Persulfidation of the zinc finger protein ZRANB2 modulates its RNA binding and alternative splicing function

Zinc finger Ran-binding domain-containing protein 2 (ZRANB2) is an RNA-binding protein that plays a key role in alternative splicing. It contains two N-terminal RanBP2-type ZF domains in which four cysteine residues coordinate Zn(II) in a tetrahedral geometry to afford proper folding and function. Persulfidation, a post-translational modification in which cysteine thiols (-SH) are converted to persulfides (-SSH) by hydrogen sulfide (H 2 S), has emerged as a means for regulating ZF activity. ZRANB2 is frequently identified as persulfidated in chemoselective proteomics screens, and here, we evaluate the direct modification of ZRANB2 by H 2 S. Using a recombinantly expressed two-domain construct (ZRANB2-2D), we report that Zn(II)-bound ZRANB2-2D undergoes persulfidation when exposed to H 2 S and oxygen with superoxide generated as an intermediate. This modification induces a loss of Zn(II)-dependent structure and abrogates binding to an RNA oligonucleotide from exon 3 of the transformer-2 protein homolog beta (TRA2B) RNA, a splicing target of ZRANB2, as well as to an optimized RNA oligonucleotide. Consistent with impaired RNA binding, cellular treatment with H 2 S leads to decreased formation of a TRA2B splice product, suggesting a connection to persulfidation of ZRANB2 in cells. Notably, addition of a reductant restores ZRANB2-2D RNA-binding activity in vitro. These results position persulfidation as a rheostat for modulating ZF protein function, exemplified here by its role in regulating ZRANB2 RNA binding and splicing. • The zinc finger protein ZRANB2 is directly persulfidated by H₂S. • Superoxide is produced as an intermediate of the persulfidation reaction. • Persulfidation of ZRANB2 disrupts zinc-dependent folding and abrogates RNA binding. • H₂S affects RNA splicing in cells, consistent with ZRANB2 persulfidation. • Reduction with TCEP reverses ZRANB2 persulfidation and restores RNA binding.