Abstract
SHAPE-JuMP is a concise strategy for identifying close-in-space interactions in RNA molecules. Nucleotides in close three-dimensional proximity are crosslinked with a bi-reactive reagent that covalently links the 2’-hydroxyl groups of the ribose moieties. The identities of crosslinked nucleotides are determined using an engineered reverse transcriptase that jumps across crosslinked sites, resulting in a deletion in the cDNA that is detected using massively parallel sequencing. Here we introduce ShapeJumper, a bioinformatics pipeline to process SHAPE-JuMP sequencing data and to accurately identify through-space interactions, as observed in complex JuMP datasets. ShapeJumper identifies proximal interactions with near-nucleotide resolution using an alignment strategy that is optimized to tolerate the unique non-templated reverse-transcription profile of the engineered crosslink-traversing reverse-transcriptase. JuMP-inspired strategies are now poised to replace adapter-ligation for detecting RNA-RNA interactions in most crosslinking experiments.
Highlights
RNA molecules form multiple levels of intra- and inter-molecular higher order structure, and these structures often have important functions
This work outlines the development and optimization of an analysis pipeline, called ShapeJumper, that substantially facilitates analysis of RNA crosslinking experiments, based on implemented JuMP technology. Both the crosslinking experiment and the analysis software described here are readily implemented by non-expert users
Secondary structures form via base pairing, and secondary structures may further fold into compact tertiary structures mediated by interactions involving canonically and non-canonically interacting nucleotides [1,2]
Summary
RNA molecules form multiple levels of intra- and inter-molecular higher order structure, and these structures often have important functions. RNA crosslinking should be able to identify short through-space interactions Chemical probes such as psoralen analogs [13,14,15,16], formaldehyde [17,18] and bis-succinimidyl esters [18], and short wavelength ultraviolet (UV) irradiation [18,19,20] have been used to crosslink interacting nucleotides. After ligation of adapter sequences to these ends, the sequences are determined by massively parallel sequencing These adapter-ligation methods yield a rough approximation of crosslink location with best-case resolution of plus-or-minus ten nucleotides [9,21], with the calculations of overall abundance biased by the complex multi-step ligation and library preparation steps required prior to sequencing [8,22]. Commonly used crosslinking reagents and UV irradiation both have strong sequence and structural selectivity, such that observed crosslinks detect only a small fraction of intermolecular RNA interactions
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