(A) Developmental expression profiles of putative lncRNA promoters (n = 3,973). (B) Heatmaps of RAMPAGE signal (left; number of reads) and DNase-seq signal (right; arbitrary uits) over individual TSCs. We are comparing TSCs that overlap FlyBase-annotated mRNA transcription start sites (top), which we use as positive controls, to the TSCs of putative lncRNAs (bottom). In each group, TSCs are sorted by total RAMPAGE signal intensity. (C) Distribution of phyloCSF scores for transcript models corresponding to putative lncRNAs (n = 1,475) and mRNAs (n = 16,105). Transcript models were built from publicly available RNA-seq data using Cufflinks. The phyloCSF metric quantifies the protein-coding potential of transcripts, based on the presence and conservation of ORFs. (D) Shannon entropy of the temporal expression profiles for lncRNA (n = 2,397) and mRNA (n = 18,067) promoters with maximum expression ≥2 RPM. Overall, the profiles of lncRNA promoters have lower entropy, reflecting more acutely stage-specific expression. (E) Number of D. melanogaster lncRNA promoters functionally preserved in other species. The grey bars represent the number of promoters for which the multiple sequence alignments passed our filtering criteria, and therefore could be interrogated. (F) Promoter sequence conservation. Considering all promoters that are functionally preserved in all five species, the sequences of lncRNA promoters (n = 631) are under comparable selective pressure to those of protein-coding genes. (G) The developmental expression profiles of functionally conserved lncRNA promoters are far more constrained than those of many categories of protein-coding genes. We considered all promoters with maximum expression ≥25 RPM and expression changes ≥5 fold (n = 55 lncRNA promoters). (H) We identified 195 D. melanogaster lncRNA promoters that are functionally preserved within the melanogaster subgroup, but not in the two outgroup species, and are therefore likely to have been recently acquired specifically in this lineage (inset, top left). Lineage-specific lncRNA promoters display a level of sequence conservation within the subgroup similar to that of conserved protein-coding gene promoters.