An intrinsically disordered RNA-binding region provides local target selectivity and is essential for LARP6 function.
Intrinsically disordered regions (IDRs) are prevalent in RNA-binding proteins (RBPs), yet their roles in RNA interactions remain poorly defined. Here, we examined the structured and disordered RNA-binding activities of LARP6, an RBP with a diverse RNA-binding repertoire. Using mass spectrometry-based RNA interaction mapping in living cells, we identified direct LARP6-RNA contacts within both the structured La-module and its flanking IDRs. Mutagenesis combined with individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) revealed the La-module, but not the IDRs, as essential for LARP6 binding to RNA. Deletion of the N-terminal IDR broadened the footprints of LARP6 on RNA, uncovering a role in RNA-binding selectivity. Mechanistically, this is achieved through restricting the conformational flexibility of the adjacent La-module. The IDR-mediated RNA-binding selectivity is critical for LARP6-driven cancer cell viability and invasion. Our findings uncover a previously unrecognised critical function for IDRs in promoting selective RBP-RNA interactions, which operates through conformational restriction.