3′ 
Regulatory element region (luciferase) A Forward 5′-GCAGGACTGTCCACAACTGTC-3′ 
Regulatory element region (luciferase) A Reverse 5′-GTGGTTACATACAAGAAGCCGTACA-3′ 
Regulatory element region (luciferase) B Forward 5′-CGCAAACTGGGTGGCCTAAA-3′ 
Regulatory element region (luciferase) B Reverse 5′-AAGTCCAGTGGCATGTGTCC-3′ 
Regulatory element region (luciferase) C Forward 5′-AACGTTGTCATGGAGACCCG-3′ 
Regulatory element region (luciferase) C Reverse 5′-CCATCCGCTACCCCAGGA-3′ 
Regulatory element region (luciferase) D Forward 5′-GGCTGAGGCACAAGAATTGC-3′ 
Regulatory element region (luciferase) D Reverse 5′-TATCCCAGCTCAGGGATGCT-3′ 
Regulatory element region (luciferase) E Forward 5′-GCAGGCCCAGGAGTGATAAG-3′ 
Regulatory element region (luciferase) E Reverse 5′-CAAGTTGGCTGTGTGACTGC-3′ 
Regulatory element region (luciferase) F Forward 5′-GGAGCCCCCAGTATTTTCC-3′ 
Regulatory element region (luciferase) F Reverse 5′-AACACCCTGTGCCTCCATT-3′ 
Regulatory element region (luciferase) G Forward 5′-GGCAGGTACGCAGGTAGTA-3′ 
Regulatory element region (luciferase) G Reverse 5′-TGTGGTCAGCATCCCTAGC-3′ 
Regulatory element region (luciferase) H Forward 5′-CCTTCCTCCATAGACCAGCG-3′ 
Regulatory element region (luciferase) H Reverse 5′-CAAAGCCCCAGCACAATACC-3′ 
Regulatory element region (luciferase) I Forward 5′-CTCTCTGTAGGGGCGAGACT-3′ 
Regulatory element region (luciferase) I Reverse 5′-GGGCGAAAACTCAAGCACTC-3′ 
Regulatory element region (luciferase) J Forward 5′-CTGGCAGAGCTGGGATTGAA-3′ 
Regulatory element region (luciferase) J Reverse 5′-CGGCATCCTAGACTGCTGTT-3′ 
Regulatory element region (luciferase) K Forward 5′-AGAGGTGGTCCCCTTAGCTC-3′ 
Regulatory element region (luciferase) K Reverse 5′-TACCGTGGTTGGTCTCCACT-3′ 
Regulatory element region (luciferase) L Forward 5′-GGAGCCCCCAGTATTTTCC-3′ 
Regulatory element region (luciferase) L Reverse 5′-AACACCCTGTGCCTCCATT-3′ 
Regulatory element region (luciferase) M Forward 5′-GGCAGGTACGCAGGTAGTA-3′ 
Regulatory element region (luciferase) M Reverse 5′-TGTGGTCAGCATCCCTAGC-3′ 
CRISPR/Cas9 target upstream gRNA 1 5′-CCGCGGGGAGGTTTCGTACC-3′ 
CRISPR/Cas9 target downstream 1 gRNA 3 5′-TGCAATCACAGTTACTCGGG-3′ 
CRISPR/Cas9 target downstream 2 gRNA 4 5′-GAGCGCGTTCCTCCTACCTA-3′ 
Genome editing intact site Forward 5′-CCCTGAAGGCTCTTGTCTGAC-3′ 
Genome editing intact site Reverse 5′-CTCAGGAAGGGTTTCCAGATTTAGG-3′ 
gRNA 1/3 deletion Forward 5′-CCCTGAAGGCTCTTGTCTGAC-3′ 
gRNA 1/3 deletion Reverse 5′-GGTATGCACTGGCGCTG-3′ 
gRNA 1/4 deletion Forward 5′-CCCTGAAGGCTCTTGTCTGAC-3′ 
gRNA 1/4 deletion Reverse 5′-CCCACAATGGTAACTTTTGTGTGC-3′ 
Control site Forward 5′-ACCCACTCCTCCACCTTTGA-3′ 
Control site Reverse 5′-GTGGTCCAGGGGTCTTACTC-3′ 
Intact site Forward 5′-GCTGCCCCTAAAAGGACAGATT-3′ 
Intact site Reverse 5′-GGTATGCACTGGCGCTG-3′ 
Gene mRNA TargetPrimerSequence
PLCG2 Forward 5′-TCAATCCGTCCATGCCTCAG-3′ 
PLCG2 Reverse 5′-CCTCGACGTAGTTGGATGGG-3′ 
AC099524.1 Forward 5′-GTCACACAGCCAACTTGCG-3′ 
AC099524.1 Reverse 5′-AGCCTCTATCTGCTTACGTGC-3′ 
U1 Forward 5′-ATACTTACCTGGCAGGGGAGA-3′ 
U1 Reverse 5′-CAGGGGGAAAGCGCGAACGCA-3′ 
18S Forward 5′-GTGTGTGGGTTGACTTCGGA-3′ 
18S Reverse 5′-AAGGCTTTTCTCACCGAGGG-3′ 
GAPDH Forward 5′-ACCCACTCCTCCACCTTTGAC-3′ 
GAPDH Reverse 5′-TGTTGCTGTAGCCAAATTCGTT-3′ 
Regulatory element region (luciferase) A Forward 5′-GCAGGACTGTCCACAACTGTC-3′ 
Regulatory element region (luciferase) A Reverse 5′-GTGGTTACATACAAGAAGCCGTACA-3′ 
Regulatory element region (luciferase) B Forward 5′-CGCAAACTGGGTGGCCTAAA-3′ 
Regulatory element region (luciferase) B Reverse 5′-AAGTCCAGTGGCATGTGTCC-3′ 
Regulatory element region (luciferase) C Forward 5′-AACGTTGTCATGGAGACCCG-3′ 
Regulatory element region (luciferase) C Reverse 5′-CCATCCGCTACCCCAGGA-3′ 
Regulatory element region (luciferase) D Forward 5′-GGCTGAGGCACAAGAATTGC-3′ 
Regulatory element region (luciferase) D Reverse 5′-TATCCCAGCTCAGGGATGCT-3′ 
Regulatory element region (luciferase) E Forward 5′-GCAGGCCCAGGAGTGATAAG-3′ 
Regulatory element region (luciferase) E Reverse 5′-CAAGTTGGCTGTGTGACTGC-3′ 
Regulatory element region (luciferase) F Forward 5′-GGAGCCCCCAGTATTTTCC-3′ 
Regulatory element region (luciferase) F Reverse 5′-AACACCCTGTGCCTCCATT-3′ 
Regulatory element region (luciferase) G Forward 5′-GGCAGGTACGCAGGTAGTA-3′ 
Regulatory element region (luciferase) G Reverse 5′-TGTGGTCAGCATCCCTAGC-3′ 
Regulatory element region (luciferase) H Forward 5′-CCTTCCTCCATAGACCAGCG-3′ 
Regulatory element region (luciferase) H Reverse 5′-CAAAGCCCCAGCACAATACC-3′ 
Regulatory element region (luciferase) I Forward 5′-CTCTCTGTAGGGGCGAGACT-3′ 
Regulatory element region (luciferase) I Reverse 5′-GGGCGAAAACTCAAGCACTC-3′ 
Regulatory element region (luciferase) J Forward 5′-CTGGCAGAGCTGGGATTGAA-3′ 
Regulatory element region (luciferase) J Reverse 5′-CGGCATCCTAGACTGCTGTT-3′ 
Regulatory element region (luciferase) K Forward 5′-AGAGGTGGTCCCCTTAGCTC-3′ 
Regulatory element region (luciferase) K Reverse 5′-TACCGTGGTTGGTCTCCACT-3′ 
Regulatory element region (luciferase) L Forward 5′-GGAGCCCCCAGTATTTTCC-3′ 
Regulatory element region (luciferase) L Reverse 5′-AACACCCTGTGCCTCCATT-3′ 
Regulatory element region (luciferase) M Forward 5′-GGCAGGTACGCAGGTAGTA-3′ 
Regulatory element region (luciferase) M Reverse 5′-TGTGGTCAGCATCCCTAGC-3′ 
CRISPR/Cas9 target upstream gRNA 1 5′-CCGCGGGGAGGTTTCGTACC-3′ 
CRISPR/Cas9 target downstream 1 gRNA 3 5′-TGCAATCACAGTTACTCGGG-3′ 
CRISPR/Cas9 target downstream 2 gRNA 4 5′-GAGCGCGTTCCTCCTACCTA-3′ 
Genome editing intact site Forward 5′-CCCTGAAGGCTCTTGTCTGAC-3′ 
Genome editing intact site Reverse 5′-CTCAGGAAGGGTTTCCAGATTTAGG-3′ 
gRNA 1/3 deletion Forward 5′-CCCTGAAGGCTCTTGTCTGAC-3′ 
gRNA 1/3 deletion Reverse 5′-GGTATGCACTGGCGCTG-3′ 
gRNA 1/4 deletion Forward 5′-CCCTGAAGGCTCTTGTCTGAC-3′ 
gRNA 1/4 deletion Reverse 5′-CCCACAATGGTAACTTTTGTGTGC-3′ 
Control site Forward 5′-ACCCACTCCTCCACCTTTGA-3′ 
Control site Reverse 5′-GTGGTCCAGGGGTCTTACTC-3′ 
Intact site Forward 5′-GCTGCCCCTAAAAGGACAGATT-3′ 
Intact site Reverse 5′-GGTATGCACTGGCGCTG-3′ 

BCALM (AC099524.1) transcript is localized primarily to the cytoplasm and shRNA knockdown of AC099524.1 does not alter the expression of neighboring genes CMIP or PLCG2 [Fig. 1D, Supplemental Fig. 1E and (15)], suggesting that cis-regulatory elements (e.g., enhancers), rather than the BCALM transcript, may control the expression of the genes in this locus. To address this question, we performed ChIP sequencing in CD19+ B cells isolated from FL, DLBCL, and CLL patient samples and tonsils. We specifically probed for H3K27ac, a chromatin mark associated with active enhancers, and H3K9/14ac (H3ac), a histone mark associated with active enhancers and promoters. Fig. 2A shows enrichment of H3K27ac and H3ac in several regions that overlap and flank AC099524.1 and its neighboring genes, CMIP and PLCG2. Coinciding with many of these regions are DNase hypersensitivity and binding sites for CTCF and EP300 (47) in B cells, which mark sites of accessible chromatin, chromatin looping, and acetylation of chromatin, respectively. These epigenetic studies identified several putative enhancers in the region, as well as the promoter of AC099524.1 (Fig. 2A). To test the transcriptional regulatory activity of these elements, we cloned them into luciferase reporter vectors and assayed them in a B cell line. We assayed the promoter of AC099524.1 because lncRNA promoters often coincide with enhancer elements (5, 7, 48, 49), but not the promoter region of PLCG2 as it is a known gene promoter. Nearly all of the regions tested demonstrate transcriptional regulatory activity that is significantly higher than the empty vector control (Fig. 2B). The elements with the highest activity are located between CMIP and AC099524.1, in the first intron of AC099524.1 and in the second intron of PLCG2. These results confirm that genomic elements in this locus exhibit cis-regulatory activity and suggest that they may regulate the transcription of PLCG2, CMIP, and/or AC099524.1.

FIGURE 2.

Active regulatory elements are distributed throughout the CMIP/AC099524.1/PLCG2 locus. (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 below show CTCF and EP300 peaks [GM12878 B cells, ENCODE (47)]. Lower tracks show histone acetylation peaks (H3K27ac, H3ac) in representative WU lymphoma and nonmalignant B cell samples (ChIP sequencing, normalized reads per million). (B) Bar graphs show luciferase reporter activity normalized to empty vector for each of the indicated regions in (A). Mean ± SD for two to three experiments. Unpaired two-tailed t test with Welch correction. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. EV, empty vector.

FIGURE 2.

Active regulatory elements are distributed throughout the CMIP/AC099524.1/PLCG2 locus. (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 below show CTCF and EP300 peaks [GM12878 B cells, ENCODE (47)]. Lower tracks show histone acetylation peaks (H3K27ac, H3ac) in representative WU lymphoma and nonmalignant B cell samples (ChIP sequencing, normalized reads per million). (B) Bar graphs show luciferase reporter activity normalized to empty vector for each of the indicated regions in (A). Mean ± SD for two to three experiments. Unpaired two-tailed t test with Welch correction. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. EV, empty vector.

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To generate a model with complete loss of BCALM, we designed CRISPR/Cas9 gene–editing guides to delete the promoter and first exon or promoter and first two exons in two additional B cell lines (OCI-Ly7 and U2932, Fig. 3A, Supplemental Fig. 2A–C). Heterozygous KO reduced BCALM expression 25–50%, and homozygous KO completely abrogated BCALM expression (Fig. 3B, 3D). As expected, there was no change in PLCG2 protein levels measured by Western blot (Fig. 3C, 3E). PLCG2 is phosphorylated and activated by BCR-associated kinases upon binding of Ag to the BCR (16). KO of BCALM (AC099524.1) did not alter phospho-PLCG2 levels at multiple timepoints after BCR stimulation with anti-IgM treatment (Fig. 3C, 3E). These findings provide further evidence that BCALM transcript does not regulate RNA or protein levels of neighboring gene PLCG2 in B cells.

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 indicated. Mean ± SD; homozygous (KO); genomic PCR in Supplemental Fig. 2A–C. (C) Western blots show levels of phosphorylated and total PLCG2 protein after BCR stimulation with anti-IgM in U2932 WT and KO cells. GAPDH is a loading control. (D) Expression of AC099524.1 in OCI-Ly7 lymphoma cell line subclones post–gene editing, as in (B). Heterozygous (Het); genomic PCR in Supplemental Fig. 2A–C. (E) Western blots of OCI-Ly7 cells treated as in (C). Representative of at least two independent experiments (B–E).

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 indicated. Mean ± SD; homozygous (KO); genomic PCR in Supplemental Fig. 2A–C. (C) Western blots show levels of phosphorylated and total PLCG2 protein after BCR stimulation with anti-IgM in U2932 WT and KO cells. GAPDH is a loading control. (D) Expression of AC099524.1 in OCI-Ly7 lymphoma cell line subclones post–gene editing, as in (B). Heterozygous (Het); genomic PCR in Supplemental Fig. 2A–C. (E) Western blots of OCI-Ly7 cells treated as in (C). Representative of at least two independent experiments (B–E).

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Some lncRNAs act as scaffolds to regulate protein/protein interactions or to directly inhibit or activate their protein targets in the cytoplasm (4, 5, 5053). To identify BCALM-interacting proteins, we performed an RNA pull-down using in vitro–transcribed, biotinylated BCALM sense (experimental) and antisense (control) transcripts (Fig. 4A). Biotinylated sense and antisense transcripts were incubated with whole-cell lysates from B lymphoma cell lines and streptavidin beads, washed, and the bound proteins were eluted, separated by SDS-PAGE, and visualized by silver stain. Fig. 4B shows a representative silver-stained gel comparing proteins bound by biotinylated sense or antisense BCALM transcript to input or streptavidin beads alone. Asterisks mark protein bands that are present in sense but not antisense lanes. We submitted sense and antisense lanes from a similar gel and from a high m.w. gel for MS, which identified peptides and quantified signal intensity (Fig. 4C, 4D, Supplemental Table I). Some of these were previously reported to interact with BCALM by RBP immunoprecipitation and sequencing (eCLIP sequencing) in myeloid and hepatic cell lines (15, 4345). However, none of these coincided with the most enriched proteins in the RNA pull-down in B cells, suggesting that lncRNA-protein interactions may vary in different cell types.

FIGURE 4.

BCALM (AC099524.1) interacts with RNA binding and signaling transduction proteins. (A) Diagram 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. Asterisks 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 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) 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 immunoprecipitation of indicated proteins from RIP in (F). Representative of at least two independent experiments, (B–G).

FIGURE 4.

BCALM (AC099524.1) interacts with RNA binding and signaling transduction proteins. (A) Diagram 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. Asterisks 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 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) 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 immunoprecipitation of indicated proteins from RIP in (F). Representative of at least two independent experiments, (B–G).

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Plotting by number of peptides and signal intensity demonstrates that several proteins were substantially enriched in sense BCALM pull-down compared with the antisense control (Fig. 4C). Several of those with the highest enrichment in sense BCALM pull-down have molecular weights that are consistent with bands in the silver stain gel, including DHX9 and PLD1 (Fig. 4C, Supplemental Table I). DHX9, a DEAH-containing RNA helicase, and DICER1, a DEXH-containing RNA helicase, both are known to bind RNA molecules, including lncRNA (5458). PLD1, a phospholipase that hydrolyzes phosphatidylcholine to produce PA, is a component of multiple signal transduction pathways, including AgR activation, and is a key player in tumorigenesis but has not been previously reported to interact with RNA (24, 31, 32, 5961). The high m.w. bands were determined to be AKAP9 (AKAP450) and AKAP13 (AKAP-Lbc), scaffold proteins that assemble kinase complexes, including PKA and PKC (2123, 29, 30), both of which phosphorylate and activate PLD1 (27, 28, 31, 32, 62) (Fig. 4D). We confirmed the MS findings by Western blot, demonstrating that both PLD1 and DHX9 were enriched in sense compared with antisense AC099524.1 pull-down. DICER1 showed modest enrichment in sense AC099524.1 and as expected PLCG2 showed no enrichment (Fig. 4E). To validate the BCALM-protein interactions identified by RNA pull-down, we performed an orthogonal assay, RIP, in which Abs against PLD1, DHX9, and DICER1 were used to assay interacting RNA transcripts. Results from the RIP assays show that BCALM transcript was significantly enriched compared with IgG control for each of the three proteins, with PLD1 and DICER1 having 2-fold or greater enrichment and DXH9 with 1.4-fold enrichment (Fig. 4F). Western blot confirmed immunoprecipitation of the target proteins (Fig. 4G). Taken together, these results identify interactions between BCALM and cytosolic signal transduction proteins, suggesting that BCALM may play a role in signaling pathways in B cells.

Given its specificity for B cells, interaction with signaling pathway proteins, and proximity to PLCG2, we next assessed the effect of BCALM KO on signaling downstream of BCR activation. Although PLD1’s role in BCR signaling has not been previously examined, PLD1 is involved in TCR activation and chemotaxis in T cells and in Ag stimulation of mast cells (27, 28, 61, 6367). We treated wild-type (WT) and KO B cell lines (OCI-Ly7) with anti-IgM to stimulate the BCR and collected at 0, 2, 5, 10, 30, 60, and 240 min poststimulation. Fig. 5A and 5B show that PLD1 phosphorylation increases 2- to 3-fold over the time course in WT cells, whereas in KO cells, phosphorylation levels show minimal change at all timepoints. IgM stimulation did not alter the levels of BCALM transcript in WT or KO cells over the course of the experiment (Supplemental Fig. 2D).

FIGURE 5.

Loss of BCALM (AC099524.1) decreased PLD1 phosphorylation 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 loading control. (B) Line graph shows the 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 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 shading indicate the mean, 10th, and 90th percentiles of three replicates. Representative of at least two independent experiments (A–D).

FIGURE 5.

Loss of BCALM (AC099524.1) decreased PLD1 phosphorylation 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 loading control. (B) Line graph shows the 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 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 shading indicate the mean, 10th, and 90th percentiles of three replicates. Representative of at least two independent experiments (A–D).

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BCALM associates with PLD1 and AKAP9, a scaffold protein that associates with PKA and PKC, which phosphorylates PLD1. Because loss of BCALM resulted in reduced levels of phospho-PLD1, we hypothesized that BCALM may bring together PLD1 and an AKAP kinase complex, thereby promoting PLD1 phosphorylation. We tested this by immunoprecipitation of PLD1 in WT and KO B cell lines (OCI-Ly7). As shown in Fig. 5C, PLD1 coprecipitates AKAP9 in WT, but not in BCALM KO lymphoma cell lines, suggesting that BCALM is necessary for the association of PLD1 with the PKA/PKC kinase scaffold protein AKAP9.

We next assessed the effect of BCALM deficiency on calcium flux downstream of BCR stimulation. In these assays, anti-IgM stimulation of the BCR causes assembly of the BCR signalosome, in which BCR-associated kinases activate PLCG2, which hydrolyzes PIP2 to DAG and IP3. IP3 stimulates IP3 receptor channels on endoplasmic reticulum to release calcium into the cytoplasm (33, 34). Indo-1 is a fluorescent calcium indicator with emission at 475 nm when unbound that shifts to 400 nm when bound to Ca+, enabling accurate measurement of intracellular calcium concentrations with flow cytometry (68). For these studies, we preincubated WT, heterozygous or homozygous AC099524.1 (BCALM)–deficient cells with Indo-1 prior to BCR stimulation with anti-IgM. The resulting calcium release into the cytoplasm caused a shift in the ratio of bound/unbound Indo-1, which was detectable by flow cytometry. Compared with WT, heterozygous and homozygous AC099524.1 (BCALM)–deficient OCI-Ly7 lymphoma cells generated from two different guide RNA (gRNA) pairs exhibited higher levels of calcium flux after BCR stimulation as shown by higher ratios of Ca+–bound/unbound Indo-1 (Fig. 5D). A similar increase in calcium flux was observed in two AC099524.1 (BCALM) KO U2932 lymphoma cell lines compared with WT (Supplemental Fig. 2E) and in a nonmalignant B cell line treated with BCALM shRNAs (GM12878, Supplemental Fig. 2F). Taken together, these data support a role for BCALM in calcium flux downstream of BCR stimulation, perhaps through modulation of PLD1 phosphorylation (summarized in Fig. 6; see Discussion).

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 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.

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 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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In mature B lymphocytes, binding of Ag to the BCR activates signaling pathways and transcription factors that change gene expression, driving differentiation, survival, and proliferation (69, 70). Perturbation of these pathways is the hallmark of B cell cancers, but the underlying mechanisms remain incompletely defined (7175). In normal hematopoietic cells and non–B cell cancers, lncRNAs have emerged as key players in modulating gene expression, protein translation, and signaling pathways (46, 9, 11, 51, 53, 7686); however, little is known regarding their functional roles in B cell activation or lymphoma (6, 13, 8790). To address this critical knowledge gap, we performed global epigenome and transcriptome studies in human B cell cancers and normal B lymphocytes and identified lncRNAs as potential actors in lymphocyte activation pathways.

We used an integrative bioinformatic tool we developed [PLAIDOH (15)] to select one of these lncRNAs, AC099524.1, for functional experimental studies. AC099524.1 (BCALM) had high potential for activity in B cells because of its specific and high expression in B cell cancers and normal B cells and its proximity to the gene that encodes PLCG2, a critical regulator of downstream BCR signaling. In addition, BCALM was intriguing because of its cytoplasmic localization, which was less common for lncRNAs but in agreement with PLAIDOH’s prediction (15). Also in agreement with its functional prediction, BCALM transcript itself does not regulate the expression of neighboring genes CMIP or PLCG2, but enhancers in this locus are active and may regulate the expression of AC099524.1 (BCALM), CMIP, and/or PLCG2. These results are consistent with reports that, for some enhancer lncRNAs, the transcripts themselves are dispensable for “cis”-regulation of neighboring gene transcription and instead function in “trans” in other pathways. Indeed, we identified BCALM-interacting proteins that are involved in nontranscriptional cell processes, including signal transduction proteins PLD1 and AKAP9. Our initial RNA pull-down studies used an in vitro–transcribed BCALM that might lack posttranscriptional modifications present on the endogenous lncRNA, and thus could have missed protein interactions that require such modifications. Nonetheless, subsequent RIP, Co-IP, B cell activation, and calcium flux assays were performed in B cells and demonstrate that endogenous BCALM interacts with PLD1 and AKAP9 and modulates their activity.

Other cytosolic lncRNAs have been shown to modulate signaling pathways. For example, lncRNA AK023948 positively regulates AKT activity in breast cancer by stabilizing PI3K subunit p85 and is associated with poorer prognosis (52). In triple-negative breast cancer, lncRNA LINK-A associates with BRK and promotes the phosphorylation and stabilization of HIFα, leading to activation of HIF1α transcriptional programs under normoxic conditions (53). In macrophages, LPS-induced lncRNA Mirt2 inhibits K63-linked ubiquitination of TRAF6, thereby attenuating NF-κB and MAPK pathways and providing negative feedback regulation of inflammation (77). Many other studies in cancer, immune cells, and other organ systems also demonstrate diverse roles for cytoplasmic lncRNAs in metabolic, signal transduction, and immune response pathways (47, 51, 53, 80, 85, 9193).

Similar to the lncRNAs in the above studies, BCALM appears to act as a modulator of downstream BCR-mediated signaling and calcium flux. Fig. 6 depicts a model for how this modulation may occur. Ag binding to the BCR activates BCR-associated kinases and PLCG2, which hydrolyzes PIP2 to second messengers DAG and IP3. IP3 stimulates IP3 receptor Ca+ channels on the endoplasmic reticulum to release Ca+ into the cytoplasm, which, along with DAG, stimulates downstream signaling proteins, including PKC, which promotes cell growth and survival in normal and lymphoma B cells (33, 34, 69, 70, 7275, 9496). PKC also phosphorylates and activates PLD1 (27, 28, 31, 32). BCALM interacts with PLD1, AKAP9 (AKAP450), and AKAP13 (AKAP-Lbc). The AKAP proteins form signaling complexes with PKA and PKC (2123, 29, 30), which phosphorylate and activate PLD1 (27, 28, 31, 32). PLD1 produces PA, which activates SHP-1 (26, 97), a phosphatase that inhibits BCR-associated kinases and downregulates BCR signaling (71, 72, 75, 98, 99) (Fig. 6A). Without BCALM, interaction of PLD1 with the AKAP kinase complex is abrogated, resulting in decreased PLD1 phosphorylation and increased intracellular Ca+ concentrations in response to BCR stimulation, in a manner commensurate with BCALM transcript level (i.e., homozygous loss increased calcium flux more than heterozygous loss). These data suggest that BCALM acts as a scaffold and brings together PLD1 and AKAP-associated kinases to promote PLD1 phosphorylation and activation, resulting in increased SHP-1 activity and downregulation of BCR signaling and calcium flux in B cells. In the absence of BCALM, the interaction of PLD1 and AKAP9 is reduced and PLD1 phosphorylation is decreased, which reduces the PLD1 activity and PA production. Lower levels of PA would result in decreased SHP-1 activity and less inhibition of BCR signaling, and thus higher levels of intracellular calcium (Fig. 6B). Together, these data suggest that BCALM transcript acts to promote negative feedback of BCR-mediated signaling and calcium flux in response to BCR stimulation.

AgR stimulation and downstream PLD and PKC signaling play essential roles in diverse immune response pathways. Phospholipase D is a critical component of lipid second messenger signaling, which is required for movement of cells (chemotaxis) and cellular components (e.g., secretory vesicles, lysosomes). In T lymphocytes, PLD1 deficiency impairs TCR-mediated signaling, proliferation, cell adhesion, and chemotaxis in autoimmune and infectious models (61, 6466, 100). In mast cells, PLD1 is activated by Ag stimulation and is required for exocytosis of secretory granules (27, 28, 60, 101). In neutrophils and phagocytes, PLD1 is involved in chemotaxis, cell adhesion, and migration, and its activation promotes antimicrobial defense by facilitating phagolysosomal maturation (66, 67, 102, 103). There has been less study of PLD enzymes in B cells; however, reports in human CLL and FL highlight the importance of PLD1 in CXCL12-mediated adhesion and in mTOR activation downstream of the BCR-associated Syk kinase (104, 105). During BCR activation, PKC is activated by both DAG and Ca+ and, in turn, activates CARD11/MALT1/BCL-10 and NF-κB prosurvival and progrowth pathways (75, 95, 96, 106108). AKAP proteins assemble kinase-containing complexes, including PKA and PKC, and are involved in multiple signaling cascades in normal and malignant cells across organ systems (21, 23, 30, 109). Relevant to these studies, AKAP9 (AKAP450) plays a key role in T cells in immunological synapse formation during Ag-dependent T cell activation (110). In addition, AKAP9-deficient T cells exhibit decreased TCR recycling to the cell surface, resulting in reduced TCR reactivation and T cell retention in peripheral inflamed tissues (111). AKAP13 (AKAP-Lbc) forms a complex with the IκB to control the production of proinflammatory cytokines in cardiac myocytes in response to adrenergic stimulation (23, 112). Taken together, these published studies and the work presented in this study suggest that BCR stimulation leads to phosphorylation and activation of PLD1 via AKAP-signaling kinase complexes, which are modulated by interaction with the lncRNA BCALM (AC099524.1). Further work is needed to determine whether loss of PLD1, AKAP9, or AKAP13 in B cells similarly impacts BCR-mediated calcium flux or functionally impacts BCR dynamics, immune synapse formation, or mTOR/NF-κB growth and survival pathways (95, 113, 114).

In summary, the results presented in this study suggest that the B cell–specific lncRNA BCALM (AC099524.1) may play an important negative regulatory role in modulating calcium signaling downstream of BCR stimulation. BCALM thus represents a new, to our knowledge, paradigm for lncRNAs in B lymphocyte activation and signaling pathways, with implications for B cell development, immune response, and lymphoma pathogenesis.

We thank the Washington University School of Medicine Lymphoma Banking Program of the Division of Medical Oncology in the Department of Medicine for support of the biopsy and banking program. We thank the patients, caregivers, and families who participated in this study.

This work was supported by National Institutes of Health (NIH), National Cancer Institute (NCI) Grants CA156690 and CA188286, Washington University Institute of Clinical and Translational Sciences (ICTS) Grant UL1 TR000448 from the National Center for Advancing Translational Sciences (NCATS), and the Alvin J. Siteman Cancer Center (CA091842). Sequencing was provided by the Genome Technology Access Center, which is partially supported by NCI Cancer Center Support Grant P30 CA91842 to the Alvin J. Siteman Cancer Center and by ICTS/Clinical and Translational Science Award (CTSA) Grant UL1 TR000448 from the National Center for Research Resources (NCRR), a component of the NIH, and the NIH Roadmap for Medical Research. The ICTS is funded by the NIH NCATS CTSA program Grant UL1 TR002345.

This publication is solely the responsibility of the authors and does not necessarily represent the official view of NCRR or NIH. None of these sources had any role in data collection, analysis, interpretation, trial design, patient recruitment, writing the manuscript, the decision to submit the manuscript, or any other aspect pertinent to the study. None of the authors were paid to write the article by a company or any other agency. The corresponding author (J.E.P.) had full access to all of the data in the study and had final responsibility for the decision to submit for publication.

The sequences presented in this article have been submitted to the Gene Expression Omnibus under accession numbers GSE62246 and GSE132053.

The online version of this article contains supplemental material.

Abbreviations used in this article:

BCALM

B cell–associated lncRNA modulator of BCR-mediated Ca+ signaling

ChIP

chromatin immunoprecipitation

CLL

chronic lymphocytic lymphoma/leukemia

Co-IP

coimmunoprecipitation

DAG

diacylglycerol

DLBCL

diffuse large B cell lymphoma

eCLIP

enhanced cross-linking immunoprecipitation

FL

follicular lymphoma

gDNA

genomic DNA

gRNA

guide RNA

IP3

inositol-1,4,5,-triphosphate

KO

knockout

lncRNA

long noncoding RNA

MS

mass spectrometry

PA

phosphatidic acid

PIP2

phosphatidylinositol-4,5-bisphosphate

PKA

protein kinase A

PKC

protein kinase C

PLAIDOH

Predicting lncRNA Activity through Integrative Data-Driven 'Omics and Heuristics

PLD1

phospholipase D 1

PLDG2

phospholipase C γ 2

qRT-PCR

real-time quantitative RT-PCR

RBP

RNA binding protein

RCI

relative concentration index

RIP

RNA immunoprecipitation

RNA-seq

RNA sequencing

shRNA

short hairpin RNA

UCSC

University of California, Santa Cruz

WT

wild-type

WU

Washington University.

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The authors have no financial conflicts of interest.

Supplementary data