Split-ADAR System to Detect in vivo Co-translational Cooperativity in Single-Molecule Resolution
Protein biogenesis is challenging in the crowded cellular environment, where off-pathway interactions can lead to protein aggregation – a hallmark of neurodegenerative diseases. To maintain protein homeostasis, cells employ an intricate network of ribosome-associated factors that regulate folding, localization, and complex assembly co-translationally. Yet, how these factors coordinate their dynamic binding to translating ribosomes remains poorly understood. Here, we developed a single-molecule approach that combines Nanopore sequencing of full-length transcripts with mRNA proximity labelling using a split-ADAR system. We have provided a proof of concept for the robustness of this approach in detecting even transient co-translational interactions at the ribosome. Using this system, we have demonstrated evidence for co-translational cooperativity between the highly conserved ribosome-associated factors Map1 and NatA along single transcripts.
