Abstract
Functional non-coding (fnc)RNAs are nucleotide sequences of varied lengths, structures, and mechanisms that ubiquitously influence gene expression and translation, genome stability and dynamics, and human health and disease. Here, to shed light on their functional determinants, we seek to exploit the evolutionary record of variation and divergence read from sequence comparisons. The approach follows the phylogenetic Evolutionary Trace (ET) paradigm, first developed and extensively validated on proteins. We assigned a relative rank of importance to every base in a study of 1070 functional RNAs, including the ribosome, and observed evolutionary patterns strikingly similar to those seen in proteins, namely, (1) the top-ranked bases clustered in secondary and tertiary structures. (2) In turn, these clusters mapped functional regions for catalysis, binding proteins and drugs, post-transcriptional modification, and deleterious mutations. (3) Moreover, the quantitative quality of these clusters correlated with the identification of functional regions. (4) As a result of this correlation, smoother structural distributions of evolutionary important nucleotides improved functional site predictions. Thus, in practice, phylogenetic analysis can broadly identify functional determinants in RNA sequences and functional sites in RNA structures, and reveal details on the basis of RNA molecular functions. As example of application, we report several previously undocumented and potentially functional ET nucleotide clusters in the ribosome. This work is broadly relevant to studies of structure-function in ribonucleic acids. Additionally, this generalization of ET shows that evolutionary constraints among sequence, structure, and function are similar in structured RNA and proteins. RNA ET is currently available as part of the ET command-line package, and will be available as a web-server.