A persistent ensemble of coding sequence structures regulates translation of the SERPINA1 mRNA

RNA molecules often populate heterogeneous ensembles of structures with potentially distinct biological roles. During protein translation, base-paired structures within mRNAs undergo dynamic unfolding during ribosome scanning and elongation, imposing energetic barriers that impact the efficiency of protein synthesis. However, the complexity and impact of mRNA structure ensembles during translation have not been observed in a native cellular context and remain poorly understood. Here we examine the SERPINA1 mRNA in cells using single-molecule correlated chemical probing and show that the early coding sequence region of the mRNA forms an ensemble of distinct and kinetically stable structures. These well-defined states are hidden to conventional analysis and involve thermodynamically suboptimal but persistent RNA conformations. During translation, we show that local structures within the ensemble are unfolded to accommodate ribosome binding. By coupling structure analysis of the native sequence and of structure-modulating point mutants, we find that wholistic unfolding of states within the mRNA ensemble regulates translation efficiency. These findings reveal that coding regions, even in highly-translated mRNAs, are not simply unfolded but populate persistent structure ensembles that significantly regulate protein expression.