RNA binding protein RBM20 regulates turtle temperature-dependent sex determination by repressing the splicing of Wt1 KTS

Accumulating correlative studies suggest a potential role of alternative splicing in vertebrate sex determination; however, the underlying mechanism remains elusive. Herein, we show that RNA binding motif protein 20 (RBM20) exhibits temperature-dependent sexually dimorphic expression in early embryonic gonads of the red-eared slider turtle Trachemys scripta , preceding the onset of gonadal differentiation. Knockdown of Rbm20 at male-producing temperature induces male-to-female sex reversal, whereas Rbm20 overexpression at female-producing temperature triggers male development. RBM20 represses the splicing of Wilms tumor suppressor WT1 at KTS sites, thereby governing the balance of +KTS and −KTS isoforms between different temperatures. In addition, functional analysis reveals that the Wt1 +KTS variant is both necessary and sufficient to drive male development in T. scripta . This study demonstrates that RBM20 activates the male pathway by repressing the Wt1 KTS splicing in T. scripta , thus establishing causality and a direct genetic link between alternative splicing mechanism and vertebrate sex determination.