Hidden Structural Modules in a Cooperative RNA Folding Transition

Abstract

SUMMARY Large-scale, cooperative rearrangements underlie the functions of RNA in RNA-protein machines and gene regulation. To understand how such rearrangements are orchestrated, we used high-throughput chemical footprinting to dissect a seemingly concerted rearrangement in P5abc RNA, a paradigm of RNA folding studies. With mutations that systematically disrupt or restore putative structural elements, we found that this transition reflects local folding of structural modules, with modest and incremental cooperativity that results in concerted behavior. First, two distant secondary structure changes are coupled through a bridging three-way junction and Mg2+-dependent tertiary structure. Second, long-range contacts are formed between modules, resulting in additional cooperativity. Given the sparseness of RNA tertiary contacts after secondary structure formation, we expect that modular folding and incremental cooperativity are generally important for specifying functional structures while also providing productive kinetic paths to these structures. Additionally, we expect our approach to be useful for uncovering modularity in other complex RNAs.

Intrinsic Regulatory Role of RNA Structural Arrangement in Alternative Splicing Control

“…Recent advances including high-throughput chemical footprinting combined with next-generation sequencing enabled to define remarkably detailed structural features of RNA in association with their biological role. Gracia and others provided a substantial body of evidence for incremental and concerted cooperativity between RNA structural motifs leading to folding of preferential RNA secondary and tertiary structures [ 66 , 67 ]. This cooperativity considers the formation of short-lived intermediates of RNA structure of less preferred thermodynamic stability which affect the kinetics of folding [ 67 ].…”

“…Gracia and others provided a substantial body of evidence for incremental and concerted cooperativity between RNA structural motifs leading to folding of preferential RNA secondary and tertiary structures [ 66 , 67 ]. This cooperativity considers the formation of short-lived intermediates of RNA structure of less preferred thermodynamic stability which affect the kinetics of folding [ 67 ]. Xue and others utilized 15 N relaxation dispersion nuclear magnetic resonance (NMR) combined with chemical probing to capture such intermediates of p5abc subdomain of the Tetrahymena group I intron ribozyme [ 68 ].…”

The Story of RNA Folding, as Told in Epochs

“…Indeed, recent NMR chemical shift exchange experiments have suggested that P5abc occupies a series of local and rare states that are favorable for tertiary structure formation, essentially breaking down a large-scale conformational process into smaller steps that can occur independently and with only modest kinetic and thermo-dynamic barriers (Xue et al 2016). This model is supported by recent work that identified folding modules within P5abc that each contribute modestly to an overall cooperativity of ∼4 kcal/mol (Gracia et al 2018). Thus, structural modules within a complex RNA "experiment with" different subconformational states, which smooths the energy landscape and speeds folding transitions.…”

Challenges and approaches to predicting RNA with multiple functional structures

“…Because RNA typically follows a hierarchical folding pathway (Tinoco and Bustamante 1999;Zhang et al 2017;Gracia et al 2018;Šponer et al 2018), prediction of RNA secondary structure followed by RNA tertiary structure is often a successful strategy. The pattern of Watson-Crick pairing provides significant constraints that reduce the complexity of tertiary structure prediction.…”