Selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis

Abstract

SHAPE chemistries exploit small electrophilic reagents that react with the 2′-hydroxyl group to interrogate RNA structure at single-nucleotide resolution. Mutational profiling (MaP) identifies modified residues based on the ability of reverse transcriptase to misread a SHAPE-modified nucleotide and then counting the resulting mutations by massively parallel sequencing. The SHAPE-MaP approach measures the structure of large and transcriptome-wide systems as accurately as for simple model RNAs. This protocol describes the experimental steps, implemented over three days, required to perform SHAPE probing and construct multiplexed SHAPE-MaP libraries suitable for deep sequencing. These steps include RNA folding and SHAPE structure probing, mutational profiling by reverse transcription, library construction, and sequencing. Automated processing of MaP sequencing data is accomplished using two software packages. ShapeMapper converts raw sequencing files into mutational profiles, creates SHAPE reactivity plots, and provides useful troubleshooting information, often within an hour. SuperFold uses these data to model RNA secondary structures, identify regions with well-defined structures, and visualize probable and alternative helices, often in under a day. We illustrate these algorithms with the E. coli thiamine pyrophosphate riboswitch, E. coli 16S rRNA, and HIV-1 genomic RNAs. SHAPE-MaP can be used to make nucleotide-resolution biophysical measurements of individual RNA motifs, rare components of complex RNA ensembles, and entire transcriptomes. The straightforward MaP strategy greatly expands the number, length, and complexity of analyzable RNA structures.

Allele-specific SHAPE-MaP assessment of the effects of somatic variation and protein binding on mRNA structure

“…We were able to obtain SHAPE data on thirty-seven selected variants within LCP1 and TPT1 using a novel, high-throughput SHAPE-MaP protocol (Siegfried et al 2014;Smola et al 2015b). Briefly, we used a clone-free, site-directed mutagenesis technique to create selected variants, transcribed the RNA variants, probed the RNA with 1M7 or the negative control, DMSO, and proceeded with error-prone reverse transcription to fix the adduct locations as mutations in the cDNA ( Fig.…”

“…We also in vitro transcribed TPT1 mRNA and compared the structural results of our protein-bound mRNA to artificially generated mRNA in the absence to any RNA binding proteins. We used the SHAPE-MaP protocol in which SHAPE adducts are converted to mutations during library preparation, and the locations of adducts are identified as mutations through next-generation sequencing (Siegfried et al 2014;Smola et al 2015b). The SHAPE-MaP datasets used to create our secondary structure models for TPT1 are available in the supplement (Supplemental Figs.…”

“…We take advantage of recent chemical probing techniques that allow us to rapidly obtain high-resolution structural information on full length transcripts (Siegfried et al 2014;Smola et al 2015b), while also facilitating a rapid, allele-specific sorting of reads for rapid and accurate riboSNitch detection. We compare the ability of traditional SHAPE-MaP to detect riboSNitches and find that using allele-specific sorting decreases the background noise and improves riboSNitch detection.…”

The predominant circular form of avocado sunblotch viroid accumulates in planta as a free RNA adopting a rod-shaped secondary structure unprotected by tightly bound host proteins

“…Between the remaining two members of Avsunviroidae, the eggplant latent viroid (ELVd) [35] and ASBVd [11], we chose the latter because despite its natural host being a woody plant, the isolate of this viroid, which we have been working with over the years (GenBank accession number J02020 with the substitution C213fiU), accumulates in vivo to levels comparable to the 5S ribosomal RNA [12,21], much higher than those reached by ELVd in eggplant. There are modifications more sensitive than conventional SHAPE [40,46], but some entail additional steps, particularly single-stranded adapter ligation, which limit the reliability of the results. These approaches, however, particularly SHAPE-MaP [46], may become indispensable with viroids reaching low titres in vivo.…”

“…There are modifications more sensitive than conventional SHAPE [40,46], but some entail additional steps, particularly single-stranded adapter ligation, which limit the reliability of the results. These approaches, however, particularly SHAPE-MaP [46], may become indispensable with viroids reaching low titres in vivo.…”