Source: College of Biological and Environmental Sciences
On November 14, the research team led by Professor Ge Chutian from our university published a research article in Science Advances titled “RNA-binding protein RBM20 regulates turtle temperature-dependent sex determination by repressing the splicing of Wt1 KTS.” The study uncovers a molecular mechanism by which the RNA-binding protein RBM20 regulates sex determination in the red-eared slider turtle. Zhejiang Wanli University is the first affiliation of the publication, with Sun Wei and Yang Hongzhe as co-first authors, and Professors Ge Chutian and Wang Zongji as co-corresponding authors. This work represents the university’s third major achievement in the field of animal sex determination, following its Science publications in 2018 and 2020.
Alternative splicing (AS) is a key regulatory mechanism of gene expression in eukaryotes—pre-mRNA can be spliced in different ways to generate diverse mRNA isoforms, significantly increasing proteomic diversity and playing essential regulatory roles in tissue development and organ formation. Sex determination is a critical biological event during early embryonic development, directing the bipotential gonads to differentiate into testes or ovaries. Traditionally, it has been believed that the sex of lower animals such as insects is controlled by alternative splicing of the same gene, whereas sex determination in vertebrates is governed by the differential expression of distinct genes. However, numerous correlative studies have identified abundant sex-biased splicing events in vertebrate embryonic gonads, strongly suggesting a role for AS in vertebrate sex determination, yet direct experimental evidence has been lacking.
To address this question, the research team used the temperature-dependent sex determination (TSD) species red-eared slider turtle (male offspring produced at 26°C, female offspring at 32°C) as the model organism. They discovered that the RNA splicing factor RBM20 is highly expressed in male-producing temperature (MPT) gonads throughout the sex-determination period. Under MPT conditions, knockdown of Rbm20 led to male-to-female sex reversal, while under female-producing temperature (FPT), overexpression of Rbm20 induced male development. The RBM20 protein directly binds to Wt1 transcripts and regulates the relative abundance of the two Wt1 splicing isoforms (+KTS and −KTS): knockdown of Rbm20 decreases +KTS and increases −KTS; overexpression of Rbm20 increases the +KTS/−KTS ratio. The +KTS isoform of Wt1 is both necessary and sufficient to initiate male development in the red-eared slider turtle.
Based on these findings, the team proposes a working model for RBM20-mediated regulation of TSD in turtles: at MPT, high RBM20 expression represses Wt1 KTS splicing, leading to retention of the +KTS isoform and initiation of male development. Conversely, at FPT, low RBM20 expression allows other splicing factors to recognize and splice at the KTS site, reducing +KTS levels and promoting ovarian differentiation. This study provides the first direct genetic evidence for the regulatory role of alternative splicing in vertebrate sex determination, offering deeper insights into the splicing-mediated regulatory mechanisms and evolutionary significance of sex determination in animals.
