Phosphorylation-mediated regulation of the essential splicing factor PUF60

PUF60 is a splicing factor related to the polypyrimidine-tract binding protein U2AF2. PUF60 is deleted in developmental disorders such as Verheij syndrome and amplified in approximately 8% of cancers. Thus, both increases and decreases in PUF60 expression can have profound physiological effects. However, little is known about how changes in PUF60 expression impact global splicing patterns. Here, we created a model system of CRISPRa/i in mouse stem cells (mESCs) to transcriptionally upregulate or downregulate Puf60. Our results uncovered extensive transcriptional, post-transcriptional, and post-translational regulation of Puf60 protein expression. We observed that Puf60 protein levels in normal mESCs drop dramatically at a critical cell density, leading to cell death. Puf60 is very essential in stem cells, and its repression causes cell death and impacts specific splicing events, including its own splicing autoregulation, providing valuable insights into the functional consequences of PUF60 dysregulation. Analysis of phosphoprotein data revealed phosphorylation of threonine at the N-terminus of PUF60. Our results showed that mutating threonine to glutamate downregulates the protein and alters its localization. Thus, our study reveals a novel regulatory mechanism of Puf60 phosphorylation that mediates its function and may be related to its frequent overexpression in cancer cells.