Deciphering Structural Selective Constraints: A Comparative Evolutionary Analysis of RNA Hairpin Structures

RNA molecules often rely on specific, stable secondary structures to fulfill their functions. Examples include microRNA (miRNA) precursors in eukaryotes and terminator signals in prokaryotes, which require stable single-stem hairpin substrates to interact with proteins or complexes. By analyzing the sequence similarity and folding patterns of highly conserved hairpin structures, we identify critical signals and features indicative of selective constraints on RNA structure formation. We classify fixed mutations according to various modes of RNA selection pressure. In this study, we introduce a comparative evolutionary approach to quantify selective pressure on RNA sequences pairing in hairpins based on their structural requirements. This approach compares substitution patterns between natural and control sequences designed to nullify some structural selection while minimizing sequence changes. We evaluate evidence of structural selective constraint by quantifying the destabilization of hairpin structures in its absence. Our analysis reveals significantly higher rates of double compensatory base-pair substitutions in functional hairpins than expected. Additionally, we observe significant differences in the frequency of structure-preserving and structure-destroying single substitutions in pre-miRNAs compared to randomized sequences or a simple theoretical model. This approach also enables the quantification of structural selective constraints in stable elements of long noncoding RNAs and mRNAs in different organisms, where such constraints are prominent.