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

Abstract

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. KeywordsCotranscriptional RNA folding, RNA, RNA structure, SHAPE-Seq, molecular dynamics, signal recognition particle, toehold strand displacement 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

DUETT quantitatively identifies known and novel events in nascent RNA structural dynamics from chemical probing data

“…This hairpin rearranges into the long helical structure, which DUETT identifies as downswing events between lengths 116 and 127 nt in bases 11, 14-15, and 17-19. These identifications are consistent with recent computational modeling (Yu et al, 2018) and single molecule optical tweezer experiments (Fukuda et al, 2018) that propose the rearrangement of H1 to occur in the window that DUETT detects.…”

“…The major cotranscriptional rearrangement event for the E. coli SRP RNA occurs when H1 refolds into the final extended structure (Yu et al, 2018;Fukuda et al, 2018). Corresponding to the formation of H1, DUETT identified upswings in bases 14-15 and 18-19 around nascent RNA length 45 nt, and upramps that conclude around length 50 nt ( Figure 2).…”

“…Another key feature of the E. coli SRP RNA cotranscriptional folding pathway is the formation of native base pairs and loops after the formation of H1, but before the rearrangement of H1 into the final native structure (Wong et al, 2007;Watters, Strobel, et al, 2016;Fukuda et al, 2018;Yu et al, 2018). DUETT identifies expected reactivity signatures of bases 26-29 and 31 through length 100 nt.…”

Global mapping of RNA homodimers in living cells

“…Homodimerization is also likely to be influenced by folding kinetics, association with other proteins and RNAs, subcellular localisation and local concentration of RNA and metal ions. RNA molecules which fold co-transcriptionally into stable secondary structures are unlikely to form extended duplexes with other RNAs (Yu, et al 2021), whereas molecules that are unfolded by helicases, or located in granules with high local concentrations of a given RNA, might be more likely to form transient or stable oligomers. Copies of RNA molecules located in close proximity may initially interact with a few nucleotides, followed by destabilisation of local structure and nucleation of longer interactions (Ganser, et al 2019).…”

Preparation ofE. coliRNA polymerase transcription elongation complexes for systematic RNA assays

“…Independent of its technical utility for linking transcribed RNA to its DNA template in RNA-HiTS assays, the cotranscriptional display of RNA is a powerful tool for studies of RNA folding for several reasons. First, RNA molecules begin to fold during transcription (3,4) and cotranscriptionally folded RNA may adopt different structures than renatured RNA (8)(9)(10). Second, experiments in which RNA is tethered to its DNA template can circumvent biases from processing steps that are typically required for RNA sequencing libraries, such as ssRNA ligation and reverse transcription, because sequence information can be recovered from the template DNA.…”