Mapping Long Non-Coding RNA Structures with Fragmentation and Next-Generation Sequencing

Abstract

Recent years have seen an explosion in our appreciation of the myriad roles that RNA plays in the cell, including the discovery of new classes of regulatory RNAs such as long non-coding RNAs (lncRNAs). The three-dimensional folded structure of many coding and non-coding RNAs plays a key role in determining their function and fate in the cell. Obtaining high quality structural information on large numbers of RNAs is therefore essential, but traditional methods such as crystallography and NMR have been limited due to RNA's rugged folding landscape, charged backbone, and the large size of many RNAs of interest. Chemical mapping, in which folded RNAs are reacted with structure-sensitive chemical probes, provides nucleotide-level resolution mapping of base pairing and backbone flexibility, and can be applied both in vitro and in living cells. Chemical mapping data can be combined with thermodynamics-based structure models to predict RNAs’ folded structure, but the confidence of these predictions decreases rapidly with increasing RNA size. Measuring the structure of both the full length RNA and multiple shorter fragments in vitro has been proposed as a route to obtaining high-confidence structures, but applying this approach to longer RNAs requires high-throughput techniques for readout. Next-generation sequencing (NGS) is well suited to this task, as the large number of reads allows simultaneous analysis of multiple sequences and fragments with multiple chemical agents and the sequencing readout avoids length-dependent inaccuracies associated with gel and capillary electrophoresis. I will present results from applying rational fragmentation with NGS readout to map the structure of the 2.4 kb trans-acting lncRNA HOTAIR and discuss implications for folding pathways of large RNAs and possibilities for in vivo measurement.