An Evolutionary Trace method defines functionally important bases and sites common to RNA families

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.
Author summary Traditionally, RNA has been delegated to the role of an intermediate between DNA and proteins. However, we now recognize that RNAs are broadly functional beyond their role in translation, and that a number of diverse classes exist. Because functional, non-coding RNAs are prevalent in biology and impact human health, it is important to better understand their functional determinants. However, the classical solution to this problem, targeted mutagenesis, is time-consuming and scales poorly. We propose an alternative computational approach to this problem, the Evolutionary Trace method. Previously developed and validated for proteins, Evolutionary Trace examines evolutionary history of a molecule and predicts evolutionarily important residues in the sequence. We apply Evolutionary Trace to a set of diverse RNAs, and find that the evolutionarily important nucleotides cluster on the three-dimensional structure, and that these clusters closely overlap functional sites. We also find that the clustering property can be used to refine and improve predictions. These findings are in close agreement with our observations of Evolutionary Trace in proteins, and suggest that structured functional RNAs and proteins evolve under similar constraints. In practice, the approach is to be used by RNA researches seeking insight into their molecule of interest, and the Evolutionary Trace program, along with a working example, is available at https://github.com/LichtargeLab/RNA_ET_ms.