Arsenite Exposure Displaces Zinc from ZRANB2 Leading to Altered Splicing

Exposure to arsenic, a class I carcinogen, affects 200 million people globally. Skin is the major target organ, but the molecular etiology of arsenic-induced skin carcinogenesis remains unclear. Arsenite (As³⁺)-induced disruption of alternative splicing could be involved, but the mechanism is unknown. Zinc finger proteins play key roles in alternative splicing. As³⁺ can displace zinc (Zn²⁺) from C3H1 and C4 zinc finger motifs (zfm's), affecting protein function. ZRANB2, an alternative splicing regulator with two C4 zfm's integral to its structure and splicing function, was chosen as a candidate for this study. We hypothesized that As³⁺ could displace Zn²⁺ from ZRANB2, altering its structure, expression, and splicing function. As³⁺/Zn²⁺ binding and mutual displacement experiments were performed with synthetic apo-peptides corresponding to each ZRANB2 zfm, employing a combination of intrinsic fluorescence, ultraviolet spectrophotometry, zinc colorimetric assay, and liquid chromatography-tandem mass spectrometry. ZRANB2 expression in HaCaT cells acutely exposed to As³⁺ (0 or 5 μM, 0-72 h; or 0-5 μM, 6 h) was examined by RT-qPCR and immunoblotting. ZRANB2-dependent splicing of TRA2B mRNA, a known ZRANB2 target, was monitored by reverse transcription-polymerase chain reaction. As³⁺ bound to, as well as displaced Zn²⁺ from, each zfm. Also, Zn²⁺ displaced As³⁺ from As³⁺-bound zfm's acutely, albeit transiently. As³⁺ exposure induced ZRANB2 protein expression between 3 and 24 h and at all exposures tested but not ZRANB2 mRNA expression. ZRANB2-directed TRA2B splicing was impaired between 3 and 24 h post-exposure. Furthermore, ZRANB2 splicing function was also compromised at all As³⁺ exposures, starting at 100 nm. We conclude that As³⁺ exposure displaces Zn²⁺ from ZRANB2 zfm's, changing its structure and compromising splicing of its targets, and increases ZRANB2 protein expression as a homeostatic response both at environmental/toxicological exposures and therapeutically relevant doses.