RNA binding protein RBM20 regulates turtle temperature-dependent sex determination by repressing the splicing of <i>Wt1</i> 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 <i>Trachemys scripta</i>, preceding the onset of gonadal differentiation. Knockdown of <i>Rbm20</i> at male-producing temperature induces male-to-female sex reversal, whereas <i>Rbm20</i> 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 <i>Wt1</i> +KTS variant is both necessary and sufficient to drive male development in <i>T. scripta</i>. This study demonstrates that RBM20 activates the male pathway by repressing the <i>Wt1</i> KTS splicing in <i>T. scripta</i>, thus establishing causality and a direct genetic link between alternative splicing mechanism and vertebrate sex determination.