PUF60-Mediated Splicing Is a Key Driver of Triple Negative Breast Cancer [eCLIP]

RNA-binding proteins (RBPs) regulate post-transcriptional gene expression, influencing key cancer pathways. Understanding how RBPs control these processes can uncover new treatment strategies, which is crucial for cancers that lack effective targeted therapies such as triple negative breast cancer (TNBC). Here, we employ dual in vitro and in vivo CRISPR/Cas9 screens to investigate the role of RBPs in TNBC, revealing the poly(U)-binding splicing factor 60 (PUF60) as a key modulator of TNBC cell survival. Integrated eCLIP and RNA-sequencing analysis identify that PUF60 drives exon inclusion within transcripts associated with cell cycle and genomic stability, thereby promoting proliferation. Furthermore, disrupting PUF60 splice site activity via a substitution in its RNA-binding domain causes widespread exon skipping, leading to the downregulation of proliferation-associated mRNAs and inducing apoptosis in TNBC cells. We demonstrate that this RNA-binding disruption inhibits TNBC cell proliferation and shrinks tumor xenografts, highlighting the molecular mechanism through which PUF60 supports cancer progression. Our work demonstrates functional in vivo screening of RBPs as an effective strategy for identifying unexpected cancer regulators. Here, we reveal a crucial role for PUF60-mediated splicing activity in supporting oncogenic proliferation rates and highlight its potential as a therapeutic target in triple negative breast cancer. Overall design: eCLIP of PUF60 in MDA-MB-231, MDA-MB-436, SUM-149, and MCF10A cells and eCLIP of V5-tagged L140P mutant PUF60 in MDA-MB-231 cells, in duplicate