Alternative Splicing of the Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) Is Regulated by RBFOX2 in Lymphoid Malignancies

Abstract

Aberrant alternative splicing (AS) of pre-mRNAs promotes the development and proliferation of cancerous cells. Accordingly, we had previously observed higher levels of the aryl hydrocarbon receptor nuclear translocator (ARNT) spliced variant isoform 1 in human lymphoid malignancies compared to that in normal lymphoid cells, which is a consequence of increased inclusion of alternative exon 5. ARNT is a transcription factor that has been implicated in the survival of various cancers. Notably, we found that ARNT isoform 1 promoted the growth and survival of lymphoid malignancies, but the regulatory mechanism controlling ARNT AS is unclear. Here, we report cis- and trans-regulatory elements which are important for the inclusion of ARNT exon 5. Specifically, we identified recognition motifs for the RNA-binding protein RBFOX2, which are required for RBFOX2-mediated exon 5 inclusion. RBFOX2 upregulation was observed in lymphoid malignancies, correlating with the observed increase in ARNT exon 5 inclusion. Moreover, suppression of RBFOX2 significantly reduced ARNT isoform 1 levels and cell growth. These observations reveal RBFOX2 as a critical regulator of ARNT AS in lymphoid malignancies and suggest that blocking the ARNT-specific RBFOX2 motifs to decrease ARNT isoform 1 levels is a viable option for targeting the growth of lymphoid malignancies.

Keywords: ARNT; RBFOX2; alternative splicing; lymphoid malignancies.

FIG 1

RBFOX2 binding sites in ARNT introns 4 and 5 correlate with RBFOX2-mediated ARNT exon 5 inclusion. (A) Schematic showing RBFOX2 binding motifs “GCAUG” in the upstream and downstream introns of ARNT exon 5. (B) RBFOX2 binding peaks in introns flanking ARNT exon 5 from two independent eCLIP experiments (rep1 and rep2), as found in the ENCODE database (GEO: GSE92030). (C) Representative reverse-transcription PCR (RT-PCR) analysis of ARNT AS in HEK293 cells after introduction of the indicated siRNAs (top), and combined densitometry analysis to determine percent spliced in (PSI) of three independent experiments performed in duplicate (bottom). (D) Representative RT-PCR analysis of ARNT AS in murine heart tissue at the indicated developmental stage (top), and combined densitometry analysis to determine PSI of n = 3 (E18 and Newborn) or n = 4 (Adult) (bottom). Data in graphs are presented as means ± standard error of the mean (SEM). E5 = exon 5. P values are derived using a two-tailed unpaired Student’s t test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

FIG 2

RBFOX2 promotes ARNT exon 5 inclusion through recognition of cis-regulatory elements in ARNT intron 5. (A) Schematic of ARNT-specific splicing vector with the indicated GCATG motifs that were targeted for deletion. (B) HEK293T cells were transfected with wild-type (WT) ARNT splicing vector or a GCATG deletion mutant ARNT splicing vector (ARNTΔp, ARNTΔd, or ARNTΔpd) plus empty vector control or WT RBFOX2. A nonfunctional (NF) RBFOX2 mutant was used as a negative control. Total RNA was isolated at 72 h posttransfection and analyzed by RT-PCR for splicing changes; a representative image is shown below the graph. The densitometry analysis to determine percent spliced in (PSI) results presented in the graph are means ± SEM of three independent experiments performed in triplicate. P values are derived using a two-tailed unpaired Student’s t test. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 compared to empty vector control; †††, P < 0.001; ††††, P < 0.0001 compared to WT ARNT + WT RBFOX2. E5 = exon 5. (C) Immunoblot analysis of cells from panel B, showing equal levels of ectopically expressed FLAG-RBFOX2 between samples.

FIG 3

RBFOX2 promotes the inclusion of ARNT exon 5, and higher ARNT isoform 1 levels, for optimal growth of lymphoid malignancies. (A) RT-qPCR analysis of RBFOX2 expression in primary T cells, Karpas 299 cells, and Jurkat cells (left), with representative RT-PCR images to show relative expression levels of ARNT and RBFOX2 (right). (B) Validation of RNAi-mediated RBFOX2 suppression by RT-qPCR analysis (left) and immunoblotting with antibodies against RBFOX2 and β-actin (right), in Karpas 299 and Jurkat cells. RT-qPCR data are means ± SEM of three independent experiments performed in triplicate. P values are derived using a two-tailed unpaired Student’s t test. **, P < 0.01; ***, P < 0.001. (C) Representative RT-PCR analysis of ARNT AS in Karpas 299 and Jurkat cells after introduction of the indicated siRNAs (top), percent spliced in (PSI) as determined by combined densitometry analysis (data are means ± SEM of three independent experiments) (middle), and immunoblot analysis using antibodies against ARNT and β-actin (bottom). E5 = exon 5. (D) Twenty-four hours post-electroporation with siControl, siRBFOX2, or siARNT-1, Karpas 299 cells were normalized to 1.7 × 10⁵ cells/mL and counted every 24 h for a total of 72 h (left). P values are derived using two-way analysis of variance. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to siControl. †, P < 0.05; ††, P < 0.01 compared to siRBFOX2. Immunoblot analysis was performed at 48 h posttransfection using antibodies against RBFOX2, ARNT, and β-actin (right).