Mechanisms of long noncoding RNA function in development and disease (original) (raw)
Introduction
It has long been known that several classes of non-protein-coding RNA molecules exert important cellular functions. For instance, ribosomal RNAs (rRNAs) are essential elements of the translation machinery and small nuclear RNAs (snRNAs) are required for splicing of nascent RNA transcripts. Also, various classes of small (around 20–30 nucleotides) noncoding RNAs such as micro (mi)RNAs, small inhibitory (si)RNAs or PIWI interacting (pi)RNAs are well known as gene silencers. With the recent advent of massive parallel sequencing techniques, however, it has been observed that a tremendously high portion, approximately 70 %, of the genome is transcribed in various contexts and cell types [[1](/article/10.1007/s00018-016-2174-5#ref-CR1 "Okazaki Y, Furuno M, Kasukawa T et al (2002) Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs. Nature 420:563–573. doi: 10.1038/nature01266
"), [2](/article/10.1007/s00018-016-2174-5#ref-CR2 "Djebali S, Davis CA, Merkel A et al (2012) Landscape of transcription in human cells. Nature 489:101–108. doi:
10.1038/nature11233
")\]. A large proportion of these newly detected RNA transcripts are structurally indistinguishable from protein-coding and processed messenger RNAs (mRNAs). They tend to be expressed at a very low level and have little to no protein-coding potential. This subclass of noncoding transcripts of variable length and function is collectively referred to as long noncoding RNAs (lncRNAs).A plethora of biological tissues, organs, pathological samples and cultured cells have been analyzed for noncoding RNA expression, and it is clear that these molecules are omnipresent. Apparently, defining noncoding RNA function has proven more challenging than detecting them, as the number of reports showing comprehensive functional data is far smaller than those describing their identification in various contexts. The flexibility of RNA transcripts and their ability to fold into complex 3D-conformations enables them to form specific interactions with proteins. They can interact with RNA or DNA molecules via base pairing, even with double-stranded DNA, and form networks with DNA, protein complexes and RNA molecules, illustrating their large potential as an important player with many biological functions. In this review, we will discuss mechanisms of lncRNA functions with a focus on their role in development and disease (Table 1).
Table 1 List of lncRNAs and their main features mentioned in this review
Molecular and genetic structure of LncRNAs
Like mRNAs, lncRNAs are transcribed by Polymerase II, mostly 5′-capped, polyadenylated and spliced, though on average they contain a lower number of exons than mRNAs and their expression level assessed across different tissues is lower [[3](/article/10.1007/s00018-016-2174-5#ref-CR3 "Derrien T, Johnson R, Bussotti G et al (2012) The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome Res 22:1775–1789. doi: 10.1101/gr.132159.111
"), [4](/article/10.1007/s00018-016-2174-5#ref-CR4 "Cabili MN, Trapnell C, Goff L et al (2011) Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses. Genes Dev 25:1915–1927. doi:
10.1101/gad.17446611
")\]. There are various algorithms calculating the coding probability based on the length of a potential open reading frame (ORF), the similarity of such an ORF to known protein-coding genes, frequency of in-frame nucleotide hexamers or other empirical sequence features \[[5](/article/10.1007/s00018-016-2174-5#ref-CR5 "Guo X, Gao L, Wang Y et al (2015) Advances in long noncoding RNAs: identification, structure prediction and function annotation. Brief Funct Genomics. doi:
10.1093/bfgp/elv022
")\]. In general, RNA transcripts containing short (<100 nt) non-conserved ORFs, which have no homology to known peptide sequences and do not match to peptides identified in mass spectrometry screens are considered noncoding \[[6](/article/10.1007/s00018-016-2174-5#ref-CR6 "Harrow J, Frankish A, Gonzalez JM et al (2012) GENCODE: the reference human genome annotation for The ENCODE Project. Genome Res 22:1760–1774. doi:
10.1101/gr.135350.111
")\]. Interestingly, the majority of lncRNAs are associated with ribosomes \[[7](/article/10.1007/s00018-016-2174-5#ref-CR7 "Ingolia NT, Lareau LF, Weissman JS (2011) Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147:789–802. doi:
10.1016/j.cell.2011.10.002
"), [8](/article/10.1007/s00018-016-2174-5#ref-CR8 "Ruiz-Orera J, Messeguer X, Subirana JA, Alba MM (2014) Long non-coding RNAs as a source of new peptides. elife 3:e03523. doi:
10.7554/eLife.03523
")\], though they do not show the characteristic release of ribosomes \[[9](/article/10.1007/s00018-016-2174-5#ref-CR9 "Guttman M, Russell P, Ingolia NT et al (2013) Ribosome profiling provides evidence that large noncoding RNAs do not encode proteins. Cell 154:240–251. doi:
10.1016/j.cell.2013.06.009
")\] or the typical 3-nucleotide phasing corresponding to codons of an ORF \[[10](/article/10.1007/s00018-016-2174-5#ref-CR10 "Bánfai B, Jia H, Khatun J et al (2012) Long noncoding RNAs are rarely translated in two human cell lines. Genome Res 22:1646–1657. doi:
10.1101/gr.134767.111
"), [11](/article/10.1007/s00018-016-2174-5#ref-CR11 "Bazzini AA, Johnstone TG, Christiano R et al (2014) Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation. EMBO J. doi:
10.1002/embj.201488411
")\]. However, in rare instances, functional oligopeptides have been found to be translated from putative lncRNAs \[[12](/article/10.1007/s00018-016-2174-5#ref-CR12 "Frith MC, Forrest AR, Nourbakhsh E et al (2006) The abundance of short proteins in the mammalian proteome. PLoS Genet 2:e52. doi:
10.1371/journal.pgen.0020052
")–[14](/article/10.1007/s00018-016-2174-5#ref-CR14 "Pauli A, Norris ML, Valen E et al (2014) Toddler: an embryonic signal that promotes cell movement via Apelin receptors. Science 343:1248636. doi:
10.1126/science.1248636
")\].Deep-sequencing experiments revealed many examples of genes producing both protein-coding and noncoding transcripts by alternative splicing. However, so far, only very few reports demonstrate a functional role for both the noncoding RNA(s) and the protein(s) encoded by transcripts derived from the same gene [[15](/article/10.1007/s00018-016-2174-5#ref-CR15 "Yan Y, Cooper C, Hamedani MK et al (2015) The steroid receptor RNA activator protein (SRAP) controls cancer cell migration/motility. FEBS Lett 589:4010–4018. doi: 10.1016/j.febslet.2015.11.007
"), [16](/article/10.1007/s00018-016-2174-5#ref-CR16 "Rupaimoole R, Lee J, Haemmerle M et al (2015) Long noncoding RNA ceruloplasmin promotes cancer growth by altering glycolysis. Cell Rep 13:2395–2402. doi:
10.1016/j.celrep.2015.11.047
")\]. It is tempting to speculate that such dual usage of transcripts is more frequent than anticipated.RNA molecules have the potential to form highly structured macromolecules by folding into double-stranded stems, single-stranded loops and bulges, which again can fold further into three-dimensional structures, allowing for the potential formation of complex shapes. So far, the structure of only a few RNAs has been experimentally determined using a combination of chemical assays, and by determining the accessibility of base-paired or single-stranded RNA by various RNases [[17](/article/10.1007/s00018-016-2174-5#ref-CR17 "Novikova IV, Hennelly SP, Tung C-S, Sanbonmatsu KY (2013) Rise of the RNA machines: exploring the structure of long non-coding RNAs. J Mol Biol 425:3731–3746. doi: 10.1016/j.jmb.2013.02.030
"), [18](/article/10.1007/s00018-016-2174-5#ref-CR18 "Weeks KM (2015) Review toward all RNA structures, concisely. Biopolymers 103:438–448. doi:
10.1002/bip.22601
")\]. However, these methods still have the limitation that they can only reveal the secondary, but not the tertiary (3D) structure. In addition, computational predictions are only beginning to provide reliable results, but program learning from experimental data might improve the predictions and more closely mirror experimental observations. This process is accelerated by recently developed techniques combined with high-throughput sequencing, such as SHAPE-MaP and icSHAPE \[[19](/article/10.1007/s00018-016-2174-5#ref-CR19 "Smola MJ, Rice GM, Busan S et al (2015) Selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis. Nat Protoc 10:1643–1669. doi:
10.1038/nprot.2015.103
"), [20](/article/10.1007/s00018-016-2174-5#ref-CR20 "Spitale RC, Flynn RA, Zhang QC et al (2015) Structural imprints in vivo decode RNA regulatory mechanisms. Nature. doi:
10.1038/nature14263
")\].The similarity between mRNA-encoding and lncRNA genes is furthermore reflected by the chromatin signatures at the genomic regions from where they are transcribed. Their transcriptional start site is, in most cases, marked by the H3K4me3 histone modification and the transcribed region by the H3K36me3 mark, no matter if the lncRNA originates from its own promoter or from an enhancer. However, these histone marks are less prominent than observed at mRNA coding genes, whereas H3K4me1, a characteristic mark of enhancers, tends to be more prominent at genomic regions encoding lncRNAs [[2](/article/10.1007/s00018-016-2174-5#ref-CR2 "Djebali S, Davis CA, Merkel A et al (2012) Landscape of transcription in human cells. Nature 489:101–108. doi: 10.1038/nature11233
"), [21](/article/10.1007/s00018-016-2174-5#ref-CR21 "Guttman M, Amit I, Garber M et al (2009) Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature 458:223–227. doi:
10.1038/nature07672
")–[23](/article/10.1007/s00018-016-2174-5#ref-CR23 "Marques AC, Hughes J, Graham B et al (2013) Chromatin signatures at transcriptional start sites separate two equally populated yet distinct classes of intergenic long noncoding RNAs. Genome Biol 14:R131. doi:
10.1186/gb-2013-14-11-r131
")\]. As expected for transcribed regions, lncRNA promoters correspond with DNaseI hypersensitive sites, which indicates accessible chromatin \[[24](/article/10.1007/s00018-016-2174-5#ref-CR24 "ENCODE Project Consortium (2012) An integrated encyclopedia of DNA elements in the human genome. Nature 489:57–74. doi:
10.1038/nature11247
"), [25](/article/10.1007/s00018-016-2174-5#ref-CR25 "Iyer MK, Niknafs YS, Malik R et al (2015) The landscape of long noncoding RNAs in the human transcriptome. Nat Genet 47:199–208. doi:
10.1038/ng.3192
")\].So far, the most commonly used categorization of the highly heterogeneous class of lncRNAs is their position in the genome relative to protein-coding genes (Fig. 1). LncRNAs can be intergenic (lincRNAs) or divergently transcribed from the same promoter as a protein-coding gene (pancRNAs). LincRNAs expressed from a promoter (with high H3K4me3 to H3K4me1 ratio) are classified as promoter-associated lncRNAs (plncRNAs). However, lncRNAs can also be transcribed from enhancers and are then termed eRNAs. They are mostly transcribed in both directions, in contrast to enhancer-associated lncRNAs (elncRNAs), which are unidirectionally transcribed from a promoter with low H3K4me3 to H3K4me1 ratio [[23](/article/10.1007/s00018-016-2174-5#ref-CR23 "Marques AC, Hughes J, Graham B et al (2013) Chromatin signatures at transcriptional start sites separate two equally populated yet distinct classes of intergenic long noncoding RNAs. Genome Biol 14:R131. doi: 10.1186/gb-2013-14-11-r131
")\]. Transcription of lncRNAs can also originate from within introns, or overlap with other transcripts in sense or antisense orientation \[[3](/article/10.1007/s00018-016-2174-5#ref-CR3 "Derrien T, Johnson R, Bussotti G et al (2012) The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome Res 22:1775–1789. doi:
10.1101/gr.132159.111
")\]. Some lncRNAs are generated by backsplicing from introns of mRNAs or other lncRNAs and are thus circular (circRNAs) \[[26](/article/10.1007/s00018-016-2174-5#ref-CR26 "Hansen TB, Jensen TI, Clausen BH et al (2013) Natural RNA circles function as efficient microRNA sponges. Nature 495:384–388. doi:
10.1038/nature11993
"), [27](/article/10.1007/s00018-016-2174-5#ref-CR27 "Memczak S, Jens M, Elefsinioti A et al (2013) Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 495:333–338. doi:
10.1038/nature11928
")\].Fig. 1
Classification of lncRNAs according to their position relative to neighboring gene(s). a Divergently transcribed lncRNA originating from the same promoter region as the adjacent (usually protein coding) gene, but from the opposite strand; b convergently transcribed genes encoded on opposite strands and facing each other; c intergenic (or intervening) lncRNA (or lincRNA) located distant from other genes (usually >10 kb); d examples for various cases of lncRNAs overlapping with other genes on the same or the opposite strand; e enhancer RNAs expressed as uni- or bidirectional transcripts; f LncRNA transcribed from an intron of another gene; g lncRNA hosting a miRNA. Noncoding genes are shown in green, protein-coding genes in orange
Genome-wide sequencing of RNA species isolated from cytosolic or nuclear fractions of cells has shown that the majority of lncRNAs tend to be localized in the nucleus or are associated with chromatin, while a considerable fraction localizes to the cytoplasm [[3](/article/10.1007/s00018-016-2174-5#ref-CR3 "Derrien T, Johnson R, Bussotti G et al (2012) The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome Res 22:1775–1789. doi: 10.1101/gr.132159.111
"), [28](/article/10.1007/s00018-016-2174-5#ref-CR28 "Werner MS, Ruthenburg AJ (2015) Nuclear fractionation reveals thousands of chromatin-tethered noncoding RNAs adjacent to active genes. Cell Rep 12:1089–1098. doi:
10.1016/j.celrep.2015.07.033
"), [29](/article/10.1007/s00018-016-2174-5#ref-CR29 "Herman RC, Williams JG, Penman S (1976) Message and non-message sequences adjacent to poly(A) in steady state heterogeneous nuclear RNA of HeLa cells. Cell 7:429–437")\]. The subcellular localization is a good indication of the putative function of a lncRNA, since in contrast to protein-coding mRNAs, lncRNAs can already function while transcription is occurring. In fact, many nuclear and, in particular, chromatin-retained lncRNAs co-regulate transcription and/or chromatin structure at or close to their site of transcription, i.e. in _cis_.Conservation and evolutionary aspects of LncRNA
The genomic sequences of lncRNAs are, in general, less conserved than exons, but more than introns of protein-coding genes, pointing towards a rapid evolution with moderate constraints [[21](/article/10.1007/s00018-016-2174-5#ref-CR21 "Guttman M, Amit I, Garber M et al (2009) Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature 458:223–227. doi: 10.1038/nature07672
"), [30](/article/10.1007/s00018-016-2174-5#ref-CR30 "Yue F, Cheng Y, Breschi A et al (2014) A comparative encyclopedia of DNA elements in the mouse genome. Nature 515:355–364. doi:
10.1038/nature13992
")\]. In contrast to the actual sequence, splice sites of lncRNAs have been found to be more stable during evolution \[[31](/article/10.1007/s00018-016-2174-5#ref-CR31 "Haerty W, Ponting CP (2015) Unexpected selection to retain high GC content and splicing enhancers within exons of multiexonic lncRNA loci. RNA 21:333–346. doi:
10.1261/rna.047324.114
"), [32](/article/10.1007/s00018-016-2174-5#ref-CR32 "Nitsche A, Rose D, Fasold M et al (2015) Comparison of splice sites reveals that long noncoding RNAs are evolutionarily well conserved. RNA 21:801–812. doi:
10.1261/rna.046342.114
")\]. Despite poor RNA sequence conservation, lncRNAs have frequently been identified across species in syntenic genomic regions \[[33](/article/10.1007/s00018-016-2174-5#ref-CR33 "Ulitsky I, Shkumatava A, Jan CH et al (2011) Conserved function of lincRNAs in vertebrate embryonic development despite rapid sequence evolution. Cell 147:1537–1550. doi:
10.1016/j.cell.2011.11.055
"), [34](/article/10.1007/s00018-016-2174-5#ref-CR34 "Necsulea A, Soumillon M, Warnefors M et al (2014) The evolution of lncRNA repertoires and expression patterns in tetrapods. Nature 505:635–640. doi:
10.1038/nature12943
")\]. It is therefore quite likely that evolutionary conservation of lncRNAs is embodied by a conserved 3D structure, although this is difficult to assess with current methods. A further layer of conservation is functional conservation between lncRNAs playing equivalent roles in particular biological settings \[[35](/article/10.1007/s00018-016-2174-5#ref-CR35 "Diederichs S (2014) The four dimensions of noncoding RNA conservation. Trends Genet 30:121–123. doi:
10.1016/j.tig.2014.01.004
")\].A different explanation for low conservation of lncRNAs is the increasing number of such transcripts in increasingly complex species. This finding, in combination with the observed highly tissue-specific expression of many lncRNAs, suggests that lncRNAs might be key molecules promoting species-specific features and organ complexity [[36](/article/10.1007/s00018-016-2174-5#ref-CR36 "Mercer TR, Dinger ME, Sunkin SM et al (2008) Specific expression of long noncoding RNAs in the mouse brain. Proc Natl Acad Sci USA 105:716–721. doi: 10.1073/pnas.0706729105
")–[38](/article/10.1007/s00018-016-2174-5#ref-CR38 "Grote P, Herrmann BG (2015) Long noncoding RNAs in organogenesis: making the difference. Trends Genet 31:329–335. doi:
10.1016/j.tig.2015.02.002
")\]. Thus, lncRNAs very likely have played important roles in the evolution of complex organisms.LncRNAs regulate gene and genome activity
The best-described function for lncRNAs in the nucleus is their role in regulating gene and genome activity on various levels (Fig. 2a–e). Many possible mechanisms by which lncRNAs influence chromatin modifications and chromatin structure, and thereby influence transcription or other chromatin-related functions by epigenetic mechanisms have been investigated. In the following section we will address different levels of gene and genome regulation separately.
Fig. 2
Schematic representation of cellular mechanisms involving lncRNAs. a LncRNA transcripts evicting proteins from chromatin; here, pancRNAs prevent DNMT from methylating DNA in their promoter region, thereby ensuring mRNA transcription. b LncRNAs recruiting the Mediator complex to an enhancer region, stabilizing loop formation and transcription of the associated gene. c LncRNAs transcribed from an enhancer region interfering with enhancer-promoter contact, thereby inhibiting transcription of the protein-coding gene. d LncRNA recruiting proteins, such as chromatin-modifying complexes to specific target sites in the genome, e.g. via DNA-RNA triplex formation. e LncRNA acting as scaffold linking different proteins required for concerted action. f LncRNA binding and sequestering proteins to prevent or attenuate their action, e.g. binding to mRNAs (left); circRNA sequestering miRNAs to prevent their binding to mRNAs (right). g Example of a lncRNA changing the splicing pattern by binding to a primary RNA transcript. h LncRNA stabilizing a mRNA by recruiting proteins such as STAU1, thereby preventing degradation
Histone modifications regulated by LncRNAs
Prominent examples of histone-modifying complexes interacting with lncRNAs are the two polycomb repressive complexes, PRC1 and, in particular PRC2, which mediates methylation of lysine 27 on histone 3 (H3K27me), a histone mark associated with repressed or poised genetic loci. The first report of an interaction of PRC2 with a noncoding RNA came from studies on X-chromosome inactivation in mammals, which involves X-inactive specific transcript (Xist), a lncRNA that is highly expressed from the inactive X-chromosomes in females (Xi) and recruits PRC2 to the Xi to silence gene expression [[39](/article/10.1007/s00018-016-2174-5#ref-CR39 "Plath K, Fang J, Mlynarczyk-Evans SK et al (2003) Role of histone H3 lysine 27 methylation in X inactivation. Science 300:131–135. doi: 10.1126/science.1084274
")–[41](/article/10.1007/s00018-016-2174-5#ref-CR41 "Zhao J, Sun BK, Erwin JA et al (2008) Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome. Science 322:750–756. doi:
10.1126/science.1163045
")\].Another prominent example is Hotair, a conserved lncRNA that is transcribed from within the HoxC gene cluster. Hotair was shown to play a repressive role at the HoxD locus by interacting with PRC2. In addition, Hotair also interacts with the histone H3K4me1/2 demethylase LSD1 (KDM1), which removes a histone mark of active chromatin and thus reinforces the establishment of a repressive chromatin environment on target loci [[42](/article/10.1007/s00018-016-2174-5#ref-CR42 "Rinn JL, Kertesz M, Wang JK et al (2007) Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129:1311–1323. doi: 10.1016/j.cell.2007.05.022
")\] (Fig. [2](/article/10.1007/s00018-016-2174-5#Fig2)d, e). Targeted inactivation of _Hotair_ in mice leads to de-repression of imprinted genes and of _HoxD_ genes, which lose H3K27-methylation and gain H3K4-methylation marks, as expected from loss of the enzymatic activity of PRC2 and LSD1 at these loci. Furthermore, _Hotair_ knockout mice show skeletal homeotic transformation phenotypes, which are typical for mutations affecting Polycomb mediated repression \[[43](/article/10.1007/s00018-016-2174-5#ref-CR43 "Li L, Liu B, Wapinski OL et al (2013) Targeted disruption of Hotair leads to homeotic transformation and gene derepression. Cell Rep 5:3–12. doi:
10.1016/j.celrep.2013.09.003
")\]. Interestingly, mice lacking most of the _HoxC_ cluster including _Hotair_ do not display these phenotypes, suggesting a complex interplay of multiple genomic regions in regulating _HoxC_ cluster gene expression \[[44](/article/10.1007/s00018-016-2174-5#ref-CR44 "Schorderet P, Duboule D (2011) Structural and functional differences in the long non-coding RNA hotair in mouse and human. PLoS Genet 7:e1002071. doi:
10.1371/journal.pgen.1002071
")\].Braveheart (Bvht) is a lncRNA that is activated during early cardiac differentiation and acts upstream of MESP1, a basic Helix-loop-helix transcription factor involved in early heart development. Bvht also exerts its function via an interaction with PRC2 [[45](/article/10.1007/s00018-016-2174-5#ref-CR45 "Klattenhoff CA, Scheuermann JC, Surface LE et al (2013) Braveheart, a long noncoding RNA required for cardiovascular lineage commitment. Cell 152:570–583. doi: 10.1016/j.cell.2013.01.003
")\]. The lncRNA Fetal-lethal developmental regulatory RNA (_Fendrr_; or _Foxf1_ adjacent noncoding developmental regulatory RNA) is likewise involved in cardiac development and heart function and interacts with PRC2\. _Fendrr_ also interacts with WDR5, which is well known for its presence in the MLL complexes that mediate H3K4 methylation, a mark that is thought to oppose H3K27me. _Fendrr_ might play a role at poised genes, or is involved in tuning the balance of active and repressive marks at its target gene promoters, thus adjusting the correct expression level of its targets \[[46](/article/10.1007/s00018-016-2174-5#ref-CR46 "Grote P, Herrmann BG (2013) The long non-coding RNA Fendrr links epigenetic control mechanisms to gene regulatory networks in mammalian embryogenesis. RNA Biol 10:1579–1585. doi:
10.4161/rna.26165
"), [47](/article/10.1007/s00018-016-2174-5#ref-CR47 "Grote P, Wittler L, Hendrix D et al (2013) The tissue-specific lncRNA Fendrr is an essential regulator of heart and body wall development in the mouse. Dev Cell 24:206–214. doi:
10.1016/j.devcel.2012.12.012
")\]. These examples are only a small selection and many more lncRNAs seem to function in concert with the PRC2 complex; for more examples, see \[[48](/article/10.1007/s00018-016-2174-5#ref-CR48 "Marín-Béjar O, Marchese FP, Athie A et al (2013) Pint lincRNA connects the p53 pathway with epigenetic silencing by the Polycomb repressive complex 2. Genome Biol 14:R104. doi:
10.1186/gb-2013-14-9-r104
")–[52](/article/10.1007/s00018-016-2174-5#ref-CR52 "Zhao J, Ohsumi TK, Kung JT et al (2010) Genome-wide identification of polycomb-associated RNAs by RIP-seq. Mol Cell 40:939–953. doi:
10.1016/j.molcel.2010.12.011
")\].An intriguing mechanism as to how lncRNAs might recruit their protein interaction partners to specific genomic loci is DNA-RNA triplex formation [[53](/article/10.1007/s00018-016-2174-5#ref-CR53 "Schmitz K-M, Mayer C, Postepska A, Grummt I (2010) Interaction of noncoding RNA with the rDNA promoter mediates recruitment of DNMT3b and silencing of rRNA genes. Genes Dev 24:2264–2269. doi: 10.1101/gad.590910
")\], which has been proposed for several lncRNAs forming a complex with PRC2 \[[46](/article/10.1007/s00018-016-2174-5#ref-CR46 "Grote P, Herrmann BG (2013) The long non-coding RNA Fendrr links epigenetic control mechanisms to gene regulatory networks in mammalian embryogenesis. RNA Biol 10:1579–1585. doi:
10.4161/rna.26165
"), [54](/article/10.1007/s00018-016-2174-5#ref-CR54 "O’Leary VB, Ovsepian SV, Carrascosa LG et al (2015) PARTICLE, a triplex-forming long ncRNA, regulates locus-specific methylation in response to low-dose irradiation. Cell Rep 11:474–485. doi:
10.1016/j.celrep.2015.03.043
"), [55](/article/10.1007/s00018-016-2174-5#ref-CR55 "Mondal T, Subhash S, Vaid R et al (2015) MEG3 long noncoding RNA regulates the TGF-β pathway genes through formation of RNA-DNA triplex structures. Nat Comms 6:7743. doi:
10.1038/ncomms8743
")\] (Fig. [2](/article/10.1007/s00018-016-2174-5#Fig2)d). In this manner, lncRNAs might direct chromatin or transcriptional modulators to specific genomic sites. This could explain, to some extent, the site-specific action of chromatin-modifying complexes, which do not themselves bind to DNA in a sequence specific manner \[[38](/article/10.1007/s00018-016-2174-5#ref-CR38 "Grote P, Herrmann BG (2015) Long noncoding RNAs in organogenesis: making the difference. Trends Genet 31:329–335. doi:
10.1016/j.tig.2015.02.002
"), [56](/article/10.1007/s00018-016-2174-5#ref-CR56 "Batista PJ, Chang HY (2013) Long noncoding RNAs: cellular address codes in development and disease. Cell 152:1298–1307. doi:
10.1016/j.cell.2013.02.012
")\].An example of a lncRNA interacting with the PRC1 complex is Focally amplified lncRNA on Chromosome 1 (FAL1). The interaction of FAL1 with BMI1, an essential subunit of PRC1, regulates its protein stability [[57](/article/10.1007/s00018-016-2174-5#ref-CR57 "Hu X, Feng Y, Zhang D et al (2014) A functional genomic approach identifies FAL1 as an oncogenic long noncoding RNA that associates with BMI1 and represses p21 expression in cancer. Cancer Cell 26:344–357. doi: 10.1016/j.ccr.2014.07.009
")\]. The knockdown of _FAL1_ in an ovarian cancer cell line led to gene expression changes, slower cell cycle progression and induction of senescence, similar to the effects of the _BMI1_ knockdown. This latter phenotype could be partially rescued by knockdown of the senescence-promoting factor _CDKN1A_ (p21). Additional ChIP data suggested that BMI1 binds to the promoter of _CDKN1A_ and, together with _FAL1_, suppresses the expression of this target and of many other genes.Whereas FAL1 acts in trans on PRC1, ANRIL, a lncRNA transcribed from the _INK4B_-_ARF_-INK4A tumor suppressor locus, acts in cis. The ANRIL transcript was discovered in a family with inherited melanoma-neural system tumors and assumed to play a role in tumorigenesis [[58](/article/10.1007/s00018-016-2174-5#ref-CR58 "Pasmant E, Laurendeau I, Héron D et al (2007) Characterization of a germ-line deletion, including the entire INK4/ARF locus, in a melanoma-neural system tumor family: identification of ANRIL, an antisense noncoding RNA whose expression coclusters with ARF. Cancer Res 67:3963–3969. doi: 10.1158/0008-5472.CAN-06-2004
")\]. _ANRIL_ recruits CBX7 (a PRC1 component) via a POLII-dependent mechanism to its locus in order to repress the neighboring _INK4B_\-_ARF_\-_INK4A_ genes, antagonize cellular senescence and indirectly promote cell cycle activity \[[58](/article/10.1007/s00018-016-2174-5#ref-CR58 "Pasmant E, Laurendeau I, Héron D et al (2007) Characterization of a germ-line deletion, including the entire INK4/ARF locus, in a melanoma-neural system tumor family: identification of ANRIL, an antisense noncoding RNA whose expression coclusters with ARF. Cancer Res 67:3963–3969. doi:
10.1158/0008-5472.CAN-06-2004
"), [59](/article/10.1007/s00018-016-2174-5#ref-CR59 "Yap KL, Li S, Muñoz-Cabello AM et al (2010) Molecular interplay of the noncoding RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a. Mol Cell 38:662–674. doi:
10.1016/j.molcel.2010.03.021
")\].The repressive H3K27-methylation, catalyzed by EZH2 in the PRC2 complex, is opposed by MLL complexes, which mediate methylation of H3K4me2/3, a mark associated with loci that are actively transcribed or primed for activation. WDR5 is an integral part of all MLL complexes, but also interacts with several other protein complexes [[60](/article/10.1007/s00018-016-2174-5#ref-CR60 "Trievel RC, Shilatifard A (2009) WDR5, a complexed protein. Nat Struct Mol Biol 16:678–680. doi: 10.1038/nsmb0709-678
")\]. WDR5 has been found to interact with more than 200 lncRNAs in mouse embryonic stem cells (mESCs) \[[61](/article/10.1007/s00018-016-2174-5#ref-CR61 "Yang YW, Flynn RA, Chen Y et al (2014) Essential role of lncRNA binding for WDR5 maintenance of active chromatin and embryonic stem cell pluripotency. elife 3:e02046. doi:
10.7554/eLife.02046
")\]. These interactions are important for WDR5 binding to chromatin since a point mutant that cannot bind RNA also fails to stably associate with chromatin. More specifically, two lncRNAs, _HOTTIP_ and _NeST_ have been described to recruit WDR5 to their neighboring genes and thus enhance their transcription \[[62](/article/10.1007/s00018-016-2174-5#ref-CR62 "Wang KC, Yang YW, Liu B et al (2011) A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression. Nature 472:120–124. doi:
10.1038/nature09819
"), [63](/article/10.1007/s00018-016-2174-5#ref-CR63 "Gomez JA, Wapinski OL, Yang YW et al (2013) The NeST long ncRNA controls microbial susceptibility and epigenetic activation of the interferon-γ locus. Cell 152:743–754. doi:
10.1016/j.cell.2013.01.015
")\].LncRNAs modulate DNA methylation
Whereas many lncRNAs associating with Polycomb complexes act by promoting PRC occupancy at genomic target sites, lncRNAs described in the context of DNA methylation have mostly been found to oppose this epigenetic mark. Frequently, transcription of noncoding RNAs, which interact with DNMTs, keeps a locus free of DNA methylation [[54](/article/10.1007/s00018-016-2174-5#ref-CR54 "O’Leary VB, Ovsepian SV, Carrascosa LG et al (2015) PARTICLE, a triplex-forming long ncRNA, regulates locus-specific methylation in response to low-dose irradiation. Cell Rep 11:474–485. doi: 10.1016/j.celrep.2015.03.043
"), [64](/article/10.1007/s00018-016-2174-5#ref-CR64 "Di Ruscio A, Ebralidze AK, Benoukraf T et al (2013) DNMT1-interacting RNAs block gene-specific DNA methylation. Nature 503:371–376. doi:
10.1038/nature12598
")–[66](/article/10.1007/s00018-016-2174-5#ref-CR66 "Hamazaki N, Uesaka M, Nakashima K et al (2015) Gene activation-associated long noncoding RNAs function in mouse preimplantation development. Development 142:910–920. doi:
10.1242/dev.116996
")\] (Fig. [2](/article/10.1007/s00018-016-2174-5#Fig2)a). Initially, it was observed that knockdown of extracoding CEBPA (_ecCEBPA_), a transcript starting upstream of the _CEBPA_ gene, leads to down-regulation of _CEBPA_ and increased DNA methylation of the locus. A subsequent global analysis of all lncRNAs bound to DNMT1 \[[64](/article/10.1007/s00018-016-2174-5#ref-CR64 "Di Ruscio A, Ebralidze AK, Benoukraf T et al (2013) DNMT1-interacting RNAs block gene-specific DNA methylation. Nature 503:371–376. doi:
10.1038/nature12598
")\] revealed that loci whose RNA products interact with DNMT1 show lower levels of DNA methylation than other loci. A similar observation was made in oocytes and two-cell stage embryos \[[66](/article/10.1007/s00018-016-2174-5#ref-CR66 "Hamazaki N, Uesaka M, Nakashima K et al (2015) Gene activation-associated long noncoding RNAs function in mouse preimplantation development. Development 142:910–920. doi:
10.1242/dev.116996
")\], in which the down-regulation of several divergently transcribed promoter-associated ncRNA (pancRNA) led to lower expression of the adjacent protein-coding gene and higher DNA methylation. In the case of one particular gene, _IL17b_, the pancRNA knockdown resulted in cell death of the developing blastocyst. Supplementing the embryos with IL17b protein rescued the phenotype.In the case of Tcf21 antisense RNA reducing DNA methylation (TARID), keeping the protein-coding gene (Tcf21) on the opposite DNA strand free of DNA methylation required GADD45A and the TET proteins [[65](/article/10.1007/s00018-016-2174-5#ref-CR65 "Arab K, Park YJ, Lindroth AM et al (2014) Long noncoding RNA TARID directs demethylation and activation of the tumor suppressor TCF21 via GADD45A. Mol Cell. doi: 10.1016/j.molcel.2014.06.031
")\] pointing towards a mechanism involving active DNA demethylation.LncRNAs regulate chromatin remodeling
In addition to interaction with enzymatic complexes, which covalently modify chromatin, lncRNAs have also been shown to control chromatin remodeling complexes that can alter the nucleosome spacing in an energy dependent manner. In human prostate cancer, gene expression analyses showed that SWI/SNF and the lncRNA SChLAP1 have opposing roles. SChlAP1 interacts with the SNF5 subunit of the chromatin remodeling complex SWI/SNF, and globally inhibits binding of SWI/SNF to chromatin, subsequently leading to genome-wide de-repression of gene activity [[67](/article/10.1007/s00018-016-2174-5#ref-CR67 "Prensner JR, Iyer MK, Sahu A et al (2013) The long noncoding RNA SChLAP1 promotes aggressive prostate cancer and antagonizes the SWI/SNF complex. Nat Genet 45:1392–1398. doi: 10.1038/ng.2771
")\]. Similarly, the lncRNA _Myheart_ (_Mhrt_), a lncRNA transcribed divergently to _Myh6_ and overlapping with _Myh7_ in antisense direction, binds to BRG1, the ATPase subunit of the SWI/SNF complex, and excludes it from the _Myh6/7_ locus, thus preventing chromatin remodeling \[[68](/article/10.1007/s00018-016-2174-5#ref-CR68 "Han P, Li W, Lin C-H et al (2014) A long noncoding RNA protects the heart from pathological hypertrophy. Nature 514:102–106. doi:
10.1038/nature13596
")\]. BRG1, in turn, down-regulates _Mhrt._ Cardiac stress leads to upregulation of BRG1 to a level allowing it to overcome the repulsion of the SWI/SNF complex from chromatin by _Mhrt_ and to bind to the _Myh6_ locus.The Evf2 lncRNA, transcribed from the genomic region between Dlx5 and Dlx6, promotes SWI/SNF binding to the enhancers of these genes. However, Evf2 inhibits the remodeling activity of SWI/SNF and thus interferes with upregulation of Dlx5/Dlx6. Accordingly, in Evf2 mutant mice, Dlx6 and, to a lesser extent, Dlx5 are upregulated [[69](/article/10.1007/s00018-016-2174-5#ref-CR69 "Bond AM, Vangompel MJW, Sametsky EA et al (2009) Balanced gene regulation by an embryonic brain ncRNA is critical for adult hippocampal GABA circuitry. Nat Neurosci 12:1020–1027. doi: 10.1038/nn.2371
"), [70](/article/10.1007/s00018-016-2174-5#ref-CR70 "Cajigas I, Leib DE, Cochrane J et al (2015) Evf2 lncRNA/BRG1/DLX1 interactions reveal RNA-dependent chromatin remodeling inhibition. Development 142:2641–2652. doi:
10.1242/dev.126318
")\]. In a more recent study, it was observed that the binding of _Evf2_ to BRG1 can be out-competed by other RNAs of similar length and results in reduced remodeling activity, suggesting that the binding of lncRNAs to the SWI/SNF complex is promiscuous. _Xist_, on the other hand, binds to components of the SWI/SNF complex, but excludes the complex from the Xi, rather than recruiting it \[[71](/article/10.1007/s00018-016-2174-5#ref-CR71 "Minajigi A, Froberg JE, Wei C et al (2015) A comprehensive Xist interactome reveals cohesin repulsion and an RNA-directed chromosome conformation. Science 349:aab2276. doi:
10.1126/science.aab2276
")\]. The SWI/SNF complex can also be recruited by lncRNAs and mediate gene activation, as has been found in hepatocellular carcinoma cancer stem cells. A lncRNA, _lncTCF7_ or _WNT signaling pathway activating non_\-_coding RNA_ (_WSPAR_), transcribed 200 kb upstream of _TCF7_, can recruit SWI/SNF to the _TCF7_ promoter and thus activate _TCF7_ expression and WNT signaling \[[72](/article/10.1007/s00018-016-2174-5#ref-CR72 "Wang Y, He L, Du Y et al (2015) The long noncoding RNA lncTCF7 promotes self-renewal of human liver cancer stem cells through activation of Wnt signaling. Cell Stem Cell 16:413–425. doi:
10.1016/j.stem.2015.03.003
")\].Is the observed binding of numerous lncRNAs to chromatin modifiers specific or promiscuous?
The specificity of lncRNA interactions with chromatin-modifying complexes has been questioned. For instance, a large number of lncRNAs have been shown to co-purify with PRC2, possibly suggesting non-specific binding [[50](/article/10.1007/s00018-016-2174-5#ref-CR50 "Khalil AM, Guttman M, Huarte M et al (2009) Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc Natl Acad Sci USA 106:11667–11672. doi: 10.1073/pnas.0904715106
"), [52](/article/10.1007/s00018-016-2174-5#ref-CR52 "Zhao J, Ohsumi TK, Kung JT et al (2010) Genome-wide identification of polycomb-associated RNAs by RIP-seq. Mol Cell 40:939–953. doi:
10.1016/j.molcel.2010.12.011
"), [73](/article/10.1007/s00018-016-2174-5#ref-CR73 "Kaneko S, Bonasio R, Saldaña-Meyer R et al (2013) Interactions between JARID2 and noncoding RNAs regulate PRC2 recruitment to chromatin. Mol Cell. doi:
10.1016/j.molcel.2013.11.012
")\]. Similarly, 15 % of all lncRNAs present in a particular cellular context were shown to be bound by DNMT1 \[[64](/article/10.1007/s00018-016-2174-5#ref-CR64 "Di Ruscio A, Ebralidze AK, Benoukraf T et al (2013) DNMT1-interacting RNAs block gene-specific DNA methylation. Nature 503:371–376. doi:
10.1038/nature12598
"), [74](/article/10.1007/s00018-016-2174-5#ref-CR74 "Merry CR, Forrest ME, Sabers JN et al (2015) DNMT1-associated long non-coding RNAs regulate global gene expression and DNA methylation in colon cancer. Hum Mol Genet. doi:
10.1093/hmg/ddv343
")\]. Likewise, WDR5 binds to more than a thousand RNAs in mESCs, of which 20 % are lncRNAs \[[61](/article/10.1007/s00018-016-2174-5#ref-CR61 "Yang YW, Flynn RA, Chen Y et al (2014) Essential role of lncRNA binding for WDR5 maintenance of active chromatin and embryonic stem cell pluripotency. elife 3:e02046. doi:
10.7554/eLife.02046
")\]. This issue of promiscuity was assessed exemplarily for PRC2 in a systematic biochemical study \[[75](/article/10.1007/s00018-016-2174-5#ref-CR75 "Davidovich C, Wang X, Cifuentes-Rojas C et al (2015) Toward a Consensus on the Binding Specificity and Promiscuity of PRC2 for RNA. Mol Cell. doi:
10.1016/j.molcel.2014.12.017
")\], where the authors demonstrate that PRC2 binds RNA in a promiscuous manner, but has remarkably higher affinity for specific lncRNAs than for e.g. bacterial mRNA. Furthermore, they provide evidence that the binding strength correlates with the length of the RNA. Though these studies were performed in vitro at non-physiological conditions, they suggest that the chromatin-modifying complexes can sufficiently differentiate between specific and non-specific interactors. Moreover, since most lncRNA transcripts are expressed at few copies per cell, it is likely that the number of PCR2 molecules per cell is in excess to the number of lncRNA transcripts, which contain a high affinity site for PRC2\. Overall, it is not unexpected that a chromatin-modifying complex acting at numerous genomic loci binds many different individual lncRNAs, which stabilize it or recruit it to distinct genomic loci.Interactions of lncRNAs with transcription factors
Besides chromatin modifiers, transcription factors have also been found to interact with lncRNAs. The lncRNA definitive _endoderm_-associated lncRNA1 (DEANR1) (also known as ALIEN), for instance, is transcribed downstream of FOXA2 and is highly induced during differentiation of ESCs towards definitive endoderm [[76](/article/10.1007/s00018-016-2174-5#ref-CR76 "Jiang W, Liu Y, Liu R et al (2015) The lncRNA DEANR1 facilitates human endoderm differentiation by activating FOXA2 expression. Cell Rep 11:137–148. doi: 10.1016/j.celrep.2015.03.008
"), [77](/article/10.1007/s00018-016-2174-5#ref-CR77 "Kurian L, Aguirre A, Sancho-Martinez I et al (2015) Identification of novel long noncoding RNAs underlying vertebrate cardiovascular development. Circulation 131:1278–1290. doi:
10.1161/CIRCULATIONAHA.114.013303
")\]. Knockdown of _DEANR1_ inhibits endodermal differentiation due to the reduction of _FOXA2_ expression. This interpretation is strongly supported by the fact that endoderm differentiation is rescued in _DEANR1_ knockdown cells upon forced _FOXA2_ expression. Mechanistically, _DEANR1_ was proposed to interact with SMAD2/3 and to recruit it to the _FOXA2_ promoter.The lncRNA rhabdomyosarcoma associated transcript (RMST) is required for neuronal differentiation by mediating binding of the transcription factor SOX2 to approximately half of its binding sites [[78](/article/10.1007/s00018-016-2174-5#ref-CR78 "Ng S-Y, Bogu GK, Soh BS, Stanton LW (2013) The long noncoding RNA RMST interacts with SOX2 to regulate neurogenesis. Mol Cell 51:349–359. doi: 10.1016/j.molcel.2013.07.017
")\]. Certain lncRNAs have been linked in different ways to transcription factors of the NF-κB pathway. _Lethe_ (_after the ‘river of forgetfulness’_ in Greek mythology) interacts with RelA and prevents it from binding to DNA \[[79](/article/10.1007/s00018-016-2174-5#ref-CR79 "Rapicavoli NA, Qu K, Zhang J et al (2013) A mammalian pseudogene lncRNA at the interface of inflammation and anti-inflammatory therapeutics. elife 2:e00762. doi:
10.7554/eLife.00762
")\]. _p50_\-_associated cyclooxygenase_\-_2 (COX_\-_2) extragenic RNA_ (_PACER_), on the other hand, sequesters p50, which forms homodimers and is repressive at high concentration, thereby lowering its concentration and allowing it to form activating heterodimers with p65 \[[80](/article/10.1007/s00018-016-2174-5#ref-CR80 "Krawczyk M, Emerson BM (2014) p50-associated COX-2 extragenic RNA (PACER) activates COX-2 gene expression by occluding repressive NF-κB complexes. elife 3:e01776")\]. Another lncRNA, _lnc dendritic cells_ (_lnc_\-_DC_) binds to the transcription factor STAT3 in the cytoplasm and prevents its dephosphorylation by SHP1, thereby activating STAT3 and thus dendritic cell differentiation \[[81](/article/10.1007/s00018-016-2174-5#ref-CR81 "Wang P, Xue Y, Han Y et al (2014) The STAT3-binding long noncoding RNA lnc-DC controls human dendritic cell differentiation. Science 344:310–313. doi:
10.1126/science.1251456
")\].An elaborate mechanism linking transcription factor binding and chromatin modifications has been found for breast cancer anti-estrogen resistance 4 (BCAR4). This lncRNA binds to the SMAD nuclear interacting protein 1 (SNIP1) and a phosphatase (PNUTS) involved in RNA polymerase II regulation. In response to cytokine stimulation, BCAR4 lifts the inhibitory effect of SNIP1 on p300, a histone acetyl transferase and transcriptional activator. Acetylated histones are necessary for BCAR4 mediated recruitment of PNUTS, which in turn leads to active polymerase II at GLI2 controlled genes [[82](/article/10.1007/s00018-016-2174-5#ref-CR82 "Xing Z, Lin A, Li C et al (2014) lncRNA directs cooperative epigenetic regulation downstream of chemokine signals. Cell 159:1110–1125. doi: 10.1016/j.cell.2014.10.013
")\].Also CTCF, a protein with the dual function of a transcriptional regulator and a boundary factor, has been shown to interact with numerous noncoding RNAs [[83](/article/10.1007/s00018-016-2174-5#ref-CR83 "Kung JT, Kesner B, An JY et al (2015) Locus-specific targeting to the X chromosome revealed by the RNA interactome of CTCF. Mol Cell. doi: 10.1016/j.molcel.2014.12.006
")\]. An example is the interplay between CTCF and the lncRNAs _Jpx_, _Tsix_ and _Xite_, which is important for X-chromosome inactivation \[[83](/article/10.1007/s00018-016-2174-5#ref-CR83 "Kung JT, Kesner B, An JY et al (2015) Locus-specific targeting to the X chromosome revealed by the RNA interactome of CTCF. Mol Cell. doi:
10.1016/j.molcel.2014.12.006
"), [84](/article/10.1007/s00018-016-2174-5#ref-CR84 "Sun S, Del Rosario BC, Szanto A et al (2013) Jpx RNA activates Xist by evicting CTCF. Cell 153:1537–1551. doi:
10.1016/j.cell.2013.05.028
")\]. Prior to inactivation, _Tsix_ and _Xite_ recruit CTCF in _cis_ to the X-inactivation center promoting homologous X-chromosome pairing at the Xic \[[83](/article/10.1007/s00018-016-2174-5#ref-CR83 "Kung JT, Kesner B, An JY et al (2015) Locus-specific targeting to the X chromosome revealed by the RNA interactome of CTCF. Mol Cell. doi:
10.1016/j.molcel.2014.12.006
")\]. During X-chromosome inactivation, _Jpx_ evicts CTCF in _trans_ from the _Xist_ locus and thereby prevents promoter blockage by CTCF \[[83](/article/10.1007/s00018-016-2174-5#ref-CR83 "Kung JT, Kesner B, An JY et al (2015) Locus-specific targeting to the X chromosome revealed by the RNA interactome of CTCF. Mol Cell. doi:
10.1016/j.molcel.2014.12.006
")\]. These examples illustrate the dynamic interaction between lncRNAs and transcription factors in regulating chromatin-mediated cellular functions.Regulation of genome organization by looping of enhancers
Previously, transcripts found at enhancers had been interpreted as byproducts of transcription from promoters contacted by enhancers. Now, it is well established that RNA transcripts produced at active enhancers are functionally important [[85](/article/10.1007/s00018-016-2174-5#ref-CR85 "Kim T-K, Hemberg M, Gray JM et al (2010) Widespread transcription at neuronal activity-regulated enhancers. Nature 465:182–187. doi: 10.1038/nature09033
")–[87](/article/10.1007/s00018-016-2174-5#ref-CR87 "Natoli G, Andrau J-C (2012) Noncoding transcription at enhancers: general principles and functional models. Annu Rev Genet 46:1–19. doi:
10.1146/annurev-genet-110711-155459
")\]. An example of enhancer-linked transcripts, already identified two decades ago, has been described for the locus control region (LCR) of the _β_\-_globin_ cluster \[[88](/article/10.1007/s00018-016-2174-5#ref-CR88 "Collis P, Antoniou M, Grosveld F (1990) Definition of the minimal requirements within the human beta-globin gene and the dominant control region for high level expression. EMBO J 9:233–240"), [89](/article/10.1007/s00018-016-2174-5#ref-CR89 "Ashe HL, Monks J, Wijgerde M et al (1997) Intergenic transcription and transinduction of the human beta-globin locus. Genes Dev 11:2494–2509")\]. To date, numerous studies have assigned functions to enhancer-associated RNAs \[[90](/article/10.1007/s00018-016-2174-5#ref-CR90 "Orom UA, Shiekhattar R (2011) Long non-coding RNAs and enhancers. Curr Opin Genet Dev 21:194–198. doi:
10.1016/j.gde.2011.01.020
")\] (Fig. [2](/article/10.1007/s00018-016-2174-5#Fig2)b). There are different classes of RNAs observed at enhancers. The classical enhancer RNAs (eRNAs) are derived from bidirectional transcription, are unspliced and rather short transcripts \[[85](/article/10.1007/s00018-016-2174-5#ref-CR85 "Kim T-K, Hemberg M, Gray JM et al (2010) Widespread transcription at neuronal activity-regulated enhancers. Nature 465:182–187. doi:
10.1038/nature09033
"), [91](/article/10.1007/s00018-016-2174-5#ref-CR91 "Andersson R, Gebhard C, Miguel-Escalada I et al (2014) An atlas of active enhancers across human cell types and tissues. Nature 507:455–461. doi:
10.1038/nature12787
")\]. Additionally, there are also unidirectional, often spliced and processed transcripts, which are indistinguishable from canonical lncRNAs, but happen to be transcribed from an enhancer \[[23](/article/10.1007/s00018-016-2174-5#ref-CR23 "Marques AC, Hughes J, Graham B et al (2013) Chromatin signatures at transcriptional start sites separate two equally populated yet distinct classes of intergenic long noncoding RNAs. Genome Biol 14:R131. doi:
10.1186/gb-2013-14-11-r131
"), [92](/article/10.1007/s00018-016-2174-5#ref-CR92 "Ounzain S, Pezzuto I, Micheletti R et al (2014) Functional importance of cardiac enhancer-associated noncoding RNAs in heart development and disease. J Mol Cell Cardiol 76:55–70. doi:
10.1016/j.yjmcc.2014.08.009
")\]. These transcripts often function in _cis_. Finally, there are lncRNAs with enhancer-like functions (also called ncRNA-a) \[[93](/article/10.1007/s00018-016-2174-5#ref-CR93 "Orom UA, Derrien T, Beringer M et al (2010) Long noncoding RNAs with enhancer-like function in human cells. Cell 143:46–58. doi:
10.1016/j.cell.2010.09.001
")\]. Whether this classification reflects different functional classes of RNAs remains to be investigated.In general, eRNAs are co-regulated with their neighboring gene(s) [[91](/article/10.1007/s00018-016-2174-5#ref-CR91 "Andersson R, Gebhard C, Miguel-Escalada I et al (2014) An atlas of active enhancers across human cell types and tissues. Nature 507:455–461. doi: 10.1038/nature12787
")\] and presumed to be important for gene expression, possibly by supporting cohesin binding \[[94](/article/10.1007/s00018-016-2174-5#ref-CR94 "Li W, Notani D, Ma Q et al (2013) Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation. Nature 498:516–520. doi:
10.1038/nature12210
")\]. However, experimental support for the latter is rather mild. While eRNAs in some studies were shown to stabilize chromosome loops \[[94](/article/10.1007/s00018-016-2174-5#ref-CR94 "Li W, Notani D, Ma Q et al (2013) Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation. Nature 498:516–520. doi:
10.1038/nature12210
"), [95](/article/10.1007/s00018-016-2174-5#ref-CR95 "Lai F, Orom UA, Cesaroni M et al (2013) Activating RNAs associate with Mediator to enhance chromatin architecture and transcription. Nature 494:497–501. doi:
10.1038/nature11884
")\], they appeared to be dispensable for promoter-enhancer contact in others, raising doubts about the role of eRNAs in chromosome looping \[[96](/article/10.1007/s00018-016-2174-5#ref-CR96 "Schaukowitch K, Joo J-Y, Liu X et al (2014) Enhancer RNA facilitates NELF release from immediate early genes. Mol Cell 56:29–42. doi:
10.1016/j.molcel.2014.08.023
"), [97](/article/10.1007/s00018-016-2174-5#ref-CR97 "Mousavi K, Zare H, Dell’orso S et al (2013) eRNAs promote transcription by establishing chromatin accessibility at defined genomic loci. Mol Cell 51:606–617. doi:
10.1016/j.molcel.2013.07.022
")\].LncRNAs transcribed from enhancer regions can also have inhibitory effects on their target gene. The promoter deletion of the lncRNA Haunt (also known as _linc_-Hoxa1), for instance, has been shown to lead to upregulation of several genes of the neighboring HoxA gene cluster. However, larger deletions in the Haunt locus impair HoxA expression [[98](/article/10.1007/s00018-016-2174-5#ref-CR98 "Yin Y, Yan P, Lu J et al (2015) Opposing roles for the lncRNA haunt and its genomic locus in regulating HOXA Gene activation during embryonic stem cell differentiation. Cell Stem Cell 16:504–516. doi: 10.1016/j.stem.2015.03.007
")\], demonstrating that the genomic locus can have opposing functions to the RNA product encoded by it. The repressive effect of _Haunt_ was furthermore observed at the single cell level confirming that the expression of _HoxA1_ and _Haunt_ are anti-correlated \[[99](/article/10.1007/s00018-016-2174-5#ref-CR99 "Maamar H, Cabili MN, Rinn J, Raj A (2013) linc-HOXA1 is a noncoding RNA that represses Hoxa1 transcription in cis. Genes Dev 27:1260–1271. doi:
10.1101/gad.217018.113
")\].A similar mechanism was found for Playrr (D030025E07Rik), a lncRNA encoded upstream of the homeodomain transcription factor Pitx2 [[100](/article/10.1007/s00018-016-2174-5#ref-CR100 "Welsh IC, Kwak H, Chen FL et al (2015) Chromatin architecture of the Pitx2 locus requires CTCF- and Pitx2-dependent asymmetry that mirrors embryonic gut laterality. Cell Rep. doi: 10.1016/j.celrep.2015.08.075
")\]. _Pitx2_ expression is constrained to the left side of the gut dorsal mesentery, whereas expression of _Playrr_ is detected on the right, but spreads to both sides in _Pitx2_ deletion mutants. A CRISPR/Cas9 generated mutation resulting in _Playrr_ RNA decay caused upregulation of _Pitx2_ expression \[[100](/article/10.1007/s00018-016-2174-5#ref-CR100 "Welsh IC, Kwak H, Chen FL et al (2015) Chromatin architecture of the Pitx2 locus requires CTCF- and Pitx2-dependent asymmetry that mirrors embryonic gut laterality. Cell Rep. doi:
10.1016/j.celrep.2015.08.075
")\]. Both experiments reveal a mutual inhibitory interaction between _Pitx2_ and _Playrr_. An enhancer involved in _Pitx2_ regulation in the gut overlaps the TSS of _Playrr_. In the cells on the left-hand side, where _Pitx2_ is expressed, the _Pitx2_ locus was found to be in closer proximity to the _Playrr_ locus than in cells on the right side and to cause down-regulation of _Playrr_ by an unknown mechanism. In cells on the right, on the other hand, _Playrr_ RNA is thought to cause a separation of the _Pitx2_ locus from the enhancer at the _Playrr_ locus suggesting that _Playrr_ expression interferes with the looping of the _Pitx2_ promoter to its enhancer at the _Playrr_ locus and thus with _Pitx2_ gene activation (Fig. [2](/article/10.1007/s00018-016-2174-5#Fig2)c).Furthermore, the lncRNAs PRNCR1 and PCGEM1 promote enhancer-promoter looping in prostate cancer cells [[101](/article/10.1007/s00018-016-2174-5#ref-CR101 "Yang L, Lin C, Jin C et al (2013) lncRNA-dependent mechanisms of androgen-receptor-regulated gene activation programs. Nature 500:598–602. doi: 10.1038/nature12451
")\]. Other lncRNAs have been suggested to assist chromosome conformation more globally. A particular situation can be observed at the inactive X-chromosome (Xi). The 3D-structure of Xi is dependent on _Xist_, supposedly because _Xist_ excludes cohesin binding from Xi \[[71](/article/10.1007/s00018-016-2174-5#ref-CR71 "Minajigi A, Froberg JE, Wei C et al (2015) A comprehensive Xist interactome reveals cohesin repulsion and an RNA-directed chromosome conformation. Science 349:aab2276. doi:
10.1126/science.aab2276
")\]. Deletion of _Xist_ led to loss of the compact chromosome structure of the Xi and reorganization into chromosome domains as they are found on the active X-chromosome. Recently, another lncRNA, functional intergenic repeating RNA element (_Firre_), which escapes X-chromosome inactivation was suggested to stabilize the perinucleolar location of the Xi \[[102](/article/10.1007/s00018-016-2174-5#ref-CR102 "Yang F, Deng X, Ma W et al (2015) The lncRNA Firre anchors the inactive X chromosome to the nucleolus by binding CTCF and maintains H3K27me3 methylation. Genome Biol 16:52. doi:
10.1186/s13059-015-0618-0
")\]. Additionally, _Firre_ was proposed to mediate focal trans-chromosomal contacts \[[103](/article/10.1007/s00018-016-2174-5#ref-CR103 "Hacisuleyman E, Goff LA, Trapnell C et al (2014) Topological organization of multichromosomal regions by the long intergenic noncoding RNA Firre. Nat Struct Mol Biol 21:198–206. doi:
10.1038/nsmb.2764
")\]. This finding was questioned by a different study, which reported a dispersed nuclear distribution of _Firre_ and a role in the control of factors involved in RNA processing \[[104](/article/10.1007/s00018-016-2174-5#ref-CR104 "Bergmann JH, Li J, Eckersley-Maslin MA et al (2015) Regulation of the ESC transcriptome by nuclear long non-coding RNAs. Genome Res 25:1336–1346. doi:
10.1101/gr.189027.114
")\]. These examples show that lncRNAs can play important roles in controlling local intra- and inter-chromosomal genomic structure and interactions.Regulation at the posttranscriptional level
LncRNAs primarily found in the cytosol are thought to be involved in gene regulation on the posttranscriptional level (Fig. 2e–h). Accordingly, several lncRNAs have been shown to influence splicing patterns of either specific genes or globally by interacting with splicing factors. The lncRNA Pnky (divergent to Pou3f1) binds to the splicing factor PTBP1 and thereby regulates the splicing patterns of a subset of genes involved in neurogenesis [[105](/article/10.1007/s00018-016-2174-5#ref-CR105 "Ramos AD, Andersen RE, Liu SJ et al (2015) The long noncoding RNA Pnky regulates neuronal differentiation of embryonic and postnatal neural stem cells. Cell Stem Cell 16:439–447. doi: 10.1016/j.stem.2015.02.007
")\]. Whether _TUNA_ (_megamind_), which interacts with PTBP1 as well, functions via the same mechanism remains to be investigated \[[33](/article/10.1007/s00018-016-2174-5#ref-CR33 "Ulitsky I, Shkumatava A, Jan CH et al (2011) Conserved function of lincRNAs in vertebrate embryonic development despite rapid sequence evolution. Cell 147:1537–1550. doi:
10.1016/j.cell.2011.11.055
"), [106](/article/10.1007/s00018-016-2174-5#ref-CR106 "Lin N, Chang K-Y, Li Z et al (2014) An evolutionarily conserved long noncoding RNA TUNA controls pluripotency and neural lineage commitment. Mol Cell 53:1005–1019. doi:
10.1016/j.molcel.2014.01.021
")\]. In addition, myocardial infarction associated transcript (_MIAT_) has been found to influence splicing in different systems. In embryonic neurogenesis, _MIAT_ is involved in controlling the splicing pattern of _Wnt7b_ \[[107](/article/10.1007/s00018-016-2174-5#ref-CR107 "Aprea J, Prenninger S, Dori M et al (2013) Transcriptome sequencing during mouse brain development identifies long non-coding RNAs functionally involved in neurogenic commitment. EMBO J 32:3145–3160. doi:
10.1038/emboj.2013.245
")\]. In iPS cells and mouse primary neurons, _MIAT_ binds to splicing factors, and in post-mortem brains of schizophrenia patients, it was found to be down-regulated, suggesting that it might contribute to this pathological phenotype \[[108](/article/10.1007/s00018-016-2174-5#ref-CR108 "Barry G, Briggs JA, Vanichkina DP et al (2014) The long non-coding RNA Gomafu is acutely regulated in response to neuronal activation and involved in schizophrenia-associated alternative splicing. Mol Psychiatry 19:486–494. doi:
10.1038/mp.2013.45
")\]. _MALAT1_, a highly expressed lncRNA enriched in nuclear speckles, has been suggested to be a general component of the splicing machinery \[[109](/article/10.1007/s00018-016-2174-5#ref-CR109 "Tripathi V, Ellis JD, Shen Z et al (2010) The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation. Mol Cell 39:925–938. doi:
10.1016/j.molcel.2010.08.011
")\]. However, this view has been challenged since the _MALAT1_ knockout did not show disintegration of splicing speckles \[[110](/article/10.1007/s00018-016-2174-5#ref-CR110 "Eißmann M, Gutschner T, Hämmerle M et al (2012) Loss of the abundant nuclear non-coding RNA MALAT1 is compatible with life and development. RNA Biol 9:1076–1087. doi:
10.4161/rna.21089
")\]. A very specific role in the cytoplasm was found for the lncRNA non-coding RNA activated by DNA damage (_NORAD_) \[[111](/article/10.1007/s00018-016-2174-5#ref-CR111 "Lee S, Kopp F, Chang T-C et al (2015) Noncoding RNA NORAD regulates genomic stability by sequestering PUMILIO proteins. Cell. doi:
10.1016/j.cell.2015.12.017
")\]. _NORAD_ sequesters PUMILIO proteins and thereby promotes genomic stability (Fig. [2](/article/10.1007/s00018-016-2174-5#Fig2)f). Ablation of _NORAD_ leads to hyperactivity of PUMILIO causing chromosome instability and aneuploidy due to repression of PUMILIO target mRNAs that function in DNA replication, mitosis and DNA damage repair.The most recently described class of lncRNAs that came into focus are the circRNAs. Due to their circular structure, they are resistant to degradation by exonucleases and are therefore thought to be highly stable within a cell. The brain specific circRNA CDR1as was shown to act as a sponge for the miRNA _let_-7 [[26](/article/10.1007/s00018-016-2174-5#ref-CR26 "Hansen TB, Jensen TI, Clausen BH et al (2013) Natural RNA circles function as efficient microRNA sponges. Nature 495:384–388. doi: 10.1038/nature11993
"), [27](/article/10.1007/s00018-016-2174-5#ref-CR27 "Memczak S, Jens M, Elefsinioti A et al (2013) Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 495:333–338. doi:
10.1038/nature11928
")\] (Fig. [2](/article/10.1007/s00018-016-2174-5#Fig2)f). Accordingly, overexpression of _CDR1as_ in zebrafish caused impaired midbrain development, similarly to the effect of a _let_\-_7_ inhibitor. It was also shown that small polypeptides can be derived from some circRNAs, extending the functional repertoire of these stable RNA molecules \[[112](/article/10.1007/s00018-016-2174-5#ref-CR112 "Chen CY, Sarnow P (1995) Initiation of protein synthesis by the eukaryotic translational apparatus on circular RNAs. Science 268:415–417. doi:
10.1126/science.7536344
")\]. Most likely, circRNAs do not only interact with other transcripts, but also with proteins and thus may function in a wide range of different processes.Functions of lncRNAs in development and differentiation
In the following section, we will focus on the physiological effects of lncRNAs in the organism, during embryonic development and in in vitro differentiation systems. There are still few known examples of lncRNAs shown to play important roles in vivo, and thus we will revisit some of the lncRNAs already mentioned above.
Maternal transcripts present in oocytes and zygotes contribute to early differentiation processes. In a single-cell sequencing study examining oocytes, zygotes and cells from the first cell divisions of the early mouse embryo, lncRNA transcripts were found at all stages examined [[113](/article/10.1007/s00018-016-2174-5#ref-CR113 "Yan L, Yang M, Guo H et al (2013) Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells. Nat Struct Mol Biol 20:1131–1139. doi: 10.1038/nsmb.2660
")\]. One subclass of lncRNAs, the pancRNAs, was compared between murine two-cell embryos and oocytes \[[66](/article/10.1007/s00018-016-2174-5#ref-CR66 "Hamazaki N, Uesaka M, Nakashima K et al (2015) Gene activation-associated long noncoding RNAs function in mouse preimplantation development. Development 142:910–920. doi:
10.1242/dev.116996
")\]. A subset of pancRNAs found to be expressed in two-cell embryos but not in oocytes was found to be important for counteracting DNA methylation at the adjacent promoters thereby ensuring expression of the neighboring gene.The mechanisms first found to be linked to lncRNAs were imprinting and X-chromosome inactivation. Accordingly, the first lncRNA that was genetically targeted and deleted was H19, an imprinted gene that was already suspected to be a lncRNA 25 years ago [114]. H19 is exclusively expressed from the maternal allele and is inhibitory for the adjacent gene, Insulin-like growth factor 2 (Igf2) [114–[116](/article/10.1007/s00018-016-2174-5#ref-CR116 "Leighton PA, Ingram RS, Eggenschwiler J et al (1995) Disruption of imprinting caused by deletion of the H19 gene region in mice. Nature 375:34–39. doi: 10.1038/375034a0
")\]. An _H19_ deletion on the maternally inherited chromosome led to an increase in _Igf2_ expression and thus to increased bodyweight. This phenotype could be rescued by deletion of one _Igf2_ allele. _H19_ knockout mice are viable and fertile \[[116](/article/10.1007/s00018-016-2174-5#ref-CR116 "Leighton PA, Ingram RS, Eggenschwiler J et al (1995) Disruption of imprinting caused by deletion of the H19 gene region in mice. Nature 375:34–39. doi:
10.1038/375034a0
")\], but have reduced muscle regeneration capacities due to loss of two miRNAs encoded within the _H19_ transcript \[[117](/article/10.1007/s00018-016-2174-5#ref-CR117 "Dey BK, Pfeifer K, Dutta A (2014) The H19 long noncoding RNA gives rise to microRNAs miR-675-3p and miR-675-5p to promote skeletal muscle differentiation and regeneration. Genes Dev 28:491–501. doi:
10.1101/gad.234419.113
")\]. The imprinted _Igf2r_ gene is silenced by the lncRNA _Airn_, which is transcribed from the opposite strand, overlapping the 5-prime region of _Igf2r_. Here, silencing is not achieved by the lncRNA transcripts as such, but by transcription through the _Igf2r_ promoter, which interferes with RNA PolII recruitment \[[118](/article/10.1007/s00018-016-2174-5#ref-CR118 "Latos PA, Pauler FM, Koerner MV et al (2012) Airn transcriptional overlap, but not its lncRNA products, induces imprinted Igf2r silencing. Science 338:1469–1472. doi:
10.1126/science.1228110
")\]. Several further lncRNAs with a well-established role in imprinting have been reported. For details we would like to refer to a recent review \[[119](/article/10.1007/s00018-016-2174-5#ref-CR119 "Kanduri C (2016) Long noncoding RNAs: lessons from genomic imprinting. Biochim Biophys Acta 1859:102–111. doi:
10.1016/j.bbagrm.2015.05.006
")\].Insight into the developmental function of Xist was obtained two decades ago when Xist knockout mice were generated and analyzed [[120](/article/10.1007/s00018-016-2174-5#ref-CR120 "Penny GD, Kay GF, Sheardown SA et al (1996) Requirement for Xist in X chromosome inactivation. Nature 379:131–137. doi: 10.1038/379131a0
"), [121](/article/10.1007/s00018-016-2174-5#ref-CR121 "Marahrens Y, Panning B, Dausman J et al (1997) Xist-deficient mice are defective in dosage compensation but not spermatogenesis. Genes Dev 11:156–166")\]. Male mice lacking _Xist_ are unaffected, whereas females die during the first half of gestation. Interestingly, knockout females with a single X-chromosome (XO) lacking _Xist_ are healthy, strongly suggesting that it is not the lack of _Xist_, but the failure to adjust the X-chromosome gene dosage that causes embryonic lethality.In a more recent example, the deletion of another imprinted lncRNA gene in the mouse, Gtl2 (Meg3, maternally expressed 3), showed clear _cis_-acting effects on neighboring genes resulting in perinatal lethality [[122](/article/10.1007/s00018-016-2174-5#ref-CR122 "Zhou Y, Cheunsuchon P, Nakayama Y et al (2010) Activation of paternally expressed genes and perinatal death caused by deletion of the Gtl2 gene. Development 137:2643–2652. doi: 10.1242/dev.045724
")\].Many other mouse models in which lncRNAs have been removed by deletion of genomic fragments did not show overt phenotypes, but after detailed analyses, some specific defects have been observed [[110](/article/10.1007/s00018-016-2174-5#ref-CR110 "Eißmann M, Gutschner T, Hämmerle M et al (2012) Loss of the abundant nuclear non-coding RNA MALAT1 is compatible with life and development. RNA Biol 9:1076–1087. doi: 10.4161/rna.21089
"), [123](/article/10.1007/s00018-016-2174-5#ref-CR123 "Sauvageau M, Goff LA, Lodato S et al (2013) Multiple knockout mouse models reveal lincRNAs are required for life and brain development. elife 2:e01749. doi:
10.7554/eLife.01749
")–[126](/article/10.1007/s00018-016-2174-5#ref-CR126 "Li L, Chang HY (2014) Physiological roles of long noncoding RNAs: insight from knockout mice. Trends Cell Biol. doi:
10.1016/j.tcb.2014.06.003
")\]. For instance, deletion of _NEAT1_ resulted in viable and fertile mice, however, knockout females showed about 50 % reduced fertility due to reduced progesterone production and corpus luteum formation \[[127](/article/10.1007/s00018-016-2174-5#ref-CR127 "Nakagawa S, Shimada M, Yanaka K et al (2014) The lncRNA Neat1 is required for corpus luteum formation and the establishment of pregnancy in a subpopulation of mice. Development. doi:
10.1242/dev.110544
")\]. Ovary transplantation or progesterone administration rescued offspring rates, confirming the cause of the phenotype, though the molecular mechanism remains to be determined.Fendrr is one of few rare examples of lncRNAs demonstrated to play an essential role in organ development and embryo survival. Fendrr, which is divergently transcribed from the Foxf1 promoter, was analyzed by two different strategies [[47](/article/10.1007/s00018-016-2174-5#ref-CR47 "Grote P, Wittler L, Hendrix D et al (2013) The tissue-specific lncRNA Fendrr is an essential regulator of heart and body wall development in the mouse. Dev Cell 24:206–214. doi: 10.1016/j.devcel.2012.12.012
"), [123](/article/10.1007/s00018-016-2174-5#ref-CR123 "Sauvageau M, Goff LA, Lodato S et al (2013) Multiple knockout mouse models reveal lincRNAs are required for life and brain development. elife 2:e01749. doi:
10.7554/eLife.01749
")\]. In one study, the insertion of a transcriptional stop cassette replacing the first exon of _Fendrr_ leaving most of the genomic context undisturbed resulted in impaired heart function, omphalocele (defects in body wall development), and embryonic death \[[47](/article/10.1007/s00018-016-2174-5#ref-CR47 "Grote P, Wittler L, Hendrix D et al (2013) The tissue-specific lncRNA Fendrr is an essential regulator of heart and body wall development in the mouse. Dev Cell 24:206–214. doi:
10.1016/j.devcel.2012.12.012
")\]. _Fendrr_ interacts with chromatin-modifying complexes and with DNA, possibly through triplex formation, and thereby changes the chromatin landscape of specific target promoters in _trans_, in particular those of mesodermal transcription factors \[[46](/article/10.1007/s00018-016-2174-5#ref-CR46 "Grote P, Herrmann BG (2013) The long non-coding RNA Fendrr links epigenetic control mechanisms to gene regulatory networks in mammalian embryogenesis. RNA Biol 10:1579–1585. doi:
10.4161/rna.26165
")\]. A BAC-transgene expressing a single dose of _Fendrr_ rescued the developmental defects caused by the loss of _Fendrr_, strongly suggesting that the observed phenotype is caused by lack of _Fendrr_ RNA, and not by disruption of genomic sites or promoter elements of the adjacent _Foxf1_ gene. This conclusion was supported by a different knockout strategy, in which the first exon was left intact and which resulted in perinatal death \[[123](/article/10.1007/s00018-016-2174-5#ref-CR123 "Sauvageau M, Goff LA, Lodato S et al (2013) Multiple knockout mouse models reveal lincRNAs are required for life and brain development. elife 2:e01749. doi:
10.7554/eLife.01749
")\]. In the early embryo, _Fendrr_ is only transiently expressed in nascent lateral mesoderm \[[47](/article/10.1007/s00018-016-2174-5#ref-CR47 "Grote P, Wittler L, Hendrix D et al (2013) The tissue-specific lncRNA Fendrr is an essential regulator of heart and body wall development in the mouse. Dev Cell 24:206–214. doi:
10.1016/j.devcel.2012.12.012
")\]. However, later during development it is expressed in the lungs, a derivate of endoderm and lateral mesoderm, where it again might play an essential role \[[123](/article/10.1007/s00018-016-2174-5#ref-CR123 "Sauvageau M, Goff LA, Lodato S et al (2013) Multiple knockout mouse models reveal lincRNAs are required for life and brain development. elife 2:e01749. doi:
10.7554/eLife.01749
"), [128](/article/10.1007/s00018-016-2174-5#ref-CR128 "Herriges MJ, Swarr DT, Morley MP et al (2014) Long noncoding RNAs are spatially correlated with transcription factors and regulate lung development. Genes Dev 28:1363–1379. doi:
10.1101/gad.238782.114
")\].More lncRNAs involved in mesoderm and endoderm development have been reported. For instance DEANR1 (also known as ALIEN) is expressed downstream of the endoderm master regulator FOXA2 [[76](/article/10.1007/s00018-016-2174-5#ref-CR76 "Jiang W, Liu Y, Liu R et al (2015) The lncRNA DEANR1 facilitates human endoderm differentiation by activating FOXA2 expression. Cell Rep 11:137–148. doi: 10.1016/j.celrep.2015.03.008
"), [77](/article/10.1007/s00018-016-2174-5#ref-CR77 "Kurian L, Aguirre A, Sancho-Martinez I et al (2015) Identification of novel long noncoding RNAs underlying vertebrate cardiovascular development. Circulation 131:1278–1290. doi:
10.1161/CIRCULATIONAHA.114.013303
")\]. _DEANR1_ depletion results in reduced expression of _FOXA2_ and accordingly to reduced differentiation of hESCs towards definitive endoderm. Interestingly, this defect can be rescued by restoring normal levels of _FOXA2_ expression, suggesting that _DEANR1_ functions mainly in _cis_. Another study described the same lncRNA, here called _ALIEN_, in cardiovascular progenitor cells in mouse and zebrafish. Depletion of _ALIEN_ interfered with cardiac development, although the functional mechanism remains to be investigated further. _DEANR1_ was described to be localized in the nucleus, whereas _ALIEN_ was seen in the perinuclear space and in the cytoplasm, even though both were analyzed in differentiating human stem cells. The discrepancy between the two studies might be a consequence of modifications in the differentiation method \[[76](/article/10.1007/s00018-016-2174-5#ref-CR76 "Jiang W, Liu Y, Liu R et al (2015) The lncRNA DEANR1 facilitates human endoderm differentiation by activating FOXA2 expression. Cell Rep 11:137–148. doi:
10.1016/j.celrep.2015.03.008
"), [77](/article/10.1007/s00018-016-2174-5#ref-CR77 "Kurian L, Aguirre A, Sancho-Martinez I et al (2015) Identification of novel long noncoding RNAs underlying vertebrate cardiovascular development. Circulation 131:1278–1290. doi:
10.1161/CIRCULATIONAHA.114.013303
")\].Also, the transcription factor Pitx2, which is essential for the development of many different organs, especially during the establishment of left–right asymmetry, is regulated by a lncRNA in the embryonic gut [[100](/article/10.1007/s00018-016-2174-5#ref-CR100 "Welsh IC, Kwak H, Chen FL et al (2015) Chromatin architecture of the Pitx2 locus requires CTCF- and Pitx2-dependent asymmetry that mirrors embryonic gut laterality. Cell Rep. doi: 10.1016/j.celrep.2015.08.075
")\]. In this case, the lncRNA _Playrr_ is transcribed from a distant known enhancer region. The expression patterns of _Pitx2_ and _Playrr_ are negatively correlated due to interference of _Playrr_ with _Pitx2_ activation (for details see above).LncRNAs expressed during neuronal and brain development have attracted particular attention. As described earlier, many lncRNAs are highly specifically expressed in distinct cell types, as illustratively shown in a targeted LacZ reporter screen [[37](/article/10.1007/s00018-016-2174-5#ref-CR37 "Goff LA, Groff AF, Sauvageau M et al (2015) Spatiotemporal expression and transcriptional perturbations by long noncoding RNAs in the mouse brain. Proc Natl Acad Sci USA 112:6855–6862. doi: 10.1073/pnas.1411263112
")\] and in expression screens \[[36](/article/10.1007/s00018-016-2174-5#ref-CR36 "Mercer TR, Dinger ME, Sunkin SM et al (2008) Specific expression of long noncoding RNAs in the mouse brain. Proc Natl Acad Sci USA 105:716–721. doi:
10.1073/pnas.0706729105
"), [107](/article/10.1007/s00018-016-2174-5#ref-CR107 "Aprea J, Prenninger S, Dori M et al (2013) Transcriptome sequencing during mouse brain development identifies long non-coding RNAs functionally involved in neurogenic commitment. EMBO J 32:3145–3160. doi:
10.1038/emboj.2013.245
"), [129](/article/10.1007/s00018-016-2174-5#ref-CR129 "Ng S-Y, Johnson R, Stanton LW (2012) Human long non-coding RNAs promote pluripotency and neuronal differentiation by association with chromatin modifiers and transcription factors. EMBO J 31:522–533. doi:
10.1038/emboj.2011.459
"), [130](/article/10.1007/s00018-016-2174-5#ref-CR130 "Ramos AD, Diaz A, Nellore A et al (2013) Integration of genome-wide approaches identifies lncRNAs of adult neural stem cells and their progeny in vivo. Cell Stem Cell 12:616–628. doi:
10.1016/j.stem.2013.03.003
")\]. A number of lncRNAs located close to the _PouIII_ genes _Pou3f3_ and _Pou3f2_ (_Brn1_ and _Brn2_) encoding important neuronal transcription factors, were also found to play roles in neuronal development \[[37](/article/10.1007/s00018-016-2174-5#ref-CR37 "Goff LA, Groff AF, Sauvageau M et al (2015) Spatiotemporal expression and transcriptional perturbations by long noncoding RNAs in the mouse brain. Proc Natl Acad Sci USA 112:6855–6862. doi:
10.1073/pnas.1411263112
"), [105](/article/10.1007/s00018-016-2174-5#ref-CR105 "Ramos AD, Andersen RE, Liu SJ et al (2015) The long noncoding RNA Pnky regulates neuronal differentiation of embryonic and postnatal neural stem cells. Cell Stem Cell 16:439–447. doi:
10.1016/j.stem.2015.02.007
"), [123](/article/10.1007/s00018-016-2174-5#ref-CR123 "Sauvageau M, Goff LA, Lodato S et al (2013) Multiple knockout mouse models reveal lincRNAs are required for life and brain development. elife 2:e01749. doi:
10.7554/eLife.01749
"), [131](/article/10.1007/s00018-016-2174-5#ref-CR131 "Chalei V, Sansom SN, Kong L et al (2014) The long non-coding RNA Dali is an epigenetic regulator of neural differentiation. elife 3:e04530. doi:
10.7554/eLife.04530
")\]. The lncRNA _Dali_ (or _Dalir_) (DNMT1-Associated Long Intergenic RNA) is transcribed 50 kb downstream of _Pou3f3._ The depletion of _Dali_ leads to a reduction of _Pou3f3_ expression and to impaired neuronal differentiation \[[131](/article/10.1007/s00018-016-2174-5#ref-CR131 "Chalei V, Sansom SN, Kong L et al (2014) The long non-coding RNA Dali is an epigenetic regulator of neural differentiation. elife 3:e04530. doi:
10.7554/eLife.04530
")\]. Gene expression analysis upon knockdown and genome-wide localization (CHART) revealed POU3F3 dependent and independent targets of _Dali_. Furthermore, _Dali_ physically interacts not only with various chromatin modifiers, but also with POU3F3 itself suggesting a multilayered association between _Dali_ and POU3F3\. The replacement of _Pantr1_ (_POU domain_, _class 3_, _transcription factor 3 adjacent noncoding transcript_), which is divergently transcribed to _Pou3f3_, by insertion of the lacZ gene did not influence _Pou3f3_ transcription itself, but modulated other _Pou3f_ transcription factors in _trans_ \[[37](/article/10.1007/s00018-016-2174-5#ref-CR37 "Goff LA, Groff AF, Sauvageau M et al (2015) Spatiotemporal expression and transcriptional perturbations by long noncoding RNAs in the mouse brain. Proc Natl Acad Sci USA 112:6855–6862. doi:
10.1073/pnas.1411263112
")\]. _Pantr2_ (or _Linc_\-_Brn1b_), expressed convergent with _Pou3f3_, did not have this effect on _Pou3f_ factors, and upon deletion led to fewer intermediate progenitors in the developing cortex. Thus, each of these three different lncRNAs, which are all transcribed near the _Pou3f3_ locus, affect neuronal development yet through distinct mechanisms.Furthermore, another lncRNA located in a region important for neural development is Evf2 in the Dlx5/6 locus [[69](/article/10.1007/s00018-016-2174-5#ref-CR69 "Bond AM, Vangompel MJW, Sametsky EA et al (2009) Balanced gene regulation by an embryonic brain ncRNA is critical for adult hippocampal GABA circuitry. Nat Neurosci 12:1020–1027. doi: 10.1038/nn.2371
")\]. _Evf2_ was targeted by inserting a polyadenylation signal close to the promoter without deleting genomic DNA. _Evf2_ −/− embryos have less GABAergic neurons than wild type controls. This mutant effect could be rescued by in utero electroporation of _Evf2_ RNA, confirming that the phenotype is indeed dependent on this lncRNA.RMST, also termed non-coding rhabdomyosarcoma (NCRMS) in humans, is a noncoding transcript expressed in rhabdomyosarcomas [[132](/article/10.1007/s00018-016-2174-5#ref-CR132 "Chan AS, Thorner PS, Squire JA, Zielenska M (2002) Identification of a novel gene NCRMS on chromosome 12q21 with differential expression between rhabdomyosarcoma subtypes. Oncogene 21:3029–3037. doi: 10.1038/sj.onc.1205460
")\] and in developing dopaminergic neurons during murine development \[[133](/article/10.1007/s00018-016-2174-5#ref-CR133 "Uhde CW, Vives J, Jaeger I, Li M (2010) Rmst is a novel marker for the mouse ventral mesencephalic floor plate and the anterior dorsal midline cells. PLoS One 5:e8641. doi:
10.1371/journal.pone.0008641
")\]. _RMST_ is induced during neuronal differentiation of hESCs, and its depletion interferes with neuronal differentiation via its binding partner SOX2 (see above) \[[78](/article/10.1007/s00018-016-2174-5#ref-CR78 "Ng S-Y, Bogu GK, Soh BS, Stanton LW (2013) The long noncoding RNA RMST interacts with SOX2 to regulate neurogenesis. Mol Cell 51:349–359. doi:
10.1016/j.molcel.2013.07.017
"), [107](/article/10.1007/s00018-016-2174-5#ref-CR107 "Aprea J, Prenninger S, Dori M et al (2013) Transcriptome sequencing during mouse brain development identifies long non-coding RNAs functionally involved in neurogenic commitment. EMBO J 32:3145–3160. doi:
10.1038/emboj.2013.245
"), [129](/article/10.1007/s00018-016-2174-5#ref-CR129 "Ng S-Y, Johnson R, Stanton LW (2012) Human long non-coding RNAs promote pluripotency and neuronal differentiation by association with chromatin modifiers and transcription factors. EMBO J 31:522–533. doi:
10.1038/emboj.2011.459
")\]. Interestingly, _RMST_ was found associated with obesity in a GWAS study, possibly pointing to a role of _RMST_ in a neuronal regulatory network influencing energy balance \[[134](/article/10.1007/s00018-016-2174-5#ref-CR134 "Wheeler E, Huang N, Bochukova EG et al (2013) Genome-wide SNP and CNV analysis identifies common and low-frequency variants associated with severe early-onset obesity. Nat Genet 45:513–517. doi:
10.1038/ng.2607
")\].Two more lncRNAs involved in nervous system development are the retinal noncoding RNA2 and 4 (RNCR2 and RNCR4). They were first found to be expressed in the developing retina and later also assigned a functional role in this process [[135](/article/10.1007/s00018-016-2174-5#ref-CR135 "Blackshaw S, Harpavat S, Trimarchi J et al (2004) Genomic analysis of mouse retinal development. PLoS Biol 2:E247. doi: 10.1371/journal.pbio.0020247
")\]. _RNCR4_ is part of a network including the RNA helicase DDX3X and the _miR_\-_183/96/182_ that is essential for retinal architecture \[[136](/article/10.1007/s00018-016-2174-5#ref-CR136 "Krol J, Krol I, Alvarez CPP et al (2015) A network comprising short and long noncoding RNAs and RNA helicase controls mouse retina architecture. Nat Comms 6:7305. doi:
10.1038/ncomms8305
")\]. _RNCR2_, also known as _MIAT_ or _Gomafu_, interferes with retinal cell differentiation when overexpressed or depleted \[[135](/article/10.1007/s00018-016-2174-5#ref-CR135 "Blackshaw S, Harpavat S, Trimarchi J et al (2004) Genomic analysis of mouse retinal development. PLoS Biol 2:E247. doi:
10.1371/journal.pbio.0020247
"), [137](/article/10.1007/s00018-016-2174-5#ref-CR137 "Rapicavoli NA, Poth EM, Blackshaw S (2010) The long noncoding RNA RNCR2 directs mouse retinal cell specification. BMC Dev Biol 10:49. doi:
10.1186/1471-213X-10-49
")\]. Furthermore _MIAT_ is expressed earlier in the developing brain, where it localizes to the nuclear matrix \[[138](/article/10.1007/s00018-016-2174-5#ref-CR138 "Sone M, Hayashi T, Tarui H et al (2007) The mRNA-like noncoding RNA Gomafu constitutes a novel nuclear domain in a subset of neurons. J Cell Sci 120:2498–2506. doi:
10.1242/jcs.009357
")\]. Purifying distinct subpopulations of the developing brain at E14.5 revealed that _MIAT_ is highly abundant in differentiating progenitors, where it is involved in corticogenesis by regulating the splicing pattern of _Wnt7b_ \[[107](/article/10.1007/s00018-016-2174-5#ref-CR107 "Aprea J, Prenninger S, Dori M et al (2013) Transcriptome sequencing during mouse brain development identifies long non-coding RNAs functionally involved in neurogenic commitment. EMBO J 32:3145–3160. doi:
10.1038/emboj.2013.245
")\] (Fig. [2](/article/10.1007/s00018-016-2174-5#Fig2)g).Another ectodermal derivative, the developing epidermis, has also been shown to be regulated by lncRNAs. During epidermal differentiation and maintenance, the nuclear lncRNA ANCR (anti-differentiation ncRNA) is essential for maintaining the proliferating progenitor pool by repressing differentiation factors, amongst them the transcription factors MAF and MAFB, probably in a PRC2 linked manner [[139](/article/10.1007/s00018-016-2174-5#ref-CR139 "Kretz M, Webster DE, Flockhart RJ et al (2012) Suppression of progenitor differentiation requires the long noncoding RNA ANCR. Genes Dev 26:338–343. doi: 10.1101/gad.182121.111
")–[141](/article/10.1007/s00018-016-2174-5#ref-CR141 "Lopez-Pajares V, Qu K, Zhang J et al (2015) A LncRNA-MAF:MAFB transcription factor network regulates epidermal differentiation. Dev Cell 32:693–706. doi:
10.1016/j.devcel.2015.01.028
")\]. Depletion of _ANCR_ leads to differentiation and loss of progenitor cells. Its differentiation promoting counterpart is a cytosolic lncRNA, terminal differentiation induced ncNRA (_TINCR_) that interacts with STAU1 and as a protein-RNA complex stabilizes mRNAs of pro-differentiation genes including _MAF_ and _MAFB_ \[[141](/article/10.1007/s00018-016-2174-5#ref-CR141 "Lopez-Pajares V, Qu K, Zhang J et al (2015) A LncRNA-MAF:MAFB transcription factor network regulates epidermal differentiation. Dev Cell 32:693–706. doi:
10.1016/j.devcel.2015.01.028
"), [142](/article/10.1007/s00018-016-2174-5#ref-CR142 "Kretz M, Siprashvili Z, Chu C et al (2013) Control of somatic tissue differentiation by the long non-coding RNA TINCR. Nature 493:231–235. doi:
10.1038/nature11661
")\] (Fig. [2](/article/10.1007/s00018-016-2174-5#Fig2)h). Ectopic expression of MAF:MAFB in _TINCR_ depleted cells restored the expression of the differentiation gene signature showing that _TINCR_ is crucial for ensuring appropriate protein levels in particular of MAF and MAFB.Functions of lncRNAs in pathological settings
LncRNAs as a “new” class of molecules have also attracted attention in pathological settings. Here, we will highlight two very prominent areas, cancer and cardiovascular disease.
Cancer
Approximately one third of publications currently listed in PubMed linked with the keyword “lncRNA” also contain the keyword “cancer”. Even before the era of next generation sequencing, a few studies found lncRNAs to be dysregulated in tumors [[35](/article/10.1007/s00018-016-2174-5#ref-CR35 "Diederichs S (2014) The four dimensions of noncoding RNA conservation. Trends Genet 30:121–123. doi: 10.1016/j.tig.2014.01.004
"), [143](/article/10.1007/s00018-016-2174-5#ref-CR143 "Ji P, Diederichs S, Wang W et al (2003) MALAT-1, a novel noncoding RNA, and thymosin beta4 predict metastasis and survival in early-stage non-small cell lung cancer. Oncogene 22:8031–8041. doi:
10.1038/sj.onc.1206928
"), [144](/article/10.1007/s00018-016-2174-5#ref-CR144 "Srikantan V, Zou Z, Petrovics G et al (2000) PCGEM1, a prostate-specific gene, is overexpressed in prostate cancer. Proc Natl Acad Sci USA 97:12216–12221. doi:
10.1073/pnas.97.22.12216
")\]. To date, numerous primary tumor samples, metastatic biopsies, the adjacent normal tissue and cancer cell lines have been profiled for their RNA expression and the results recapitulate the high degree of specificity found for noncoding RNAs in normal tissue compared to protein-coding genes \[[25](/article/10.1007/s00018-016-2174-5#ref-CR25 "Iyer MK, Niknafs YS, Malik R et al (2015) The landscape of long noncoding RNAs in the human transcriptome. Nat Genet 47:199–208. doi:
10.1038/ng.3192
"), [67](/article/10.1007/s00018-016-2174-5#ref-CR67 "Prensner JR, Iyer MK, Sahu A et al (2013) The long noncoding RNA SChLAP1 promotes aggressive prostate cancer and antagonizes the SWI/SNF complex. Nat Genet 45:1392–1398. doi:
10.1038/ng.2771
"), [145](/article/10.1007/s00018-016-2174-5#ref-CR145 "Brunner AL, Beck AH, Edris B et al (2012) Transcriptional profiling of long non-coding RNAs and novel transcribed regions across a diverse panel of archived human cancers. Genome Biol 13:R75. doi:
10.1186/gb-2012-13-8-r75
")–[147](/article/10.1007/s00018-016-2174-5#ref-CR147 "Du Z, Fei T, Verhaak RGW et al (2013) Integrative genomic analyses reveal clinically relevant long noncoding RNAs in human cancer. Nat Struct Mol Biol 20:908–913. doi:
10.1038/nsmb.2591
")\]. Clustering of lncRNA expression patterns identifies subclasses of tumor types with high significance, opening the possibility to define tumor subtypes with a high chance to respond to targeted therapy. A study that considered samples from more than 5000 tumors from the cancer genome atlas (TCGA) project, demonstrated that, while approximately the same number of protein coding and lncRNA genes were dysregulated, 60 % of lncRNAs showed specificity for only one tumor type \[[146](/article/10.1007/s00018-016-2174-5#ref-CR146 "Yan X, Hu Z, Feng Y et al (2015) Comprehensive genomic characterization of long non-coding RNAs across human cancers. Cancer Cell 28:529–540. doi:
10.1016/j.ccell.2015.09.006
")\].In addition, somatic copy number alterations (SCNA) found in cancer tissue often include lncRNA loci [[57](/article/10.1007/s00018-016-2174-5#ref-CR57 "Hu X, Feng Y, Zhang D et al (2014) A functional genomic approach identifies FAL1 as an oncogenic long noncoding RNA that associates with BMI1 and represses p21 expression in cancer. Cancer Cell 26:344–357. doi: 10.1016/j.ccr.2014.07.009
"), [146](/article/10.1007/s00018-016-2174-5#ref-CR146 "Yan X, Hu Z, Feng Y et al (2015) Comprehensive genomic characterization of long non-coding RNAs across human cancers. Cancer Cell 28:529–540. doi:
10.1016/j.ccell.2015.09.006
"), [147](/article/10.1007/s00018-016-2174-5#ref-CR147 "Du Z, Fei T, Verhaak RGW et al (2013) Integrative genomic analyses reveal clinically relevant long noncoding RNAs in human cancer. Nat Struct Mol Biol 20:908–913. doi:
10.1038/nsmb.2591
")\]. For example, the lncRNA _FAL1_, a lncRNA that was found amplified in a number of different tumor types, was shown to be important for the tumorigenic effects of different cell lines via a BMI1-mediated mechanism (see above), and its expression level and copy number significantly correlated with patient survival \[[57](/article/10.1007/s00018-016-2174-5#ref-CR57 "Hu X, Feng Y, Zhang D et al (2014) A functional genomic approach identifies FAL1 as an oncogenic long noncoding RNA that associates with BMI1 and represses p21 expression in cancer. Cancer Cell 26:344–357. doi:
10.1016/j.ccr.2014.07.009
")\]. Furthermore, several lncRNAs have been shown to be prognostic for overall or disease-free survival of patients \[[67](/article/10.1007/s00018-016-2174-5#ref-CR67 "Prensner JR, Iyer MK, Sahu A et al (2013) The long noncoding RNA SChLAP1 promotes aggressive prostate cancer and antagonizes the SWI/SNF complex. Nat Genet 45:1392–1398. doi:
10.1038/ng.2771
"), [82](/article/10.1007/s00018-016-2174-5#ref-CR82 "Xing Z, Lin A, Li C et al (2014) lncRNA directs cooperative epigenetic regulation downstream of chemokine signals. Cell 159:1110–1125. doi:
10.1016/j.cell.2014.10.013
"), [148](/article/10.1007/s00018-016-2174-5#ref-CR148 "Pandey GK, Mitra S, Subhash S et al (2014) The risk-associated long noncoding RNA NBAT-1 controls neuroblastoma progression by regulating cell proliferation and neuronal differentiation. Cancer Cell 26:722–737. doi:
10.1016/j.ccell.2014.09.014
")–[150](/article/10.1007/s00018-016-2174-5#ref-CR150 "Xue S, Li Q-W, Che J-P et al (2015) Decreased expression of long non-coding RNA NBAT-1 is associated with poor prognosis in patients with clear cell renal cell carcinoma. Int J Clin Exp Pathol 8:3765–3774")\], information that is integrated into therapeutic decisions. Depletion of selected lncRNAs led to reduced malignancy, assayed as tumor size or metastatic potential in xenograft experiments \[[67](/article/10.1007/s00018-016-2174-5#ref-CR67 "Prensner JR, Iyer MK, Sahu A et al (2013) The long noncoding RNA SChLAP1 promotes aggressive prostate cancer and antagonizes the SWI/SNF complex. Nat Genet 45:1392–1398. doi:
10.1038/ng.2771
"), [72](/article/10.1007/s00018-016-2174-5#ref-CR72 "Wang Y, He L, Du Y et al (2015) The long noncoding RNA lncTCF7 promotes self-renewal of human liver cancer stem cells through activation of Wnt signaling. Cell Stem Cell 16:413–425. doi:
10.1016/j.stem.2015.03.003
"), [82](/article/10.1007/s00018-016-2174-5#ref-CR82 "Xing Z, Lin A, Li C et al (2014) lncRNA directs cooperative epigenetic regulation downstream of chemokine signals. Cell 159:1110–1125. doi:
10.1016/j.cell.2014.10.013
"), [101](/article/10.1007/s00018-016-2174-5#ref-CR101 "Yang L, Lin C, Jin C et al (2013) lncRNA-dependent mechanisms of androgen-receptor-regulated gene activation programs. Nature 500:598–602. doi:
10.1038/nature12451
"), [148](/article/10.1007/s00018-016-2174-5#ref-CR148 "Pandey GK, Mitra S, Subhash S et al (2014) The risk-associated long noncoding RNA NBAT-1 controls neuroblastoma progression by regulating cell proliferation and neuronal differentiation. Cancer Cell 26:722–737. doi:
10.1016/j.ccell.2014.09.014
"), [149](/article/10.1007/s00018-016-2174-5#ref-CR149 "Gutschner T, Hämmerle M, Eißmann M et al (2013) The noncoding RNA MALAT1 is a critical regulator of the metastasis phenotype of lung cancer cells. Cancer Res 73:1180–1189. doi:
10.1158/0008-5472.CAN-12-2850
"), [151](/article/10.1007/s00018-016-2174-5#ref-CR151 "Trimarchi T, Bilal E, Ntziachristos P et al (2014) Genome-wide mapping and characterization of Notch-regulated long noncoding RNAs in acute leukemia. Cell 158:593–606. doi:
10.1016/j.cell.2014.05.049
")–[154](/article/10.1007/s00018-016-2174-5#ref-CR154 "Dijkstra JM, Alexander DB (2015) The “NF-κB interacting long noncoding RNA” (NKILA) transcript is antisense to cancer-associated gene PMEPA1. F1000Res 4:96. doi:
10.12688/f1000research.6400.1
")\], although these experiments were not able to distinguish between effects on tumor formation versus tumor progression. Further refinement was achieved when RNA knockdown was induced after tumor formation, mimicking therapeutic conditions. Reduced tumor growth was, for instance, observed upon _FAL1_ depletion \[[57](/article/10.1007/s00018-016-2174-5#ref-CR57 "Hu X, Feng Y, Zhang D et al (2014) A functional genomic approach identifies FAL1 as an oncogenic long noncoding RNA that associates with BMI1 and represses p21 expression in cancer. Cancer Cell 26:344–357. doi:
10.1016/j.ccr.2014.07.009
")\]. In a different study, the inhibition of _MALAT1_ after tumor induction reduced metastasis formation in a xenograft lung cancer model and in a metastasizing breast cancer model \[[149](/article/10.1007/s00018-016-2174-5#ref-CR149 "Gutschner T, Hämmerle M, Eißmann M et al (2013) The noncoding RNA MALAT1 is a critical regulator of the metastasis phenotype of lung cancer cells. Cancer Res 73:1180–1189. doi:
10.1158/0008-5472.CAN-12-2850
"), [155](/article/10.1007/s00018-016-2174-5#ref-CR155 "Arun G, Diermeier S, Akerman M et al (2015) Differentiation of mammary tumors and reduction in metastasis upon Malat1 lncRNA loss. Genes Dev 30:34–51. doi:
10.1101/gad.270959.115
")\]. _MALAT1_ knockout mice do not show overt phenotypes \[[110](/article/10.1007/s00018-016-2174-5#ref-CR110 "Eißmann M, Gutschner T, Hämmerle M et al (2012) Loss of the abundant nuclear non-coding RNA MALAT1 is compatible with life and development. RNA Biol 9:1076–1087. doi:
10.4161/rna.21089
"), [156](/article/10.1007/s00018-016-2174-5#ref-CR156 "Nakagawa S, Ip JY, Shioi G et al (2012) Malat1 is not an essential component of nuclear speckles in mice. RNA 18:1487–1499. doi:
10.1261/rna.033217.112
"), [157](/article/10.1007/s00018-016-2174-5#ref-CR157 "Zhang B, Arun G, Mao YS et al (2012) The lncRNA Malat1 is dispensable for mouse development but its transcription plays a cis-regulatory role in the adult. Cell Rep 2:111–123. doi:
10.1016/j.celrep.2012.06.003
")\] suggesting that down-regulation of _MALAT1_ in humans might also not be harmful for normal human cells, which makes it an attractive target for systemic application in patients.A special case is the noncoding pseudo gene BRAFP1, which has been shown to be either mutated or aberrantly expressed in cancers, like its coding homologue BRAF. A mouse model revealed that induced overexpression of _Braf_-rs1 (the murine homologue of BRAFP1) leads to DLBCL (diffuse large B cell lymphomas)-like tumors. However, when the transgene was first induced to form tumors and then shut down again, the tumors regressed demonstrating the functional significance of the pseudo gene [[158](/article/10.1007/s00018-016-2174-5#ref-CR158 "Karreth FA, Reschke M, Ruocco A et al (2015) The BRAF pseudogene functions as a competitive endogenous RNA and induces lymphoma in vivo. Cell. doi: 10.1016/j.cell.2015.02.043
")\]. _Braf_\-_rs1_ was shown to cause upregulation of _Braf_ by acting as a ceRNA (competing endogenous RNA) and by “sponging” miRNAs that would otherwise target _Braf_ transcripts.One mechanism that cells can employ to prevent tumor formation is oncogene-induced senescence (OIS) [[159](/article/10.1007/s00018-016-2174-5#ref-CR159 "Gil J, Peters G (2006) Regulation of the INK4b-ARF-INK4a tumour suppressor locus: all for one or one for all. Nat Rev Mol Cell Biol 7:667–677. doi: 10.1038/nrm1987
")\]. The _INK4B_\-_ARF_\-_INK4A_ locus, which plays a major role in OIS, is silenced in proliferating cells by Polycomb group proteins. This mechanism is dependent on the lncRNA _ANRIL_, which is transcribed from within the locus and acts in _cis_ \[[58](/article/10.1007/s00018-016-2174-5#ref-CR58 "Pasmant E, Laurendeau I, Héron D et al (2007) Characterization of a germ-line deletion, including the entire INK4/ARF locus, in a melanoma-neural system tumor family: identification of ANRIL, an antisense noncoding RNA whose expression coclusters with ARF. Cancer Res 67:3963–3969. doi:
10.1158/0008-5472.CAN-06-2004
"), [59](/article/10.1007/s00018-016-2174-5#ref-CR59 "Yap KL, Li S, Muñoz-Cabello AM et al (2010) Molecular interplay of the noncoding RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a. Mol Cell 38:662–674. doi:
10.1016/j.molcel.2010.03.021
"), [160](/article/10.1007/s00018-016-2174-5#ref-CR160 "Wan G, Mathur R, Hu X et al (2013) Long non-coding RNA ANRIL (CDKN2B-AS) is induced by the ATM-E2F1 signaling pathway. Cell Signal 25:1086–1095. doi:
10.1016/j.cellsig.2013.02.006
")\]. Furthermore, the _MIR31HG_ gene located 400 kb upstream of the _INK4B_\-_ARF_\-_INK4A_ locus, also encodes a lncRNA involved in the regulation of _INK4A_ \[[161](/article/10.1007/s00018-016-2174-5#ref-CR161 "Montes M, Nielsen MM, Maglieri G et al (2015) The lncRNA MIR31HG regulates p16(INK4A) expression to modulate senescence. Nat Comms 6:6967. doi:
10.1038/ncomms7967
")\]. A third lncRNA functioning in OIS, _UCA1_, stabilizes mRNAs of protein-coding genes involved in the senescence pathway, probably by sequestering hnRNPKA1 \[[162](/article/10.1007/s00018-016-2174-5#ref-CR162 "Kumar PP, Emechebe U, Smith R et al (2014) Coordinated control of senescence by lncRNA and a novel T-box3 co-repressor complex. elife. doi:
10.7554/eLife.02805
")\]. Also, the tumor suppressor p53 and its associated signaling pathways include lncRNAs. p53 binds and regulates numerous lncRNAs, such as the _linc_\-_p21_, which in turn regulates gene expression in concert with p53 and p21 \[[163](/article/10.1007/s00018-016-2174-5#ref-CR163 "Huarte M, Guttman M, Feldser D et al (2010) A large intergenic noncoding RNA induced by p53 mediates global gene repression in the p53 response. Cell 142:409–419. doi:
10.1016/j.cell.2010.06.040
")–[166](/article/10.1007/s00018-016-2174-5#ref-CR166 "Wu G, Cai J, Han Y et al (2014) LincRNA-p21 regulates neointima formation, vascular smooth muscle cell proliferation, apoptosis, and atherosclerosis by enhancing p53 activity. Circulation 130:1452–1465. doi:
10.1161/CIRCULATIONAHA.114.011675
")\].Cardiovascular diseases
LncRNAs have also been linked to cardiovascular diseases (CVD), which are a major cause of death in western societies, and have been studied both in patient data, as well as in mouse models. It was observed that several genomic regions significantly associated with myocardial infarction encode lncRNAs, for example MIAT and ANRIL [[167](/article/10.1007/s00018-016-2174-5#ref-CR167 "Ishii N, Ozaki K, Sato H et al (2006) Identification of a novel non-coding RNA, MIAT, that confers risk of myocardial infarction. J Hum Genet 51:1087–1099. doi: 10.1007/s10038-006-0070-9
"), [168](/article/10.1007/s00018-016-2174-5#ref-CR168 "Broadbent HM, Peden JF, Lorkowski S et al (2008) Susceptibility to coronary artery disease and diabetes is encoded by distinct, tightly linked SNPs in the ANRIL locus on chromosome 9p. Hum Mol Genet 17:806–814. doi:
10.1093/hmg/ddm352
")\]. Deletion of a 70 kb region including a large proportion of the _ANRIL_ locus led to increased mortality in mice, particularly in combination with a high fat diet \[[169](/article/10.1007/s00018-016-2174-5#ref-CR169 "Visel A, Zhu Y, May D et al (2010) Targeted deletion of the 9p21 non-coding coronary artery disease risk interval in mice. Nature 464:409–412. doi:
10.1038/nature08801
")\].In other studies, massive parallel RNA sequencing revealed numerous lncRNAs displaying altered expression following surgically-induced myocardial infarction in mice [[170](/article/10.1007/s00018-016-2174-5#ref-CR170 "Matkovich SJ, Edwards JR, Grossenheider TC et al (2014) Epigenetic coordination of embryonic heart transcription by dynamically regulated long noncoding RNAs. Proc Natl Acad Sci USA 111:12264–12269. doi: 10.1073/pnas.1410622111
"), [171](/article/10.1007/s00018-016-2174-5#ref-CR171 "Ounzain S, Micheletti R, Beckmann T et al (2015) Genome-wide profiling of the cardiac transcriptome after myocardial infarction identifies novel heart-specific long non-coding RNAs. Eur Heart J 36:353–368a. doi:
10.1093/eurheartj/ehu180
")\]. In cellular differentiation models, the depletion of selected lncRNAs impaired cardiomyocyte formation making them promising candidates for in vivo assessment of their functions \[[172](/article/10.1007/s00018-016-2174-5#ref-CR172 "Ounzain S, Micheletti R, Arnan C et al (2015) CARMEN, a human super enhancer-associated long noncoding RNA controlling cardiac specification, differentiation and homeostasis. J Mol Cell Cardiol. doi:
10.1016/j.yjmcc.2015.09.016
")\]. Analysis of the noncoding transcripts originating from the _myosin heavy chain 7 (Myh7_) locus, an important structural protein required for heart contractions, revealed that one, the lncRNA _Mhrt_, was down-regulated after induced myocardial infarction. Ectopic expression of _Mhrt_ in mice, which had been subjected to this operation reduced the severity of symptoms observed in control mice during recovery \[[68](/article/10.1007/s00018-016-2174-5#ref-CR68 "Han P, Li W, Lin C-H et al (2014) A long noncoding RNA protects the heart from pathological hypertrophy. Nature 514:102–106. doi:
10.1038/nature13596
")\]. When lncRNAs in plasma from myocardial infarction patients were profiled, a mitochondrial lncRNA named long intergenic noncoding RNA predicting cardiac remodeling (_LIPCAR_) was found to significantly correlate with mortality caused by CVD and could thus be considered as a biomarker \[[173](/article/10.1007/s00018-016-2174-5#ref-CR173 "Kumarswamy R, Bauters C, Volkmann I et al (2014) Circulating long noncoding RNA, LIPCAR, predicts survival in patients with heart failure. Circ Res 114:1569–1575. doi:
10.1161/CIRCRESAHA.114.303915
")\]. Whether this lncRNA also plays a causative role in infarction or heart failure remains to be investigated.LncRNAs have been widely suggested as biomarkers and therapeutic targets in certain pathological settings. One reason for this is the extremely specific expression of many lncRNAs, opening the potential of targeting cellular subgroups rather than affecting patients systemically as in conventional treatments. Even if a lncRNA itself is not the disease driver, it can serve as drug target in cases where it acts as co-factor for a driver gene, which is more broadly expressed. Also, since lncRNAs often play quantitative roles rather than acting as switches in genetic programs, side effects of therapeutic treatments might be easier to control.
Concluding remarks
In recent years, the analysis of lncRNAs has highlighted the large diversity of cellular functions involving RNA molecules and has greatly expanded the catalog of functions previously attributed to noncoding RNAs. By now, we can conclude that noncoding RNAs play roles at all levels of gene control, including epigenetic mechanisms and nuclear organization, as well as in RNA processing, stability and translation. The recent discoveries of lncRNA functions provide good reasons for the notion that further investigation of lncRNAs bears high chances for the discovery of novel gene regulatory mechanisms in the future.
So far, only few lncRNAs playing an essential role in embryonic development, organogenesis or tissue homeostasis have been reported. Studies based on in vitro differentiation systems can provide important insights into the molecular modes of lncRNA function in local gene control or in regulatory networks. However, with regard to the roles of lncRNAs, for example in lineage decisions and cellular differentiation many findings are hampered by the lack of definitive proof in vivo. In particular, data derived from in vitro differentiated stem cells, whose genomic integrity and quality is hard to control, have to be interpreted cautiously. Also, the type of genetic change introduced into a lncRNA gene/locus for functional analysis (transcription stop, knockin, deletion etc.) needs to be considered carefully in order to draw the right conclusions [174–[176](/article/10.1007/s00018-016-2174-5#ref-CR176 "Goff LA, Rinn JL (2015) Linking RNA biology to lncRNAs. Genome Res 25:1456–1465. doi: 10.1101/gr.191122.115
")\]. Since lncRNAs do not only act as transcripts in _cis_ or _trans_, but can also be side products of transcription affecting the expression of overlapping genes or accommodate regulatory DNA elements in their genomic loci, the analysis of their exact mode of action and functional roles in development and disease processes is more complex and difficult than that of protein-coding genes. It is now up to the responsibility of journal editors and reviewers to enforce the high standards of investigation this new and exciting field of research deserves.References
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