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Chapter 1 Diverse Functions and Mechanisms of Mammalian Long Noncoding RNAs Callie R. Merry, Courtney Niland, and Ahmad M. Khalil Abstract Long noncoding RNAs are becoming increasingly appreciated as major players in gene regulation. They have been reported to play diverse roles in many biological processes. Here, we discuss their discovery, features, and known functions in cells. While not comprehensive, this chapter should serve to illustrate the power and promise of studying long noncoding RNAs. Key words Long noncoding RNA, lncRNA, X inactivation, Nuclear structure, Gene regulation, Epigenetic regulation

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Introduction

1.1 Discovery of Long Noncoding RNAs

It is now well established that mammalian genomes encode, in addition to protein-coding genes, thousands of RNA molecules that have no protein-coding capacity, and thus are referred to as noncoding RNAs (ncRNAs) [1]. In addition to ribosomal (r) RNAs, transfer (t)RNAs and other well-studied noncoding RNAs, mammalian genomes also encode a heterogeneous population of noncoding transcripts, which are currently classified into small and long noncoding RNAs. Small noncoding RNAs, such as microRNAs and piwi-associated RNAs (piRNAs), which are ~20–32 nucleotides in length, will be discussed in other chapters in this book. By contrast, noncoding RNAs that are more than 200 nucleotides in length are referred to as long (or large) noncoding RNAs (abbreviated as lncRNAs), and will be the focus of this chapter. Although one of the earliest functional lncRNAs (i.e., Xist) was discovered in the early 1990s [2, 3], it was not until the development of high-throughput methods, such as tilling arrays and RNA sequencing, that lncRNAs were discovered on a large scale. It is now estimated that mammalian genomes encode at least 15,000–20,000 lncRNAs [4–10].

Gordon G. Carmichael (ed.), Regulatory Non-Coding RNAs: Methods and Protocols, Methods in Molecular Biology, vol. 1206, DOI 10.1007/978-1-4939-1369-5_1, © Springer Science+Business Media New York 2015

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1.2 Fighting the Dogma

The discovery of thousands of lncRNAs in the mammalian genome was initially greeted by some scientists with skepticism; they argued that lncRNAs could merely be transcribed as a result of open chromatin structure without any functional significance. However, several lines of evidence have clearly demonstrated that lncRNAs are not simply “transcriptional noise” and are indeed functional (see below). First, the expression of lncRNAs changes substantially between tissues, and within various cell types in the same tissue, suggesting that the expression of lncRNAs is highly regulated [4, 9, 11]. Secondly, the expression of lncRNAs is regulated by the same set of transcription factors that regulate protein-coding genes [6]. Lastly, and most importantly, numerous studies from independent laboratories have now demonstrated that many lncRNAs are biologically functional by experimental evidence [12–21].

1.3 Classification of Long Noncoding RNAs

Long noncoding RNAs are expressed from various regions of the genome. The currently annotated lncRNAs fall into four categories, which include antisense, intronic, bidirectional, and intervening lncRNAs: (1) Antisense lncRNAs (natural antisense transcripts, NATs) are lncRNAs that are transcribed from the opposite strand of protein-coding genes. It is now estimated that 70 % of mammalian protein-coding genes have an overlapping NAT, and the expression of a NAT can be concordant or discordant with its overlapping protein-coding partner [7, 8]. To date, many NATs have been shown to regulate the expression of their protein-coding partners by diverse mechanisms [8, 13, 22–24]. (2) Intronic lncRNAs are transcribed completely from within a single intron of a protein-coding gene. These intronic lncRNAs were initially thought to be mere products of pre-mRNA splicing; however, gene expression and functional analyses have now demonstrated that at least some of these intronic lncRNAs are independently regulated and functionally distinct from their host protein-coding genes [25, 26]. (3) Bidirectional lncRNAs share promoters with protein-coding genes but they are transcribed in the opposite direction and therefore have no overlapping sequence [18]. (4) Intervening lncRNAs (lincRNAs) are transcribed from regions that are at least 5 kb or more from protein-coding genes. LincRNAs were initially identified in humans [18] and mouse [6] by utilizing a chromatin signature of actively transcribed genes, and subsequently by RNA deep sequencing [4, 27].

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Known Functions of lncRNAs

2.1 X Chromosome Inactivation (Xi)

X chromosome inactivation (Xi) is an epigenetic process that occurs in the somatic cells of mammalian females, and results in the silencing of most genes on one of the two X chromosomes.

Diverse Functions and Mechanisms of Mammalian Long Noncoding RNAs

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Thus, Xi equalizes the dosage of X chromosome genes between males and females [28]. A specific region on the X chromosome, termed the X inactivation center, encodes a 17 kb lncRNA termed Xist (X inactive specific transcript), which is required for the initiation of Xi [2, 28, 29]. Xist is spliced, capped, polyadenylated, completely nuclear, and coats the entire X chromosome from which it is transcribed. Although the entire mechanism of Xist-mediated Xi is not completely understood, recent studies suggest that Xist transcripts serve as “docking stations” for repressive chromatin modifiers on the inactive X chromosome [30, 31]. In addition to Xist, there are several lncRNAs that are transcribed from the X inactivation center and play various roles in Xi. For example, the expression of Tsix, an antisense lncRNA to Xist, represses Xist expression from the active X chromosome [23]. Furthermore, other lncRNAs, such as Jpx and Xite, also transcribed from the X inactivation center, regulate X chromosome counting and choice by unknown mechanisms [28, 32]. lncRNAs that regulate X chromosome inactivation in mammalian females provide an important example of how multiple lncRNAs work cooperatively to regulate gene expression. 2.2 Regulation of Imprinted Genes by lncRNAs

Genomic imprinting is an epigenetic process that leads to the differential expression of a subset of genes according to their parental origin [33]. Genomic imprints are established in germ cells and maintained in the offspring by specific epigenetic marks that help distinguish maternal from paternal alleles [34]. Moreover, imprinting control regions (ICRs), which are regulated by DNA methylation and histone modifications, play an essentials role in regulating the expression of imprinted genes. A number of studies have shown that imprinted loci express many types of ncRNAs including lncRNAs. Functional studies of these lncRNAs have demonstrated that they are critical for regulating the expression of imprinted protein-coding genes as discussed below. Example 1. The lncRNA Kcnq1ot1 is expressed from the paternal allele only, and plays an essential role in the silencing of several imprinted protein-coding genes including its antisense partner Kcnq1 [33]. Genes targeted for repression by Kcnq1ot1 exhibit repressive chromatin marks including methylation of histone H3 at lysine 9 (H3K9) and lysine 27 (H3K27) [35]. Interestingly, Kcnq1ot1 targets the chromatin-modifying complexes G9a and PRC2, which modify H3K9 and H3K27 by methylation, respectively, to the paternal Kcnq1 locus leading to heterochromatin formation and, subsequently, gene repression [35]. Example 2. The lncRNA Air is transcribed only from the paternal allele antisense to the Igf2r gene, and is required for the silencing of three imprinted protein-coding genes (i.e., Igf2r, Slc22a2, and Slc22a3) [33, 36]. Mutation of Air on the paternal chromosome,

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but not on the maternal chromosome, leads to the activation of these imprinted genes suggesting that Air functions in cis [36]. Moreover, a deletion of the Igf2r, Slc22a2, and Slc22a3 genes on the maternal chromosome, while lethal alone, can be compensated by a truncated Air transcript on the paternal allele [36]. These studies clearly demonstrate that Air is required for the parentalspecific silencing of Igf2r, Slc22a2, and Slc22a3. More recently, Air has been shown to interact with and guide the chromatinmodifying complex G9a, a H3K9 methyltransferase, to the Igf2r/ Slc22a2/Slc22a3 locus to regulate gene expression [37]. The observations above clearly demonstrate that Air and Kcnq1ot1 as well as other lncRNAs that are expressed from imprinted loci play essential roles in regulating the expression of imprinted protein-coding genes by guiding chromatin-modifying complexes to imprinted loci and, potentially, by other mechanisms as well. 2.3 Emerging Roles for lncRNAs in Development and Cellular Functions

The differentiation of pluripotent and progenitor cells in humans and other multicellular organisms into various tissues is highly regulated and requires various transcription factors that modulate specific gene expression patterns. Recent studies suggest that some lncRNAs also contribute to developmental pathways, as well as modulate cellular function, by regulating gene expression and/or the localization of proteins within the cell. Below, we will highlight two examples of lncRNAs that have been implicated in such processes: Example 1. TUG1 (Taurine Upregulated Gene 1): The lncRNA Tug1 is conserved among mammals and is expressed in the developing retina and brain as well as adult tissues [38]. Young et al. previously demonstrated that the downregulation of Tug1 in the retina leads to malformation or the loss of the outer segment of photoreceptors [38]. Although these findings suggest an essential role for Tug1 in the proper development of the retina, Tug1 mechanism of action remains unknown. A recent study found Tug1 to be one of select lncRNAs that associate with the chromatinmodifying complex PRC2 (polycomb repressive complex 2) in several human cell types, suggesting that Tug1 may potentially function, similar to HOTAIR and Xist, as a guide and/or a scaffold for PRC2 at specific genomic loci [9]. And thus, perturbation of Tug1 levels may lead to changes in PRC2 occupancy and, subsequently, gene expression, which are responsible for the phenotype observed upon Tug1 downregulation. Indeed, gene expression profiling with or without Tug1 depletion identified many cell cycle genes as potential targets for Tug1 [9, 38]. Future studies are needed to dissect the direct targets of Tug1 as a first step toward elucidating its mechanism of action.


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Example2. HOTAIR (Hox Transcript Antisense Intergenic RNA): HOX genes encode transcription factors that regulate body patterning during embryonic development. In mammals, there are four HOX gene clusters, which are thought to have arisen through gene duplication. Intriguingly, human HOX loci encode, in addition to transcription factors, over 200 lncRNAs [39]. One particular lncRNA, HOTAIR, which is transcribed from the human HOXC locus, guides the chromatin-modifying complexes PRC2 and LSD1 to HOXD genes as well as numerous other genomic loci throughout the genome to repress their expression in trans [9, 14, 21, 39, 40]. Previous studies have shown that HOXD genes are involved in the proximal-distal patterning of the limbs [41], which suggest that HOTAIR is acting upstream of this critical developmental pathway. Surprisingly however, a mouse knockout of Hotair has no effect on the expression of Hoxd genes or PRC2 localization to chromatin, and displayed no obvious phenotype suggesting that HOTAIR function may have rapidly evolved in mammals [42]. 2.4 lncRNAs in Nuclear Structure and Organization

The nucleus is a highly organized structure with several compartments that are distinguished by their contents and subcellular localization [12, 43]. Studies have shown that the lncRNA NEAT1 (nuclear-enriched autosomal transcript 1) is essential for the formation and integrity of paraspeckles, which are nuclear compartments that were previously identified by the presence of several proteins (i.e., PSP1, PSF, and p54) [12, 44–48]. These proteins are involved in pre-mRNA splicing, regulation of transcription, and retention of RNA in the nucleus [12, 45, 49]. NEAT1 localizes exclusively to paraspeckles, and depletion of NEAT1 leads to loss of paraspeckles suggesting an essential role for NEAT1 in maintaining the integrity of paraspeckles [12, 45]. It is thought that NEAT1 plays a structural role in “bridging” proteins within paraspeckles into a functional ribonucleoprotein (RNP) complex, but it is possible that NEAT1 has additional role(s) that are yet to be elucidated. Finally, it will be interesting if future research uncovers other lncRNAs that are also involved in maintaining specific structures and compartments in the cell.

2.5 lncRNAMediated Alternative Splicing

Alternative splicing of pre-mRNAs increases the complexity of the proteome by several folds by producing two or more distinct mRNAs from the same pre-mRNA, which are then translated into various protein isoforms with distinct functions [50]. Recent studies revealed that the lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript-1) is involved in alternative splicing, as the depletion of MALAT1 results in the skewing of alternative splicing products of many genes [51]. MALAT1 functions by modulating the phosphorylation of SR (serine/arginine-rich) proteins in nuclear speckles, which are nuclear compartments that coordinate the assembly and storage of the splicing machinery [51].

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Proper regulation of alternative splicing of pre-mRNAs is critical as the dysregulation of this process has been observed in cancer [50]. It is possible that MALAT1 upregulation in cancer cells [52] shifts alternative splicing of pre-mRNAs toward mRNAs that encode protein isoforms which, for example, enhance cellular proliferation and metastasis [50].

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Epigenetic Regulation of Gene Expression by lncRNAs

3.1 Epigenetic Regulation of Gene Expression

The body of a multicellular organism, such as human, is composed of trillions of cells that are genetically identical; however, these cells form various tissues that are morphologically and functionally distinct. Therefore, additional information beyond the DNA sequence itself must be guiding cellular differentiation. Both DNA methylation and histone modifications play critical roles in epigenetic regulation of gene expression, and thus contribute to tissue-specific gene expression patterns. However, many of the protein complexes that modify DNA and chromatin lack DNA binding capacity, and thus, it has been puzzling how these complexes find their target genes in the various cell types. Intriguingly, recent studies found numerous lncRNAs to be associated with such complexes, and in a few cases, it was shown that such lncRNAs are responsible for directing their associated protein complexes to specific genomic loci [9, 14, 15, 39, 53]. Accordingly, lncRNAs appear to add an additional layer of genome regulation that makes it possible for genetically identical cells to be functionally distinct. Below, we will highlight several examples of how lncRNAs contribute to the epigenetic code.

3.2 Nuclear lncRNAs Associate and Guide Chromatin-Modifying Complexes to Regulate Gene Expression

Over the past few years, a number of studies have clearly documented that numerous nuclear lncRNAs associate various chromatin-modifying complexes [9, 54]. The discovery that HOTAIR associates with the chromatin-modifying complex PRC2 was, to the best of our knowledge, the first report of a mammalian lncRNA that associate with a chromatin-modifying complex [39]. Shortly thereafter, both Xist and Kcnq1ot1 were also shown to interact with PRC2 [31, 35]. Subsequently, two high-throughput studies demonstrated that PRC2 as well as other chromatinmodifying complexes associate with numerous lncRNAs in various human and mouse cell types suggesting that such interactions are more prominent than initially thought [9, 54]. Beyond the physical association of some lncRNAs with chromatin-modifying complexes and transcription factors, some of these lncRNAs have been shown to guide these proteins to their target genes [14, 21, 31, 35, 37, 40, 55, 56]. For example, the lncRNA HOTAIR is required for the proper targeting of PRC2 and the histone demethylase LSD1 to repress numerous


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genes in trans [14, 21], the lncRNA XIST, which is involved in X chromosome inactivation, is also required for the proper targeting of PRC2 to the inactive X chromosome [31], and the lncRNA Air guides the histone methyltransferase G9a to repress the expression of several imprinted genes in cis [37]. Although the examples above focus on lncRNAs that associate with repressive chromatin-modifying complexes, other lncRNAs have been shown to guide chromatin-modifying complexes that are involved in gene activation as well. The lncRNAs Evx1as, Hoxb5/6as, HOTTIP and Mira all associate and target the H3K4 methyltransferase MLL to chromatin in mouse embryonic stem cells to activate gene expression [57–59]. In summary, an increasing number of lncRNAs has now been shown to be required for the proper targeting of chromatinmodifying complexes to chromatin in multiple human and mouse cell types suggesting that this mechanism of gene expression is widespread in mammals [60–62]. However, many questions regarding this mechanism of gene regulation remain to be addressed including: (1) How do lncRNAs specifically associate with their protein partners? (2) How do lncRNAs target proteins to chromatin? Is it via direct lncRNA–DNA interactions or via intermediate DNA binding proteins? (3) When lncRNAs become dysregulated in human disease, do they alter the occupancy of their protein partners to the genome? These key questions and others must be addressed to fully appreciate this mechanism of gene regulation.

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Long Noncoding RNAs and Human Disease A number of studies have now shown that many lncRNAs are dysregulated in a wide range of human diseases and disorders [63]. Although these findings are not entirely surprising since the expression of many mRNAs and other classes of noncoding RNAs also dramatically changes in disease states, in some cases, the dysregulation of select lncRNAs has been shown to be strongly associated with poor prognosis suggesting potential roles for these lncRNAs in the disease state that warrant further investigation [14, 25, 64, 65]. Below, we will highlight several examples of lncRNAs that may have a role in driving human disease, and try to shed light on possible mechanisms of such lncRNAs.

4.1 Dysregulation of lncRNAs in Cancer

One of the hallmarks of cancer is a genome-wide alteration of gene expression of both protein-coding genes and microRNAs [50, 66–71]. Recently, a number of studies have demonstrated that the expression of lncRNAs is also altered in various cancer types [72–74]. Perez and colleagues reported that a number of lncRNAs are abnormally expressed in breast and ovarian cancer tissues. Furthermore, they found mutations in several of these

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lncRNA genes in cancer cell lines in comparison to normal cell lines derived from similar tissues [75]. However, since the authors did not follow up these observations with functional and mechanistic studies it is difficult to assess the exact role of these lncRNAs in cancer etiology. As discussed earlier in the chapter, the lncRNA MALAT1 was originally discovered due to its altered expression in lung cancer cells [76], and subsequent studies of MALAT1 demonstrated that it plays an essential role in alternative splicing of pre-mRNAs [51]. The lncRNA HOTAIR has also been shown to be highly upregulated in several cancers, and the level of HOTAIR expression correlates with the overall projected lifespan, with low HOTAIR expression patients having a better prognosis [14]. In vitro and in vivo studies have shown that the overexpression of HOTAIR plays a role in metastasis by altering the genome-wide occupancy of the chromatin-modifying complexes PRC2 and LSD1 [14, 21]. PRC2 is a histone methyltransferase that targets histone H3 lysine 27 (H3K27) for methylation [77], and LSD1 is a histone demethylase that targets H3K4 for demethylation [78]. Both PRC2 and LSD1 play an essential role in regulating the expression of hundreds of genes genome-wide, and thus, the overexpression of HOTAIR and consequently, the mis-localization of PRC2 and LSD1 results in altering the expression of hundreds of genes. These global changes in gene expression result in abnormal cell growth and proliferation [14, 21]. Although we only highlighted a few examples of lncRNAs dysregulation in cancer, there are several other studies that suggest that other lncRNAs may also play important roles in cancer etiology. For example, the lncRNA linc-P21 was previously shown to be 1 of 40 lincRNAs that are directly regulated by the tumor suppressor p53 [17]. Once linc-P21 is activated, it binds to hnRNP K and regulates the expression of hundreds of genes in trans [17]. We anticipate that future research will uncover more examples of such lncRNAs. Also, functional and mechanistic studies of these lncRNAs will be critical to uncovering how their dysregulation contributes to cancer etiology. 4.2 lncRNAs in Neurological Disorders

Neurological disorders affect an estimated one billion people worldwide according to the World Health Organization. In many cases, the genetics and environmental factors that lead to neurological disorders remain unknown despite great efforts to identify such factors. In the cases where a genetic mutation in a proteincoding gene has been identified, there is usually significant gene expression and phenotypic variability among patients suggesting that other factors may also contribute to the disease state [79–81]. Intriguingly, recent studies have shown that the human brain has the second highest expression of lncRNAs [4, 11], and some of these lncRNAs are dysregulated in several neurological disorders [82, 83].


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Although most studies to date have lacked enough evidence to implicate lncRNAs as causative, these lncRNAs appear to play critical roles in the nervous system and should be further investigated. Below, we will highlight a few examples of lncRNAs that have been implicated in neurological disorders: Example1. Fragile X Syndrome (FXS): FXS is the most common form of inherited mental retardation, and is thought to result from an expansion of a trinucleotide CGG repeat in the 5′ UTR of the FMR1 gene [84]. The expansion of the CGG trinucleotide repeat above a certain threshold (>200 repeats) leads to lower FMR1 mRNA levels and, consequently, low or no detectable FMR1 protein (FMRP) [85]. In addition to affecting FMR1 expression, the CGG expansion also represses the lncRNAs FMR4 and ASFMR1 [18, 22]. By contrast to fragile X patients, the expression of FMR1, FMR4 and ASFMR1 is elevated in premutation carriers, which typically have a range of 50–200 CGG repeats [18, 22]. FMR4 is a 2.4 kb lncRNA that shares a bidirectional promoter with FMR1 [18], and ASFMR1 is an antisense lncRNA to FMR1 [22]. FMR4 is expressed in many fetal and adult tissues and differentially expressed in different regions of the brain, with the highest expression of FMR4 seen in the hippocampus, a region critical for shortand long-term memory [18]. In vitro studies of FMR4 demonstrated that FMR4 has an anti-apoptotic function in human cells, however, the molecular mechanisms by which FMR4 exerts its effect are still not known [18]. By contrast, neither the functions nor the mechanisms of ASFMR1 are currently known [22]. The function of FMR4 has been difficult to assess in animal models since FMR4 is primate-specific; however, mechanistic studies of FMR4 may shed light on its specific role in human biology. At this stage, we can only speculate how the dysregulation of FMR4 may contribute to fragile X syndrome and/or related disorders. It is possible that the loss of FMR4 expression and, consequently, the loss of its anti-apoptotic function may affect the survivability of neurons during development. This potential mechanism is plausible since another lncRNA, ESlncRNA, which also has an anti-apoptotic function, was recently shown to protect red blood cell progenitors from apoptosis [16]. Example 2. Alzheimer’s Disease (AD): Alzheimer’s is a devastating neurological disease that affects nearly 24 million people worldwide. Currently, the overall underlying genetic susceptibility of AD remains largely uncharacterized [86]. In addition to proteincoding genes, several lncRNAs have been shown to be dysregulated in the brain of AD patients. For example, the lncRNA BC200, which is specifically expressed in somatodendritic areas of the nervous system of the brain, is expressed at higher levels in AD patients in comparison to controls [87, 88]. Moreover, the elevated expression of BC200 is found in regions of the brain affected by AD,

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but not in other regions of the brain [87]. The BC200 gene is thought to have arisen by the duplication of an Alu element in the 7SL gene, which encodes the RNA component of the signal recognition particle (SRP) complex [89]. Intriguingly, BC200 binds the SRP complex suggesting that BC200 may play a role in translation of specific mRNAs [89]. These findings taken together suggest that BC200 may be involved in maintaining the plasticity of synapses in the brain by modulating protein synthesis and, therefore, its elevated expression in AD may contribute to the neuronal defects seen in AD [90, 91]. Another lncRNA known as BACE1-AS (BACE1 antisense) is also elevated in the brain of AD patients [13]. This particular lncRNA is transcribed antisense to BACE1, a protein-coding gene that was previously implicated in AD etiology [92]. BACE1-AS appears to increase the stability of BACE-1, by unknown mechanisms [13]. It is possible that BACE1-AS acts a “sponge” for microRNAs targeting BACE1 [93]. In summary, lncRNAs, such as BC200, BACE1-AS and others, may play significant roles in maintaining normal brain functions, and dysregulation of such lncRNAs may contribute to a number of human neurological diseases and disorders. In that regard, future studies to dissect the functions and mechanisms of such lncRNAs may provide insights into brain function, as well as provide novel strategies for therapeutic interventions.

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Potential Mechanisms of lncRNAs Although great progress has been made in discovering thousands of lncRNAs in the past several years, the mechanisms by which these molecules exert their effects are not currently well understood. Several mechanisms for lncRNAs have been proposed based on recent studies [62]. One possible mechanism involves lncRNAmediated recruitment of chromatin-modifying complexes and transcription factors to specific genomic regions. This mechanism is supported by several studies [14, 17, 21, 55, 57, 59], and could be a major mechanism for lncRNAs since a large percentage of nuclear lncRNAs associate with chromatin-modifying complexes and transcription factors in several human and mouse cell types [9, 54]. Currently, however, how a lncRNA targets a specific protein complex to the genome is not well understood. Some evidence suggests that lncRNAs may interact directly with DNA and form RNA:DNA hybrids [94], while other evidence suggest that this process is mediated by DNA binding proteins that also bind to lncRNAs [30]. In addition to targeting protein complexes to specific genomic loci, some lncRNAs serve as molecular scaffolds between two or more protein complexes that target the same genomic loci [21]. For example, the lncRNA HOTAIR, which binds to both PRC2


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and LSD1, forms a molecular scaffold for these two complexes at their shared genomic targets [21]. Experiments using various truncated forms of HOTAIR showed that the 5′ end of HOTAIR binds to PRC2, while the 3′ end binds to LSD1, and the full-length HOTAIR is required for PRC2 interaction with LSD1 [21]. Other lncRNAs may also serve as molecular scaffolds for other protein complexes that cannot interact directly via protein–protein interactions. As discussed above, both MALAT1 and NEAT1 are required for the formation of speckles and paraspeckles, respectively, by functioning as molecular scaffolds for several proteins [12, 45, 51]. It is very likely that most, if not all, lncRNAs function as part of RNPs rather than as naked RNA molecules [53]. Recent emerging evidence suggests that lncRNAs may also regulate microRNAs interaction with mRNAs [93]. In this model, lncRNAs function as “sponges” that bind to complementary microRNAs and prevent them from binding to their mRNA targets. For example, the lncRNA linc-MD1 binds to both miR-133 and miR-135 and prevents them from binding to MAML1 and MEF2C mRNAs to regulate muscle-specific gene expression [93]. It is possible that other lncRNAs function in a similar manner to linc-MD1, however, many questions regarding this mechanism of action remain including: (1) What are the factors that regulate lncRNA–miRNA interactions? (2) Do lncRNA–miRNA interactions lead to the degradation of lncRNAs? (3) Under what conditions does a lncRNA bind to a miRNA? In summary, it is clear from experimental evidence that lncRNAs play important roles in many biological processes, and lncRNAs utilize various mechanisms to exert their effects, however, our understanding of these mechanisms remains at infancy [62]. Detailed understanding of these mechanisms is likely to provide important insights into gene regulation at the transcriptional as well as the posttranscriptional levels, as well as other aspects of cell and molecular biology, which may alter our view of genome organization and regulation in profound ways. References 1. Alexander RP et al (2010) Annotating noncoding regions of the genome. Nat Rev Genet 11:559–571 2. Brockdorff N et al (1991) Conservation of position and exclusive expression of mouse Xist from the inactive X chromosome. Nature 351:329–331 3. Brown CJ et al (1991) A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome. Nature 349:38–44

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Chapter 1 - Springer Link

Chapter 1 - Springer Link

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