PRMT2 interacts with splicing factors and regulates the alternative splicing of BCL-X

Abstract

Protein arginine N-methyltransferase 2 (PRMT2) functions in JAK-STAT and Wnt/β-catenin signalling pathways, serves as a nuclear receptor-dependent transcriptional co-activator, and represses NF-κB and E2F1 transcription factor activities to promote apoptosis. We have previously demonstrated that PRMT2 interacts with PRMT1 and increases its activity. Here, we reveal associations using proteomics between the PRMT2 SH3 domain and splicing factors including Src-associated in mitosis 68 kDa protein (SAM68), a PRMT1 substrate and trans-acting factor that mediates BCL-X alternative splicing. We determined that PRMT2 interacts with SAM68 in cells and regulates its subcellular localization via the SH3 domain of PRMT2, prompting us to investigate the potential role of PRMT2 in BCL-X alternative splicing. We found that the expression of the full-length, wildtype form of PRMT2 promotes an increase in the BCL-X(L)/BCL-X(s) ratio in TNF-α or LPS stimulated cells. These results indicate that active PRMT2 may play a role during inflammation in alternative splicing regulation.

Keywords: BCL-X; PRMT2; SAM68; SH3 domain; alternative splicing.

Fig. 1

Identification of PRMT2 SH3 domain-associated proteins. HeLa cell lysates were prepared for GST pull-down experiments. Samples were separated by gel electrophoresis, and protein bands 1 − 6 were isolated from the Coomassie Blue-stained gel for proteomic analysis. GST pull-down samples were also probed for methylated proteins by the western blot using an anti-ADMA antibody.

Fig. 2

The interaction between SAM68 and PRMT2 is dependent on the PRMT2 SH3 domain. (A) Proteins bound to GST pull-downs from HeLa cell lysate were resolved by gel electrophoresis and blotted with an anti-SAM68 antibody. Whole cell lysate from HeLa cells that expressed HA-SAM68 (B), HA-PRMT2 (C), or HA-ΔSH3PRMT2 (D) were immunoprecipitated with anti-HA or mouse IgG (negative control) and blotted as indicated.

Fig. 3

PRMT2 affects the subcellular localization of SAM68. HeLa cells untransfected (A) or transfected with mCitrine-PRMT2 (B, green) or mCitrine-ΔSH3 (C, green) were treated with or without 20 μM AdOx as indicated. SAM68 was immunostained with anti-SAM68 and Alexa Fluor 546-conjugated antibodies (red). Nuclei were visualized by DAPI stain (blue). The phase contrasted images (left) and the merged images (right) are also shown. The scale bar indicates 10 µm.

Fig. 4

The effect of PRMT2 expression levels on BCL-X alternative splicing under inflammatory conditions. HEK293T cells transfected with scramble RNA or PRMT2-targeted siRNAs were harvested 48 h post-transfection and qRT-PCR was performed to show PRMT2/GAPDH (A), and the relative amounts of BCL-X transcript ratios normalized to the scramble siRNA control (B). HEK293T cells were transfected with control shRNA (shControl) or PRMT2-targeted shRNAs, and harvested for qRT-PCR to show PRMT2 normalized to GAPDH (C). The relative amounts of BCL-X transcript ratios for shRNA-A and shControl treated cells were determined by qRT-PCR and normalized to the shControl samples (D). HEK293T cells harbouring shControl or shRNA-A were treated with either TNF-α or LPS and harvested after 24 h of treatment. qRT-PCR was performed to show PRMT2 normalized to GAPDH (E), and BCL-X transcript ratios (F). Standard deviations of two replicates are shown. Statistical comparisons between experimental samples were analysed using a Student’s t-test (*P < 0.05; **P < 0.005).

Fig. 5

The effect of added PRMT2 on BCL-X alternative splicing under inflammatory conditions. HEK293T cells harbouring shControl or shRNA-A targeting PRMT2 were transfected with pcDNA3.1 (negative control), or plasmids encoding HA-PRMT2, HA-ΔSH3PRMT2, or HA-PRMT2(E220Q). After 24 h of transfection, cells were treated with TNF-α or LPS and harvested after 24 h of treatment. qRT-PCR was performed to show PRMT2 normalized to GAPDH, and BCL-X transcript ratios for TNF-α (A) or LPS (B) treatment groups. Standard deviations of two replicates are shown. Statistical comparisons between experimental samples were analysed using a Student’s t-test.