Direct Mapping of Higher-Order RNA Interactions by SHAPE-JuMP

Abstract

Higher-order structure governs function for many RNAs. However, discerning this structure for large RNA molecules in solution is an unresolved challenge. Here, we present SHAPE-JuMP (selective 2′-hydroxyl acylation analyzed by primer extension and juxtaposed merged pairs) to interrogate through-space RNA tertiary interactions. A bifunctional small molecule is used to chemically link proximal nucleotides in an RNA structure. The RNA cross-link site is then encoded into complementary DNA (cDNA) in a single, direct step using an engineered reverse transcriptase that “jumps” across cross-linked nucleotides. The resulting cDNAs contain a deletion relative to the native RNA sequence, which can be detected by sequencing, that indicates the sites of cross-linked nucleotides. SHAPE-JuMP measures RNA tertiary structure proximity concisely across large RNA molecules at nanometer resolution. SHAPE-JuMP is especially effective at measuring interactions in multihelix junctions and loop-to-helix packing, enables modeling of the global fold for RNAs up to several hundred nucleotides in length, facilitates ranking of structural models by consistency with through-space restraints, and is poised to enable solution-phase structural interrogation and modeling of complex RNAs.

Identifying proximal RNA interactions from cDNA-encoded crosslinks with ShapeJumper

“…Individual reads may or may not have a deletion, the deletions may vary in length, and the rates of occurrence of deletions vary. The RT enzyme currently used in the SHAPE-JuMP strategy has the special ability to read across crosslinked sites but also has a high non-crosslink-related per-nucleotide mutation rate of 3-4% 23 , which makes alignment challenging. No aligner has been specifically designed or optimized to operate with this type of complex data.…”

“…We evaluated the ability of these aligners to detect SHAPE-JuMP deletions using datasets of synthetic sequencing reads designed to mimic SHAPE-JuMP sequencing reads that contained known deletion locations. These datasets were designed specifically to contain sequences that mirrored those observed in experimental SHAPE-JuMP reads, performed with the RT-C8 enzyme 23 . Two synthetic read datasets were created, a deletion set and a deletion-insertion set.…”

“…After optimizing the pipeline with synthetic data, the proto-ShapeJumper pipeline was used to process experimentally generated SHAPE-JuMP reads obtained from analyses of a set of small to large RNAs (158-412 nts): the P546 group II intron domain, M-Box riboswitch, Varkud satellite ribozyme, RNase P catalytic domain, and group II intron 23 . Quality filtered and merged reads were aligned, the resulting alignments parsed to identify deletion sites, and deletion rates were normalized by the median read depth of the 5 nucleotides downstream of the 3' deletion site (Fig.…”

“…6). We note that parameter choices and optimization steps were tailored to the specific mutation and deletion characteristics of the RT-C8 25 enzyme, characterized for SHAPE-JuMP 23 . We think the algorithm developed here will be effective for alternative jumping polymerases and reagents identified in the future, with only minor modification or optimization of the ShapeJumper pipeline.…”

“…We recently introduced a strategy we call SHAPE-JuMP (for selective 2'-hydroxyl acylation analyzed by primer extension and juxtaposed merged pairs) 23 in which nucleotides in close three-dimensional proximity are crosslinked with a bi-reactive reagent (Fig. 1A, left).…”