24In comparison to protein coding sequences, the impact of mutation and natural selection 25 on the sequence and function of non-coding (ncRNA) genes is not well understood. 26Many ncRNA genes are narrowly distributed to only a few organisms, and appear to be 27 rapidly evolving. Compared to protein coding sequences, there are many challenges 28 associated with assessment of ncRNAs that are not well addressed by conventional 29 phylogenetic approaches, including: short sequence length, lack of primary sequence 30 conservation, and the importance of secondary structure for biological function. 31Riboswitches are structured ncRNAs that directly interact with small molecules to 32 regulate gene expression in bacteria. They typically consist of a ligand-binding domain 33 (aptamer) whose folding changes drive changes in gene expression. The glycine 34 riboswitch is among the most well-studied due to the widespread occurrence of a 35 tandem aptamer arrangement (tandem), wherein two homologous aptamers interact 36 with glycine and each other to regulate gene expression. However, a significant 37 proportion of glycine riboswitches are comprised of single aptamers (singleton). Here 38 we use graph clustering to circumvent the limitations of traditional phylogenetic analysis 39 when studying the relationship between the tandem and singleton glycine aptamers. 40Graph clustering enables a broader range of pairwise comparison measures to be used 41 to assess aptamer similarity. Using this approach, we show that one aptamer of the 42 tandem glycine riboswitch pair is typically much more highly conserved, and that which 43 aptamer is conserved depends on the regulated gene. Furthermore, our analysis also 44 reveals that singleton aptamers are more similar to either the first or second tandem 45 aptamer, again based on the regulated gene. Taken together, our findings suggest that 46 2 tandem glycine riboswitches degrade into functional singletons, with the regulated 47 gene(s) dictating which glycine-binding aptamer is conserved. 48 49
Author Summary 50The glycine riboswitch is a ncRNA responsible for the regulation of several distinct gene 51 sets in bacteria that is found with either one (singleton) or two (tandem) aptamers, each 52 of which directly senses glycine. Which aptamer is more important for gene-regulation, 53 and the functional difference between tandem and singleton aptamers, are long-54 standing questions in the riboswitch field. Like many biologically functional RNAs, 55 glycine aptamers require a specific 3D folded conformation. Thus, they have low 56 primary sequence similarity across distantly related homologs, and large changes in 57 sequence length that make creation and analysis of accurate multiple sequence 58 alignments challenging. To better understand the relationship between tandem and 59 singleton aptamers, we used a graph clustering approach that allows us to compare the 60 similarity of aptamers using metrics that measure both sequence and structure 61 similarity. Our investigation reveals that in ...
