Evolutionary evidence for alternative structure in RNA sequence co-variation

Sequence conservation and co-variation of base pairs are hallmarks of structured RNAs. For certain RNAs (e.g. riboswitches), a single sequence must adopt at least two alternative secondary structures to effectively regulate the message. If alternative secondary structures are important to the function of an RNA, we expect to observe evolutionary co-variation supporting multiple conformations. We set out to characterize the evolutionary co-variation supporting alternative conformations in riboswitches to determine the extent to which alternative secondary structures are conserved. We found strong co-variation support for the terminator, P1, and anti-terminator stems in the purine riboswitch by extending alignments to include terminator sequences. When we performed Boltzmann suboptimal sampling on purine riboswitch sequences with terminators we found that these sequences appear to have evolved to favor specific alternative conformations. We extended our analysis of co-variation to classic alignments of group I/II introns, tRNA, and other classes of riboswitches. In a majority of these RNAs, we found evolutionary evidence for alternative conformations that are compatible with the Boltzmann suboptimal ensemble. Our analyses suggest that alternative conformations are selected for and thus likely play functional roles in even the most structured of RNAs.
Author Summary RNA (Ribonucleic Acid) is a messenger of genetic information, master regulator, and catalyst in the cell. To carry out its function, RNA can fold into complex three-dimensional structures. Certain classes of RNAs, called riboswitches, adopt at least two alternative structures to act as a switch. We set out to detect the evolutionary signal for alternative structures in riboswitches as we hypothesize that these RNA sequences must have evolved to allow both conformations. We find that indeed such signals exist when we compare the sequences of riboswitches from multiple species. When we extend this analysis to other RNA regulators in the cell that are not thought of as switches, we detect equivalent evolutionary support for alternative structures. Viewed through the lens of evolutionary structure conservation RNA sequences appear to have adapted to adopt multiple conformations.