Computationally Reconstructing Cotranscriptional RNA Folding Pathways from Experimental Data Reveals Rearrangement of Non-Native Folding Intermediates

Summary The series of RNA folding events that occur during transcription, or a cotranscriptional folding pathway, can critically influence the functional roles of RNA in the cell. Here we present a method, Reconstructing RNA Dynamics from Data (R2D2), to uncover details of cotranscriptional folding pathways by predicting RNA secondary and tertiary structures from cotranscriptional SHAPE-Seq data. We applied R2D2 to the folding of the Escherichia coli Signal Recognition Particle (SRP) RNA sequence and show that this sequence undergoes folding through non-native intermediate structures that require significant structural rearrangement before reaching the functional native structure. Secondary structure folding pathway predictions and all-atom molecular dynamics simulations of folding intermediates suggest that this rearrangement can proceed through a toehold mediated strand displacement mechanism, which can be disrupted and rescued with point mutations. These results demonstrate that even RNAs with simple functional folds can undergo complex folding processes during synthesis, and that small variations in their sequence can drastically affect their cotranscriptional folding pathways. Highlights - Computational methods predict RNA structures from cotranscriptional SHAPE-Seq data - The E. coli SRP RNA folds into non-native structural intermediates cotranscriptionally - These structures rearrange dynamically to form an extended functional fold - Point mutations can disrupt and rescue cotranscriptional RNA folding pathways