Long Noncoding RNAs and Repetitive Elements: Junk or Intimate Evolutionary Partners?
Authors: Hyunmin Lee, Zhaolei Zhang, Henry M. Krause
Journal: Trends in Genetics (2019)

Most lncRNA genes are expressed during spermatogenesis and are localized to many different subcellular locations.Solubility issues and alternative 3ʹ end processing have resulted in underestimates of lncRNA abundance.The diversity of known lncRNA molecular and subcellular functions is growing rapidly and may approach those of proteins.lncRNA and transposable element expression are coordinated during periods when spermatocyte heterochromatin is removed and replaced by protamines.Most lncRNAs contain transposable element sequences and many are expressed under the control of transposable element promoters. Our recent ability to sequence entire genomes, along with all of their transcribed RNAs, has led to the surprising finding that only ∼1% of the human genome is used to encode proteins. This finding has led to vigorous debate over the functional importance of the transcribed but untranslated portions of the genome. Currently, scientists tend to assume coding genes are functional until proven not to be, while the opposite is true for noncoding genes. This review takes a new look at the evidence for and against widespread noncoding gene functionality. We focus in particular on long noncoding RNA (noncoding RNAs longer than 200 nucleotides) genes and their 'junk' associates, transposable elements, and satellite repeats. Taken together, the suggestion put forward is that more of this junk DNA may be functional than nonfunctional and that noncoding RNAs and transposable elements act symbiotically to drive evolution. Our recent ability to sequence entire genomes, along with all of their transcribed RNAs, has led to the surprising finding that only ∼1% of the human genome is used to encode proteins. This finding has led to vigorous debate over the functional importance of the transcribed but untranslated portions of the genome. Currently, scientists tend to assume coding genes are functional until proven not to be, while the opposite is true for noncoding genes. This review takes a new look at the evidence for and against widespread noncoding gene functionality. We focus in particular on long noncoding RNA (noncoding RNAs longer than 200 nucleotides) genes and their 'junk' associates, transposable elements, and satellite repeats. Taken together, the suggestion put forward is that more of this junk DNA may be functional than nonfunctional and that noncoding RNAs and transposable elements act symbiotically to drive evolution. There has been a sometimes quiet, sometimes loud, decades-long discussion on the roles of noncoding transcripts and transposable elements (TEs) within our genomes. This discussion escalated when the ENCODE program revealed that the majority of the human genome is transcribed (at least 76% in humans), but that only ∼1.2% of this RNA is protein-coding [1ENCODE Project ConsortiumAn integrated encyclopedia of DNA elements in the human genome.Nature. 2012; 489: 57-74Crossref PubMed Scopus (11031) Google Scholar, 2Djebali S. et al.Landscape of transcription in human cells.Nature. 2012; 489: 101-108Crossref PubMed Scopus (3618) Google Scholar]. Based on our previous knowledge of bacterial and yeast genomes, these findings were a huge surprise. For most bacteria, over 90% of their DNA is protein-coding. The Escherichia coli genome uses an average of 1 × 103 bp per coding gene, with the eukaryotic yeast genome using about twice that amount. In contrast, our genomes contain ∼1.6 × 105 bp/coding gene. Unexpectedly, humans have only approximately five times more coding genes than E. coli, three times more than yeast, and fewer than both mice and the roundworm Caenorhabditis elegans (Table 1, Key Table).Table 1Key Table. Comparative Genome ComplexitiesOrganismkbpCoding genesRatio (bp/gene)Escherichia coli4.6 × 1034.3 × 1031.1 × 103Saccharomyces cerevisiae1.2 × 1045.8 × 1032.2 × 103Caenorhabditis elegans1.0 × 1052.2 × 1044.6 × 103Drosophila melanogaster1.2 × 1051.5 × 1048.2 × 103Mouse2.8 × 1092.3 × 1041.2 × 105Human3.3 × 1092.0 × 1041.7 × 105 Open table in a new tab This new sequence information pushed most scientists to fall into one of two camps. The ENCODE team and some others surmised that the additional transcribed but untranslated portions of mammalian genomes are like the dark matter of the universe: previously unrecognized but likely a major contributor to our relatively complex body plans and cognitive abilities [1ENCODE Project ConsortiumAn integrated encyclopedia of DNA elements in the human genome.Nature. 2012; 489: 57-74Crossref PubMed Scopus (11031) Google Scholar, 2Djebali S. et al.Landscape of transcription in human cells.Nature. 2012; 489: 101-108Crossref PubMed Scopus (3618) Google Scholar]. However, based primarily on previous arguments [3Doolittle W.F. Sapienza C. Selfish genes, the phenotype paradigm and genome evolution.Nature. 1980; 284: 601-603Crossref PubMed Scopus (1232) Google Scholar, 4Orgel L.E. Crick F.H. Selfish DNA: the ultimate parasite.Nature. 1980; 284: 604-607Crossref PubMed Scopus (1372) Google Scholar], those in the other camp believed that these additional sequences are mainly composed of nonfunctional 'junk' or 'selfish' DNA (TEs, repeats, and noncoding DNA), with little or nothing to do with our physical or cognitive complexities [5Doolittle W.F. Is junk DNA bunk? A critique of ENCODE.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 5294-5300Crossref PubMed Scopus (275) Google Scholar, 6Graur D. et al.On the immortality of television sets: "function" in the human genome according to the evolution-free gospel of ENCODE.Genome Biol. Evol. 2013; 5: 578-590Crossref PubMed Scopus (330) Google Scholar, 7Palazzo A.F. Gregory T.R. The case for junk DNA.PLoS Genet. 2014; 10: e1004351Crossref PubMed Scopus (151) Google Scholar]. Before getting into the arguments supporting the two sides, it is important that we first revisit a seemingly simple but nevertheless contentious issue: what defines function? At the heart of the term 'functional' is the definition of what constitutes a gene. Scientists used to, and a surprising number still do, define a gene as a sequence that encodes a protein. However, following the discovery of noncoding RNA genes, the basic definition was changed to a DNA sequence that encodes a functional product, although more complex definitions have been put forward to deal with issues such as overlapping and unrelated products produced from the same DNA sequence [2Djebali S. et al.Landscape of transcription in human cells.Nature. 2012; 489: 101-108Crossref PubMed Scopus (3618) Google Scholar, 8Gerstein M.B. et al.What is a gene, post-ENCODE? History and updated definition.Genome Res. 2007; 17: 669-681Crossref PubMed Scopus (478) Google Scholar]. This concept has become even more confusing, as many DNA sequences previously thought of as transcription factor-binding regulatory elements are also transcribed, and in most of the cases studied, the transcribed RNAs play important roles in processes such as transcription factor recruitment, DNA looping, chromatin composition, nuclear localization, etc. [9Quinodoz S. Guttman M. Long noncoding RNAs: an emerging link between gene regulation and nuclear organization.Trends Cell Biol. 2014; 24: 651-663Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 10Jandura A. Krause H.M. The new RNA world: growing evidence for long noncoding RNA functionality.Trends Genet. 2017; 33: 665-676Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 11Amaral P.P. et al.Genomic positional conservation identifies topological anchor point RNAs linked to developmental loci.Genome Biol. 2018; 19: 32Crossref PubMed Scopus (79) Google Scholar]. So, by the definition above, these sequences would also qualify as genes. These issues are discussed further in [2Djebali S. et al.Landscape of transcription in human cells.Nature. 2012; 489: 101-108Crossref PubMed Scopus (3618) Google Scholar, 12Kellis M. et al.Defining functional DNA elements in the human genome.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 6131-6138Crossref PubMed Scopus (466) Google Scholar]. This leads us to the term 'functional'. Historically, for a gene to be considered functional there had to be an observable phenotype when it was mutated. In most cases, however, this was, and continues to be, assessed under optimal laboratory conditions. We now know that this is much more complicated, as many genes with important functions can be compensated for by other genes with related functions (genetic redundancy). In this way, cells and organisms have evolved brilliant and extensive backup systems to deal with the numerous types of genetic and environmental disruptions that they may encounter. In the laboratory, with ideal temperature, food, space, and mating conditions, these backup plans are not needed nearly as often as in the wild where issues such as temperature swings, drought, mate availability, and the presence of predators are all major issues. Most of our inbred 'wild type' and genetically altered model organism strains would never survive or propagate effectively in the wild. Yet, there has been a tendency to assume that genes without an obvious 'in-lab' phenotype are dispensable or even 'nonfunctional'. In yeast, only ∼1/3 of coding genes are required for viability under standard growth conditions. While this low number seemed surprising at first, we now know that combining the deletions of two 'nonrequired' genes can result in 'synthetic' lethality. The numbers of required and synthetically lethal genes also vary depending on factors such as growth media composition, temperature, and a multitude of other stresses. Indeed, a more in-depth analysis of the yeast gene deletion set, grown in a diverse set of conditions, revealed phenotypes for 97% of them [13Hillenmeyer M.E. et al.The chemical genomic portrait of yeast: uncovering a phenotype for all genes.Science. 2008; 320: 362-365Crossref PubMed Scopus (747) Google Scholar]. Similar to yeast, the initial estimation of essential gene numbers for Drosophila coding genes suggested that only 1/4–1/3 are required for viability under standard laboratory conditions [14Spradling A.C. et al.The Berkeley Drosophila Genome Project gene disruption project: single P-element insertions mutating 25% of vital Drosophila genes.Genetics. 1999; 153: 135-177Crossref PubMed Google Scholar, 15Chen S. et al.New genes in Drosophila quickly become essential.Science. 2010; 330: 1682-1685Crossref PubMed Scopus (203) Google Scholar], and when human cells are grown in culture, only ∼10% of coding genes were found to be essential [16Wang T. et al.Identification and characterization of essential genes in the human genome.Science. 2015; 350: 1096-1101Crossref PubMed Scopus (945) Google Scholar]. Clearly though, this does not mean that the genes not required in these utopian environments are nonfunctional. In many cases, the importance of a gene may be better gauged by the depth and sophistication of built-in redundancies. Some examples of backup redundancies include the ability of Drosophila segmentation genes to adapt to large changes in the expression of other segmentation genes [17Namba R. et al.Drosophila embryonic pattern repair: how embryos respond to bicoid dosage alteration.Development. 1997; 124: 1393-1403Crossref PubMed Google Scholar, 18Hughes S.C. Krause H.M. Establishment and maintenance of parasegmental compartments.Development. 2001; 128: 1109-1118PubMed Google Scholar], or by paralogous homologues in Hox gene clusters [19Wagner A. Genetic redundancy caused by gene duplications and its evolution in networks of transcriptional regulators.Biol. Cybern. 1996; 74: 557-567Crossref PubMed Scopus (36) Google Scholar]. There are many kinds of noncoding RNAs (ncRNAs), such as the well-studied ribosomal and transfer RNAs. Among the more recently recognized ncRNAs are the relatively short miRNAs, small interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs). Due to similarities in their sizes, structures, and molecular functions, scientists have been able to identify and assess many of these new RNA functions in a relatively quick and efficient manner, showing that the majority serve clear and important roles [20Bartel D.P. Metazoan microRNAs.Cell. 2018; 173: 20-51Abstract Full Text Full Text PDF PubMed Scopus (1777) Google Scholar]. However, the most diverse and genetically abundant class of ncRNAs, referred to as long noncoding RNAs (lncRNAs), have not been so easy to tackle. Their diversity in terms of length, numbers, structures, distributions, and functions is partly to blame for the lag in attention and understanding. However, the prior widely held perception that they are predominantly junk is also factored in. Some examples of the shadow cast by the assumption that most lncRNA genes are nonfunctional are found in early studies of pioneer lncRNAs, in which some groups concluded prematurely that the RNAs themselves were nonfunctional and that it was simply the act of transcribing them that was important. One of the first examples was the H19 lncRNA, which regulates imprinting and contributes to numerous cancers when misexpressed [21Lecerf C. et al.The long non-coding RNA H19: an active player with multiple facets to sustain the hallmarks of cancer.Cell Mol. Life Sci. 2019; (Published online July 23, 2019)https://doi.org/10.1007/s00018-019-03240-zCrossref PubMed Scopus (61) Google Scholar]. Based on a number of deletion constructs and cell-based assays, it was concluded that it was the H19 promoter and its activity that was required and not the RNA itself [22Jones B.K. et al.Igf2 imprinting does not require its own DNA methylation or H19 RNA.Genes Dev. 1998; 12: 2200-2207Crossref PubMed Scopus (90) Google Scholar]. Similar studies initially suggested that the lncRNAs Kcnq1ot1 and Airn were also not themselves required (reviewed in [23Pauler F.M. et al.Silencing by imprinted noncoding RNAs: is transcription the answer?.Trends Genet. 2007; 23: 284-292Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 24Kornienko A.E. et al.Gene regulation by the act of long non-coding RNA transcription.BMC Biol. 2013; 11: 59Crossref PubMed Scopus (562) Google Scholar]). Although a PubMed search reveals that there have since been more than a thousand publications on the functions of these lncRNAs, both in cis and trans, many are still only aware of the earlier dismissive publications. Similar studies and conclusions were made with Hox locus lncRNAs. Although it was suggested some time ago [25Nasiadka A. et al.Anterior-posterior patterning in the Drosophila embryo.Adv. Dev. Biol. Biochem. 2002; 12: 155-204Crossref Scopus (48) Google Scholar] that these lncRNAs could be used to coordinate the proximal to distal recruitment of epigenetic-regulating transcription factors, similar to the mechanism that Rox1 had just been shown to recruit chromatin components, several groups conducted experiments that, as with H19, Kcnq1ot1, and Airn, suggested that it was the act of transcription, and not the RNAs themselves, that was important [26Hogga I. Karch F. Transcription through the iab-7 cis-regulatory domain of the bithorax complex interferes with maintenance of Polycomb-mediated silencing.Development. 2002; 129: 4915-4922PubMed Google Scholar, 27Rank G. et al.Transcription through intergenic chromosomal memory elements of the Drosophila bithorax complex correlates with an epigenetic switch.Mol. Cell. Biol. 2002; 22: 8026-8034Crossref PubMed Scopus (128) Google Scholar, 28Schmitt S. et al.Intergenic transcription through a polycomb group response element counteracts silencing.Genes Dev. 2005; 19: 697-708Crossref PubMed Scopus (198) Google Scholar, 29Petruk S. et al.Transcription of bxd noncoding RNAs promoted by trithorax represses Ubx in cis by transcriptional interference.Cell. 2006; 127: 1209-1221Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 30Sessa L. et al.Noncoding RNA synthesis and loss of Polycomb group repression accompanies the colinear activation of the human HOXA cluster.RNA. 2007; 13: 223-239Crossref PubMed Scopus (105) Google Scholar]. Again, we now know that many mammalian Hox complex lncRNAs do in fact have important functions in coordinating the proximal to distal epigenetic states across these complexes with anterior to posterior Hox gene expression patterns [31De Kumar B. Krumlauf R. HOXs and lincRNAs: two sides of the same coin.Sci. Adv. 2016; 2: e1501402Crossref PubMed Scopus (38) Google Scholar]. It seems likely that this will also turn out to be true for Drosophila Hox lncRNAs, as a relatively recent study has shown that simply transcribing through Polycomb response elements (PREs) is not sufficient for mediating subsequent epigenetic states, as had previously been thought [32Erokhin M. et al.Transcriptional read-through is not sufficient to induce an epigenetic switch in the silencing activity of Polycomb response elements.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 14930-14935Crossref PubMed Scopus (27) Google Scholar]. Some of the first CRISPR-based lncRNA knockout studies have also led some authors and readers to negative takes on lncRNA gene functionality. These publications are similar, in that a number of embryonically expressed lncRNAs failed to yield obvious or overtly significant phenotypes when deleted [33Schor I.E. et al.Non-coding RNA expression, function, and variation during Drosophila embryogenesis.Curr. Biol. 2018; 28: 3547-3561Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 34Goudarzi M. et al.Individual long non-coding RNAs have no overt functions in zebrafish embryogenesis, viability and fertility.eLife. 2019; 8: e40815Crossref PubMed Scopus (48) Google Scholar]. However, as pointed out earlier, these modest effects may be due to functional redundancies and/or the nature of the assays. As discussed below, it may also be related to the fact that most lncRNAs appear to have evolved relatively recently, with the majority of their expression and functions occurring in male reproductive tissues and the brain. The basic animal body design, however, is largely controlled by ancient and highly conserved genetic pathways that were derived before the majority of lncRNAs evolved [35Domazet-Loso T. Tautz D. A phylogenetically based transcriptome age index mirrors ontogenetic divergence patterns.Nature. 2010; 468: 815-818Crossref PubMed Scopus (268) Google Scholar, 36Kalinka A.T. et al.Gene expression divergence recapitulates the developmental hourglass model.Nature. 2010; 468: 811-814Crossref PubMed Scopus (289) Google Scholar, 37Irie N. Kuratani S. Comparative transcriptome analysis reveals vertebrate phylotypic period during organogenesis.Nat. Commun. 2011; 2: 248Crossref PubMed Scopus (194) Google Scholar]. This likely explains the relative paucity of lncRNA expression and major roles during these earlier stages of development, although this is not to say that lncRNAs do not also have important functions at these times, as clearly evidenced by the roles of Rox/Xist, Hox, and let-7, for example. While ENCODE and modENCODE studies have demonstrated high numbers of lncRNAs expressed in metazoan genomes, and suggested relatively high levels of functionality, they also provided data supporting the alternative view, noting that most of the detected lncRNAs were very low in abundance. However, this latter observation can largely be attributed to issues such as the use of cultured cell lines and the isolation methods used. For example, as noted in the ENCODE publication [2Djebali S. et al.Landscape of transcription in human cells.Nature. 2012; 489: 101-108Crossref PubMed Scopus (3618) Google Scholar], transcripts annotated as very low or undetected in some cell lines could be quite abundant in others, or in tissues or stages of development not represented by the chosen cell lines. RNA-Seq approaches also tend to average out spatially and temporally restricted patterns, which locally, may be relatively high. Another key issue is that these studies made use of poly(A)-containing RNA selection, which discards the 50% or more of lncRNAs that are not polyadenylated [12Kellis M. et al.Defining functional DNA elements in the human genome.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 6131-6138Crossref PubMed Scopus (466) Google Scholar, 38Cheng J. et al.Transcriptional maps of 10 human chromosomes at 5-nucleotide resolution.Science. 2005; 308: 1149-1154Crossref PubMed Scopus (973) Google Scholar]. In situ hybridization (ISH), however, does not discriminate between RNAs with alternative 3ʹ ends. For example, an analysis of over 100 Drosophila lncRNAs, all previously annotated as very low in expression or undetected, showed that all were expressed at similar levels to coding genes, although not usually as broadly or as early in development [39Clark A.G. et al.Evolution of genes and genomes on the Drosophila phylogeny.Nature. 2007; 450: 203-218Crossref PubMed Scopus (1524) Google Scholar]. ISH-based studies also do not have to deal with RNA extraction issues; one study on the Neat1 lncRNA, which nucleates paraspeckles, showed that it aggregates in the interphase of the phenol/chloroform mixtures used to extract the RNA, and that >20 times more RNA could be extracted after repeated passage of the extract through a syringe needle [40Chujo T. et al.Unusual semi-extractability as a hallmark of nuclear body-associated architectural noncoding RNAs.EMBO J. 2017; 36: 1447-1462Crossref PubMed Scopus (68) Google Scholar]. Another recent study has shown that the use of 100 mM ammonium acetate appears to be a more simple and effective way to dissociate similar liquid–liquid phase-separated (LLPS) structures and to fully recover their RNA cargoes [41Jain A. Vale R.D. RNA phase transitions in repeat expansion disorders.Nature. 2017; 546: 243-247Crossref PubMed Scopus (410) Google Scholar]. Further analyses will be required to determine how LLPS solubility issues affect the extractability of other RNA cargoes. In terms of conservation, many junk advocates were expecting functional lncRNAs to exhibit higher levels of conservation, perhaps even approaching those of coding genes [6Graur D. et al.On the immortality of television sets: "function" in the human genome according to the evolution-free gospel of ENCODE.Genome Biol. Evol. 2013; 5: 578-590Crossref PubMed Scopus (330) Google Scholar, 42Lindblad-Toh K. et al.A high-resolution map of human evolutionary constraint using 29 mammals.Nature. 2011; 478: 476-482Crossref PubMed Scopus (757) Google Scholar], but this is not a fair expectation given that ncRNAs do not have to deal with protein coding issues such as codon usage, reading frame, folding, stability, or function. Because of this, functional elements within ncRNAs are also relatively free to shuffle around within the gene, which makes aligning their sequences much more challenging. This becomes even more challenging if the complementary sequences of conserved double-strand structures are free to drift far away from one another. Indeed, recent genome-wide RNA–RNA crosslinking studies have shown that up to 40% of double-stranded structures have counterpart sequences that are more than 200 nucleotides away, which is a larger span than what has been used until now to identify potential palindromic counterparts [43Lu Z. et al.RNA duplex map in living cells reveals higher-order transcriptome structure.Cell. 2016; 165: 1267-1279Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar]. Importantly, this problem is further magnified for the many interactions that were found in the crosslinking studies to occur in trans. This study also confirmed that many base pairs in these secondary structures are relatively free to change, but are quickly compensated for by corresponding changes in the paired nucleotides. Overall, 25% of the aligned double strand helix-forming sequences were found to be conserved between amniotes (mammals, birds, reptiles), with compensating covariations averaging 46% [43Lu Z. et al.RNA duplex map in living cells reveals higher-order transcriptome structure.Cell. 2016; 165: 1267-1279Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar]. These numbers are strikingly high given the ∼300 million years of divergence among these species. Previous studies using conventional alignment methods had found that homologous lncRNA sequences or structures are generally undetectable beyond 50 million years of species divergence [44Ulitsky I. Evolution to the rescue: using comparative genomics to understand long non-coding RNAs.Nat. Rev. Genet. 2016; 17: 601-614Crossref PubMed Scopus (336) Google Scholar]. Although homologous lncRNA gene sequences are difficult to detect beyond 50 million years, other studies have shown that syntenic lncRNA promoters and exon/intron boundaries were actually similarly or even more conserved than those of coding genes [2Djebali S. et al.Landscape of transcription in human cells.Nature. 2012; 489: 101-108Crossref PubMed Scopus (3618) Google Scholar, 44Ulitsky I. Evolution to the rescue: using comparative genomics to understand long non-coding RNAs.Nat. Rev. Genet. 2016; 17: 601-614Crossref PubMed Scopus (336) Google Scholar, 45Carninci P. et al.The transcriptional landscape of the mammalian genome.Science. 2005; 309: 1559-1563Crossref PubMed Scopus (2794) Google Scholar, 46Ponjavic J. et al.Functionality or transcriptional noise? Evidence for selection within long noncoding RNAs.Genome Res. 2007; 17: 556-565Crossref PubMed Scopus (564) Google Scholar]. Furthermore, some of these orthologs were shown to rescue one another, despite the lack of obvious sequence or structure conservation [44Ulitsky I. Evolution to the rescue: using comparative genomics to understand long non-coding RNAs.Nat. Rev. Genet. 2016; 17: 601-614Crossref PubMed Scopus (336) Google Scholar, 47Ulitsky I. et al.Conserved function of lincRNAs in vertebrate embryonic development despite rapid sequence evolution.Cell. 2011; 147: 1537-1550Abstract Full Text Full Text PDF PubMed Scopus (874) Google Scholar]. A particularly good example of lncRNAs with conserved synteny and promoters, and relatively degenerate sequences, are those referred to as topological anchor point RNAs (tapRNAs), which appear to control chromatin looping and topology [11Amaral P.P. et al.Genomic positional conservation identifies topological anchor point RNAs linked to developmental loci.Genome Biol. 2018; 19: 32Crossref PubMed Scopus (79) Google Scholar]. As with guide RNAs that have been shown to recruit transcription factors to DNA, this may not be too surprising, as sequences that bind to DNA in cis will retain complementarity no matter how much the counterpart DNA sequence changes and the sequences and structures that bind proteins appear to be relatively degenerate and promiscuous [10Jandura A. Krause H.M. The new RNA world: growing evidence for long noncoding RNA functionality.Trends Genet. 2017; 33: 665-676Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 11Amaral P.P. et al.Genomic positional conservation identifies topological anchor point RNAs linked to developmental loci.Genome Biol. 2018; 19: 32Crossref PubMed Scopus (79) Google Scholar, 48Krause H.M. New and prospective roles for lncRNAs in organelle formation and function.Trends Genet. 2018; 34: 736-745Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar]. As mentioned earlier, the linker sequences that join these functional motifs are also unlikely to be constrained by selective evolutionary pressure. While all of these rationales can be given for poor lncRNA gene collinearity and conservation, a comparison between the relatively well annotated Drosophila melanogaster coding and lncRNA genes shows that lncRNA genes are actually surprisingly well conserved between Drosophila species (Figure 1). Furthermore, as would be expected if functional, lncRNA gene exons are more conserved than introns. In some cases, the overall degree of lncRNA gene and exon conservation significantly exceed those of the average coding genes. It should be noted though that Drosophila contains relatively few lncRNA genes compared with humans, suggesting that their rate of evolution may be much slower and, thus, their conservation and functionality levels may be proportionally higher. Another possible concern about lncRNA functionality that is seldom brought up is the apparent paucity of reports on natural and forward genetic mutations in lncRNA gene loci that result in phenotypes. However, this can also be attributed to many of the considerations that affect conservation levels, such as the increased ability, relative to coding genes, to incur sequence changes without significantly affecting overall structure or function. In many cases, researchers have also likely concluded that mutations affecting functions in ncDNA were in key regulatory or structural elements associated with nearby coding genes, or simply ignored them due to our past 'coding gene-centric' ways of thinking. However, it should be pointed out that the majority of genome-wide association study-identified disease loci are within ncDNA sequences, many of which are or may be transcribed [1ENCODE Project ConsortiumAn integrated encyclopedia of DNA elements in the human genome.Nature. 2012; 489: 57-74Crossref PubMed Scopus (11031) Google Scholar, 49Khurana E. et al.Integrative annotation of variants from 1092 humans: application to cancer genomics.Science. 2013; 342: 1235587Crossref PubMed Scopus (271) Google Scholar, 50Brown J.B. et al.Diversity and dynamics of the Drosophila transcriptome.Nature. 2014; 512: 393-399Crossref PubMed Scopus (414) Google Scholar, 51Zhang F. Lupski J.R. Non-coding genetic variants in human disease.Hum Mol Genet. 2015; 24: R102-R110Crossref PubMed Scopus (286) Google Scholar, 52Schonrock N. et al.Seq and y