SELEX and SHAPE reveal that sequence motifs and an extended hairpin in the 5’ portion of Turnip crinkle virus satellite RNA C mediate fitness in plants

Noncoding RNAs use their sequence and/or structure to mediate function(s). The 5’ portion (166 nt) of the 356-nt noncoding satellite RNA C (satC) of Turnip crinkle virus (TCV) was previously modeled to contain a central region with two stem-loops (H6 and H7) and a large connecting hairpin (H2). We now report that in vivo functional selection (SELEX) experiments assessing sequence/structure requirements in H2, H6, and H7 reveal that H6 loop sequence motifs were recovered at nonrandom rates and only some residues are proposed to base-pair with accessible complementary sequences within the 5’ central region. In vitro SHAPE of SELEX winners indicates that the central region is heavily base-paired, such that along with the lower stem and H2 region, one extensive hairpin exists composing the entire 5’ region. As these SELEX winners are highly fit, these characteristics facilitate satRNA amplification in association with TCV in plants. •In vivo SELEX was used to find sequence-structure requirements within a subviral RNA.•In vitro SHAPE of SELEX winners shows the satC 5’ central region can be highly zipped.•Some WT sequence motifs were evolved, but RNA structure is more critical for fitness.