Integrative CRISPR Screening and RNA Analyses Discover an Essential Role for PUF60 Interactions with 3' Splice Sites...

Area of Science:

• Molecular Biology
• Cancer Research
• Gene Regulation

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 RBPs essential for TNBC cell survival.
• To elucidate the mechanism by which PUF60 promotes TNBC progression.

Main Methods:

• In vitro and in vivo pooled CRISPR/Cas9 screening identified 50 RBP candidates.
• Integrated eCLIP and RNA-sequencing analyzed PUF60's function.
• PUF60-RNA interaction disruption via domain substitution was tested.

Main Results:

• PUF60 drives exon inclusion in proliferation-associated transcripts, inducing cell cycle arrest and DNA damage.
• Disrupting PUF60-RNA interactions caused exon skipping and apoptosis in TNBC cells.
• PUF60 knockdown or interaction disruption inhibited TNBC proliferation and tumor xenografts.

Conclusions:

• PUF60 is a key driver of TNBC progression.
• PUF60 supports TNBC by regulating alternative splicing of proliferation-associated genes.
• Targeting PUF60-RNA interactions may offer a therapeutic strategy for TNBC.