In this study, Quinn et. al. used an integrative strategy based on matching focal and repeated RNA secondary structures and other RNA features that uncovers novel lncRNA orthologs despite limited sequence similarity. This method was applied to Drosophilia roX1 and roX2 RNAs, and 47 new roX RNAs across ∼40 million years of evolution were discovered.
Many long noncoding RNAs (lncRNAs) can regulate chromatin states, but the evolutionary origin and dynamics driving lncRNA–genome interactions are unclear. We adapted an integrative strategy that identifies lncRNA orthologs in different species despite limited sequence similarity, which is applicable to mammalian and insect lncRNAs. Analysis of the roX lncRNAs, which are essential for dosage compensation of the single X chromosome in Drosophila males, revealed 47 new roX orthologs in diverse Drosophilid species across ∼40 million years of evolution. Genetic rescue by roX orthologs and engineered synthetic lncRNAs showed that altering the number of focal, repetitive RNA structures determines roX ortholog function. Genomic occupancy maps of roX RNAs in four species revealed conserved targeting of X chromosome neighborhoods but rapid turnover of individual binding sites. Many new roX-binding sites evolved from DNA encoding a pre-existing RNA splicing signal, effectively linking dosage compensation to transcribed genes. Thus, dynamic change in lncRNAs and their genomic targets underlies conserved and essential lncRNA–genome interactions.