Unbiased RNA Degrader Identification Uncovers an LC3B-Recruiting Chimera for <i>COL15A1</i> 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 (<b>F1</b>) with a mapped transcriptome-wide binding profile was conjugated to the LC3B ligand ispinesib to generate the degrader <b>F1-ispinesib</b>. 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 (<i>COL15A1</i>) mRNA as a target selectively degraded by <b>F1-Ispinesib</b>. The induced <i>COL15A1</i> 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, <b>F1-ispinesib</b> phenocopied siRNA-mediated <i>COL15A1</i> knockdown, reducing protein levels, suppressing proliferation, and enhancing migration. A synthetic LC3B recruitment model also recapitulated the on-target <i>COL15A1</i> 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.