CLC3 incorporates and extends previous versions (CLC1, 118 lncRNAs, CLC2 374 lncRNAs) [
1,
2]. In addition to its size, CLC3 now incorporates for the first time lncRNAs involved in chemoresistance, with 10% of CLC lncRNAs exhibiting this functionality (
Figure 1A). We previously observed that CLC lncRNAs carry a range of features distinguishing them from other lncRNAs. Amongst these were elevated rates for several measures of evolutionary conservation, similar to those previously observed for protein-coding cancer genes [
1,
5]. First, we evaluated the confidence level of experimental support for CLC genes, finding these to be consistent between versions with roughly 40% of lncRNAs validated by the highest-confidence in vivo evidence (
Figure 1A). Therefore, any differences observed between CLC versions is not likely to arise from differences in confidence regarding their disease roles. Next, we comprehensively evaluated a range of features of CLC versions, comparing non-redundant gene sets to non-cancer lncRNAs (“nonCLC”) (
Figure 1B). For comparison, we also compared a collection of disease-associated lncRNAs, from which cancer genes were removed (EVlncRNA) [
6]. All CLC versions and EVlncRNAs display elevated levels of gene expression, expression ubiquity, overall gene length, spliced RNA length and proximity to nearest protein-coding genes (
Figure 1B). Surprisingly, however, we noticed that CLC3 lncRNAs are not more evolutionarily conserved compared to other non-cancer lncRNAs (arrows). This is true not only for two different measures of conservation from the widely used PhastCons measure (average base-level score and percentage of exon coverage by conserved elements), but also for the promoter (average base-level), for which particularly elevated conservation has been observed in lncRNAs [
7,
8]. A more detailed gene-level inspection supported these findings (
Figure 1C–F), showing a pronounced trend for the CLC3 lncRNAs to have comparable or even lower conservation than lncRNAs in general.To strengthen these findings, we used an alternative method to evaluate evolutionary conservation: the existence of orthologous lncRNA genes in other species. Using the tool ConnectOR [
9], we searched for orthologues of human lncRNAs in chimpanzees and mice (see Methods). Overall, we identified orthologues for 4102 and 4493 lncRNAs in chimpanzees and mice, respectively (lower rates in chimpanzees likely reflect less mature lncRNA annotations). Consistent with previous results, we observed that CLC3 lncRNAs have a significantly lower chance of having an identifiable orthologue than CLC1 and CLC2, at a level comparable to nonCLC lncRNAs (
Figure 2A). Given that CLC3 lncRNAs were collected most recently, we hypothesised that the observed trend arose from a relationship between conservation and the moment when the lncRNA was studied. Indeed, we observed a significant negative correlation between conservation and year of discovery (
Figure 2B, left). This trend appears to be specific to cancer, because EVlncRNAs from other diseases do not display this behaviour (
Figure 2B, right). In other words, as time goes on, researchers are turning their attention to less conserved lncRNAs that nevertheless play functional cancer roles.
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