Functional screening reveals novel RNA-binding proteins that drive exon inclusion in human cells, highlighting new molecular tools for programmable splicing control.
Key Points
To comprehensively evaluate the ability of human RNA-binding proteins to activate alternative exon inclusion and discover compact domains for synthetic splicing regulation.
Screened 718 human RNA-binding proteins (RBPs) using high-throughput tethered-function luciferase splicing reporters.
Characterized candidate RBPs lacking prior splicing associations using enhanced cross-linking immunoprecipitation (eCLIP), RNA sequencing, and affinity purification-mass spectrometry (AP-MS).
Engineered programmable fusion proteins using newly identified activation domains to benchmark performance against existing artificial splicing factors.
Identified uncharacterized roles for TRNAU1AP, SCAF8, and RTCA in directing hundreds of endogenous alternative splicing events.
Mapped potent and compact exon inclusion activation domains from top-performing candidate RBPs.
Constructed engineered splicing fusions that outperformed established artificial splicing factors at activating inclusion of both reporter and endogenous exons.