Developing a single molecule approach to selective ribosome profiling
mRNA-protein interactions are at the center of the translation process. Ribosome-associated factors, from modifying enzymes 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? Do they compete for binding? Is there a coordinated handover? By only observing population averages we can miss crucial mechanistic features. To address this, we are developing a single molecule approach, targeting two canonical ribosome-associated chaperones: RAC-SSB1 and NAC-alpha (Stress-Seventy subfamily B subunit of the ribosome-associated complex and Alpha subunit of the Nascent Polypeptide-Associated Complex, respectively). We have generated two chimeric constructs under an inducible promoter; Fusing the co-translationally acting factor RAC-SSB1 with N6-adenosine methyltransferase IME4, and the co-translationally acting factor NAC-alpha with RNA-specific adenosine deaminase ADAR2 catalytic domain. Recent development in Oxford nanopore sequencing platform sensitivity has allowed for the identification of enzymatically modified nucleotides. We are utilizing this advance to detect co-translational interaction in vivo, by labeling the mRNAs nucleotides in close proximity to those interactions. Using immunoprecipitation, we demonstrated that our chimeric constructs show a strong ribosomal association. Polysome profiling demonstrated that the attached RNA-modifying enzymes are in proximity to translated mRNA. RNA extraction following induction and sequencing of the entire orfs using NanoPore revealed significant changes in the electrical output of samples, strongly suggesting the presence of modified bases in the test strain. Further analysis will reveal the interplay between SSB1 and NACα and establish a platform for analysis of various co-translational factors interaction in single-molecule resolution.
The lecture will be given in English