Amaya N. Mendez-Molina

Depletion of PUF60 in TNBC cells significantly shrinks tumor volume, related to Figure 4.

CRISPR screening in isogenic cell lines and TNBC-derived cell lines identifies PUF60 as an essential RBP in TNBC, related to Figure 1.

KD RNA-sequencing reveals exon skipping events modulated by PUF60 in TNBC and normal cells, related to Figure 3.

Quality control of PUF60 eCLIP and cell line comparisons of 3' splice site targets, related to Figure 2.

RNA-binding proteins (RBP) are important regulators of posttranscriptional gene expression. Understanding which and how RBPs promote cancer progression is crucial for cancers that lack effective targeted therapies, such as triple-negative breast cancer (TNBC). In this study, we employed both in vitro and in vivo pooled CRISPR/Cas9 screening to identify 50 RBP candidates essential for TNBC cell survival. Integrated enhanced cross-linking and immunoprecipitation and RNA sequencing analysis identified that poly(U)-binding splicing factor 60 (PUF60) drives exon inclusion within proliferation-associated transcripts that, when misspliced, induce cell cycle arrest and DNA damage. Furthermore, disrupting PUF60 interactions with 3′ splice sites via a substitution in its RNA-binding domain caused widespread exon skipping, leading to downregulation of proliferation-associated mRNAs and inducing apoptosis in TNBC cells. Knockdown of PUF60 or disruption of PUF60–RNA interactions inhibited TNBC cell proliferation and shrunk tumor xenografts in multiple models. Together, these findings reveal the molecular mechanism by which PUF60 supports cancer progression.Significance:Functional screening of RNA-binding proteins is an effective strategy for identifying cancer regulators that revealed PUF60-mediated splicing activity as a driver of oncogenic proliferation and a potential therapeutic target.

Silencing of PUF60 splice site activity shrinks tumor volume, related to Figure 6.

PUF60-activated exons are excluded following L140P substitution, related to Figure 5.

Supplementary Figure 6. Squamous cell carcinoma (SCC)-positive tongues from the metformin + naproxen treatment arm. (Related to Figure 4)

Supplementary Figure 1. Genome-wide CRISPR-Cas9 screen identifies PKA signaling as a metformin resistance pathway in HNSCC. (Related to Figure 1)