Developing a single molecule approach to Ribosome Profiling

mRNA-protein interactions are at the center of the translation process. Ribosome associated proteins, from targeting factors to molecular chaperones, play a pivotal role in facilitating the folding of the emerging nascent-chain, protecting it from aberrant interactions in the crowded cytoplasm. However, the interplay between the various ribosome associated factors remains largely obscure. What is the mode of interplay? Competition? Cooperation? Coordinated hand-over? By observing only population averages, crucial mechanistic features are masked. To address this, we are developing a single-molecule ribosome profiling approach, targeting two canonical ribosome associated chaperones: SSB1 and NAC (Stress-Seventy subfamily B-subunit of the ribosome-associated complex and Alpha-subunit of the Nascent polypeptide-Associated Complex). We have generated two chimeric constructs, fusing each co-translationally acting factor with a different RNA modifying enzyme. Recent development in the nanopore platform sensitivity has allowed for direct identification of enzymatically modified RNA nucleotides. We are utilizing this advance to detect co-translational interaction in vivo, by labelling the mRNA nucleotides in close proximity to those interactions, combining A to I editing and N6-adenosine methylation. Using immunoprecipitation, we found that our chimeric constructs bind ribosomes as strong as wt. Utilizing polysome profiling we have demonstrated that the attached RNA modifying enzymes are in proximity to translated mRNA. RNA extraction following induction and sequencing of the entire orf’s using NanoPore sequencing will reveal the interplay between SSB1 and NACα and establish a platform for analysis of various co-translational factors interactions in single-molecule resolution.