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

The impact of inherited and somatic mutations on messenger RNA (mRNA) structure remains poorly understood. Recent technological advances that leverage next-generation sequencing to obtain experimental structure data, such as SHAPE-MaP, can reveal structural effects of mutations, especially when these data are incorporated into structure modeling. Here, we analyze the ability of SHAPE-MaP to detect the relatively subtle structural changes caused by single-nucleotide mutations. We find that allele-specific sorting greatly improved our detection ability. Thus, we used SHAPE-MaP with a novel combination of clone-free robotic mutagenesis and allele-specific sorting to perform a rapid, comprehensive survey of noncoding somatic and inherited riboSNitches in two cancer-associated mRNAs, TPT1 and LCP1. Using rigorous thermodynamic modeling of the Boltzmann suboptimal ensemble, we identified a subset of mutations that change TPT1 and LCP1 RNA structure, with approximately 14% of all variants identified as riboSNitches. To confirm that these in vitro structures were biologically relevant, we tested how dependent TPT1 and LCP1 mRNA structures were on their environments. We performed SHAPE-MaP on TPT1 and LCP1 mRNAs in the presence or absence of cellular proteins and found that both mRNAs have similar overall folds in all conditions. RiboSNitches identified within these mRNAs in vitro likely exist under biological conditions. Overall, these data reveal a robust mRNA structural landscape where differences in environmental conditions and most sequence variants do not significantly alter RNA structural ensembles. Finally, predicting riboSNitches in mRNAs from sequence alone remains particularly challenging; these data will provide the community with benchmarks for further algorithmic development.