Rapid Evolution of in vivo-Selected Sequences and Structures Replacing 20% of a Subviral RNA
Allison M. Murawski
Johnathan L. Nieves
Holleh F. Tajalli
Nina B. Jean-Jacques
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.
Murawski, Allison M., Johnathan L. Nieves, Maitreyi Chattopadhyay, Megan Y. Young, Christine Szarko, Holleh F. Tajalli, Tareq Azad, Nina B. Jean-Jacques, Anne E. Simon, David B. Kushner. "Rapid Evolution of in vivo-Selected Sequences and Structures Replacing 20% of a Subviral RNA." Virology 483 (2015): 149-162.