Landscape of mitochondrial RNA binding proteome [RNAseq_siDHX30_siTFam]

We combine RNA 5-ethynyluridine labelling, optimized mitochondria isolation, with click chemistry-based affinity purification to systematically capture the RBPs interacting with mitochondria-located RNAs. This methodology, termed mitochondrial RNA interactome using click chemistry (mitoRICK), unexpectedly revealed a rapid turnover of mitochondria-encoded RNAs compared to their nuclear-encoded counterparts. MitoRICK has identified 122 high-confidence and 25 lower-confidence mitochondrial RNA binding proteins, which expanded the repertoire of mitochondrial RNA binding proteome, and provided a new avenue for understanding mitochondrial RNA metabolism or inherited diseases. We found that MOV10, amongst many newly-identified RNA helicases, is a novel mitochondria-associated RBP. Functionally, MOV10 is important for MT-RNR1 expression or unwinding the mitochondrial double-strand RNAs. As a proof-of-concept, we applied mitoRICK to naïve-to-primed pluripotency transition for studying mitochondrial RBP dynamics, suggesting that intact mitochondrial RNA metabolism is essential for the cell fate transition. MitoRICK is broadly applicable to understand dynamics of mitochondrial RNA binding proteome in various cell contexts.