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

Abstract

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.

Biologically-supported structural model for a viral satellite RNA

“…Overall, 58% of the sat-Cym RNA is predicted to be involved in base pairing interactions forming secondary structures. Similarly, the secondary structure model for the helper TBSV genome predicts 60% of this RNA to be base paired ( 52 ), and comparable values were observed for CMV-associated satRNA (51%), TCV-associated satC (47%) and STMV (62%) ( 9 , 24 , 34 ). Interestingly, the structural models for all three of the aforementioned satellite species contain a large central or 5′-proximal domain comprising ∼47–60% of their sequence.…”

“…The virally-derived portion of satC has been extensively studied and includes many functionally important secondary and tertiary RNA structures, including a 3′-terminal conformational switch, which are important for satC replication ( 13 – 20 ). The activity of the 5′ half of satC corresponding to satD is less well characterized, but terminal promoters for plus-strand synthesis have been defined ( 21 – 23 ) and a large internal hairpin is known to contribute to its accumulation ( 24 ). In the satRNA of Bamboo mosaic virus (satBaMV), the 3′-terminal sequence is structurally similar to that of its helper genome, however direct structural equivalence at the functional level is not observed, indicating the presence of satRNA-specific features ( 25 ).…”

“…Secondary structure models based on chemical probing data of full-length RNAs have also been generated for STMV ( 9 – 11 ), however, biological activity has been reported for only a few substructures ( 8 ). Similarly, complete structure models based on solution probing have been reported for satC of TCV and satRNA of Cucumber mosaic virus (CMV satRNA), but not all of the predicted substructures have been assessed for biological relevance ( 24 , 34 ).…”

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

“…The 5’ portion of satC, which is derived from a second satRNA of TCV (satD), is more poorly characterized. Mfold (Zuker, 2003) and in vitro SHAPE (selective 2’-hydroxyl acylation analyzed by primer extension [Merino et al ., 2005]) analyses have suggested that the 5’ 166 nt of satC form a separate RNA domain (Murawski et al , 2015). This 5’ portion was suggested to contain (i) a lower stem; (ii) an upper connecting hairpin termed H2; and (iii) a central region composed of two opposing stem-loops (H6 and H7; Fig.…”

“…The H2 region (76 nt, positions 48-123) was extensively examined using in vivo SELEX by replacing it with either 76, 38, 19, or 0 random nt. The most fit satCs that were recovered possessed either a 38 or 39 nt H2 region that folded into a stable hairpin, with one recovered satRNA having fitness similar to WT satC for propagation with the helper virus in plants (Murawski et al ., 2015). Furthermore, consistent with prior studies examining the effect of deletions within the H2 region (Carpenter et al ., 1991; Zhang, 2006), satC with reduced-length H2 accumulated mainly in dimeric form in protoplasts unlike WT satC, which mainly accumulates as monomers along with low levels of multimeric forms (Murawski et al.…”