Structure based H/ACA guide RNA design and testing explains the structure-function relationship of H/ACA guide RNA

Abstract

H/ACA guide RNAs are a class of noncoding RNA that direct the pseudouridylation of many cellular RNA species. In most eukaryotes, H/ACA guide RNAs share a conserved hairpin-hinge-hairpin structure, where each hairpin can direct pseudouridylation when associated with evolutionarily conserved core proteins. Target selection occurs by base pairing between target RNA and single-stranded loops within each hairpin of the H/ACA guide RNA, called pseudouridylation pockets. Here, I have analyzed the structure-function relationship of H/ACA guide RNAs by applying a structure-focused approach to design H/ACA guide RNAs for pseudouridylation of novel substrates. Thereby, I designed and tested several artificial H/ACA guide RNAs that were both highly active and specific for their respective substrates in vitro. In addition, I generated multiple sub-optimal H/ACA guide RNA designs that reveal important information regarding H/ACA guide RNA features dictating productivity. My results open new avenues for evaluating, predicting/identifying, and designing cellular guide-substrate RNA combinations.