Variant induced RNA structure change in human genetic disease

SUMMARY My research program focuses on quantitatively characterizing the role of RNA structure in post-transcriptional regulatory processes. Comparisons of protein and messenger RNA (mRNA) abundance at genome scale reveal low correlation between the two gene expression levels in most human tissues and other organisms. This poor correlation suggests that a significant amount of gene regulation occurs post-transcriptionally. To discover elements in mRNAs that control their activities, we measure the effects of human disease-associated structure variants that map to non-coding regions of the transcriptome. Specifically, we integrate computational structure prediction with high-throughput allele-specific chemical structure probing in vivo to assess the functional consequences of RNA structure change. We also establish the causality of these variants by using quantitative reporter assays to measure translation efficiency, splicing, and mRNA stability. In total, these experiments provide molecular explanations of disease mechanisms. To support these goals, we develop, implement, and apply both computational and experimental approaches to study RNA structure in the cell. Our proposed research program will further develop two important technological innovations. The first is a hybrid experimental/computational approach for studying precursor and mature mRNA structure simultaneously in vivo; this approach integrates mutational profiling of SHAPE-MaP data (Selective 2’-Hydroxyl Acylation by Primer Extension – Mutational Profiling) with Boltzmann suboptimal sampling of the secondary structural ensemble. The second innovation is SHAPE-JuMP, which uses a bifunctional RNA modification reagent and a highly processive reverse transcriptase that “jumps” across chemical crosslinks to probe through-space three-dimensional contacts in RNA. We will use these technologies to establish the structures of both precursor and mature mRNAs. In addition, we will extend the biological scope of our work by using these technologies to collaboratively investigate inter- and intramolecular interactions in positive strand RNA viruses. In sum, this program will identify novel RNA structure motifs that regulate the functions of precursor and mature mRNAs and viral genomes.