Unbiased RNA Degrader Identification Uncovers an LC3B-Recruiting Chimera for COL15A1 mRNA Degradation.

Heterobifunctional RNA degraders, such as ribonuclease-targeting chimeras (RiboTACs), eliminate structured RNAs by recruiting endogenous effector proteins. Here, we expand the effector toolbox by co-opting microtubule-associated protein 1 light chain 3B (MAP1LC3B, also known as LC3B), a core autophagy component recently implicated in mRNA decay. An RNA-binding fragment (F1) with a mapped transcriptome-wide binding profile was conjugated to the LC3B ligand ispinesib to generate the degrader F1-ispinesib. Integration of RNA-seq and Chemical Cross-Linking and Isolation by Pull-down combined with NGS Sequencing (Chem-CLIP-seq) data identified collagen type XV alpha 1 chain (COL15A1) mRNA as a target selectively degraded by F1-Ispinesib. The induced COL15A1 mRNA decay was LC3B- and autophagy-dependent, confirmed by genetic and pharmacological inhibition. Cellular engagement of LC3B was validated by NanoBRET and photocatalytic proximity labeling. In human aortic smooth muscle cells, F1-ispinesib phenocopied siRNA-mediated COL15A1 knockdown, reducing protein levels, suppressing proliferation, and enhancing migration. A synthetic LC3B recruitment model also recapitulated the on-target COL15A1 mRNA decay, further supporting the degradation mechanism. These findings establish LC3B as a recruitable effector protein for small-molecule-directed RNA decay and provide a general URID framework for discovering degraders that harness diverse RNA-regulatory proteins.