Transcriptome-wide interrogation of RNA secondary structure in living cells with icSHAPE

Abstract

icSHAPE (in vivo click selective 2-hydroxyl acylation and profiling experiment) captures RNA secondary structure at a transcriptome-wide level by measuring nucleotide flexibility at base resolution. Living cells are treated with the icSHAPE chemical NAI-N3 followed by selective chemical enrichment of NAI-N3-modified RNA, which provides an improved signal-to-noise ratio compared with similar methods leveraging deep sequencing. Purified RNA is then reverse-transcribed to produce cDNA, with SHAPE-modified bases leading to truncated cDNA. After deep sequencing of cDNA, computational analysis yields flexibility scores for every base across the starting RNA population. The entire experimental procedure can be completed in ∼5 d, and the sequencing and bioinformatics data analysis take an additional 4-5 d with no extensive computational skills required. Comparing in vivo and in vitro icSHAPE measurements can reveal in vivo RNA-binding protein imprints or facilitate the dissection of RNA post-transcriptional modifications. icSHAPE reactivities can additionally be used to constrain and improve RNA secondary structure prediction models.

Figure 1

Schematic overview of icSHAPE. (a) Stepwise scheme for acylating flexible RNA nucleobases with the SHAPE reagent NAI-N₃ and subsequent ‘click’ reaction of DIBO-biotin specifically to icSHAPE-modified nucleotides. (b) Experimental and computational steps in the icSHAPE procedure. Living cells are first treated with NAI-N₃ to record the in vivo flexibility of RNA nucleotides across the transcriptome. Modified (and mock-modified) RNA is isolated, and ‘click’ reactions add DIBO-biotin specifically to modified RNAs. RNA fragmentation and RT ‘reads out’ the NAI-N₃ modification, after which only NAI-N₃–modified molecules are selectively isolated and sequenced. Deep-sequencing reads are separated for individual experiments, removed of PCR artifacts and mapped to the appropriate transcriptome build. RT stop sites and abundances are calculated and biological replicate experiments are merged to ensure reproducibility. RT stops are normalized for each transcript, and the resulting data are visualized in the UCSC genome browser or used for downstream analysis.

Figure 2

Experimental data from representative icSHAPE experiments. (a) Urea PAGE analysis of fragmented and 3′-end–ligated RNA generated in Step 31. The free RNA linkers are labeled (black arrows) and the region of the gel cut for subsequent steps is shown within a dashed red box for one sample. (b) Urea PAGE analysis of cDNAs generated in Step 55. Carry-over RNA linkers and unextended RT primers are labeled (black arrows), and the red box (dashed red line) denotes the region cut for subsequent steps. (c) Native PAGE analysis of icSHAPE library DNA generated in Step 64. Un-incorporated PCR primers and PCR primer-dimer products are labeled (black arrows). The final library DNA, above the PCR primer dimer, is highlighted (dashed red box) and subsequently cut from the gel and analyzed on an Illumina deep-sequencing platform.

Figure 3

icSHAPE reactivities of Nanog (Nanog homeobox) mRNA. A 7-kb region centered on the Nanog locus is shown with two isoforms of the Nanog mRNA in the mm10 build of the mouse genome. In vivo (red) and in vitro icSHAPE experimental data are represented as tracks with values ranging from 0 to 1. The vivo–vitro difference (VTD) score for each base is shown in gray.