Integrative CRISPR Screens and RNA-Omics Discover an Essential Role for PUF60-3' Splice Site Interactions in Cancer...

Area of Science:

• Molecular Biology
• Cancer Research
• Genetics

Background:

• RNA-binding proteins (RBPs) regulate gene expression post-transcriptionally.
• Triple-negative breast cancer (TNBC) lacks effective targeted therapies.
• Identifying RBPs driving TNBC progression is critical.

Purpose of the Study:

• To identify essential RBPs for TNBC cell survival using pooled CRISPR/Cas9 screening.
• To elucidate the mechanism by which PUF60 promotes TNBC progression.
• To evaluate PUF60 as a potential therapeutic target in TNBC.

Main Methods:

• In vitro and in vivo pooled CRISPR/Cas9 screening to identify essential RBPs.
• eCLIP and RNA-sequencing to analyze PUF60's role in splicing.
• Functional assays to assess the impact of disrupting PUF60-RNA interactions.

Main Results:

• Identified 50 RBP candidates essential for TNBC cell survival.
• PUF60 drives exon inclusion in proliferation-associated transcripts, promoting TNBC growth.
• Disrupting PUF60-RNA interactions inhibits TNBC proliferation and shrinks tumors in vivo.
• PUF60-mediated splicing is crucial for maintaining oncogenic proliferation rates.

Conclusions:

• PUF60 plays a critical role in supporting TNBC cell proliferation through specific splicing activities.
• Targeting PUF60-RNA interactions presents a promising therapeutic strategy for TNBC.
• Functional in vivo screening is effective for discovering novel cancer regulators.