Rapid evolution of in vivo-selected sequences and structures replacing 20% of a subviral RNA

•Sequence-structure-function requirements in a 76 nt subviral RNA hairpin were studied.•Subviral RNAs with hairpins of 0, 19, 38, or 76 random nt were evolved in planta.•Rapidly-evolved hairpins were half-sized (38 or 39 nt) regardless of starting size.•An evolved subviral RNA with a 39 nt hairpin was at least as functional as wt.•Subviral RNA dimer:monomer ratio increased as hairpin length decreased.

The 356 nt noncoding satellite RNA C (satC) of Turnip crinkle virus (TCV) is composed of 5′ sequences from a second TCV satRNA (satD) and 3′ sequences derived from TCV. SHAPE structure mapping revealed that 76 nt in the poorly-characterized satD-derived region form an extended hairpin (H2). Pools of satC in which H2 was replaced with 76, 38, or 19 random nt were co-inoculated with TCV helper virus onto plants and satC fitness assessed using in vivo functional selection (SELEX). The most functional progeny satCs, including one as fit as wild-type, contained a 38-39 nt H2 region that adopted a hairpin structure and exhibited an increased ratio of dimeric to monomeric molecules. Some progeny of satC with H2 deleted featured a duplication of 38 nt, partially rebuilding the deletion. Therefore, H2 can be replaced by a 38-39 nt hairpin, sufficient for overall structural stability of the 5′ end of satC.