Open access peer-reviewed chapter
Abstract
RNA or ribonucleic acid constitutes of nucleotides, which are ribose sugars coupled to nitrogenous bases and phosphate groups. Nitrogenous bases include adenine, guanine, cytosine and uracil. Messenger RNA, ribosomal RNA and Transfer RNA are three main types of RNA that are involved in protein synthesis. Apart from its primary role in synthesis of protein, RNA comes in variety of forms like snRNA, miRNA, siRNA, antisense RNA, LncRNA etc., that are involved in DNA replication, post-transcriptional modification, and gene regulation etc. LncRNAs regulate gene expression by various ways including at, transcriptional, post-transcriptional, translational, post-translational and epigenetic levels by interacting principally with mRNA, DNA, protein, and miRNA. Among other biological functions, they are involved in chromatin remodelling, transcriptional interference, transcriptional activation, mRNA translation and RNA processing. In this chapter we shall be discussing the origin of lncRNAs, their biogenesis, their mechanism of action and their role in many biological and pathological processes like epigenetics, genome imprinting, several cancers and autoimmune diseases.
Keywords
- long non-coding RNAs
- epigenetics
- chromatin remodelling
- gene expression regulation
- cancer
- autoimmune diseases
1. Introduction
The genome of higher organisms contains less than 3% of protein coding genes while rest of genome is known as junk/or non-coding. With recent advances in science and technology we are learning more about the complexity of organisms, which has led to the discovery of the remarkable complexity of RNA. Large-scale projects such as ENCODE and FANTOM, for the systematic annotation and functional depiction of genes have pointed that 80% of genomic DNA of mammals are effectively transcribed and intricately controlled with majority belonging to noncoding RNA genes [1]. The figure of ncRNA genes varies by species. Frequency of ncRNA genes but not protein-coding genes, is strongly linked with the complexity of an organism emphasising the growing relevance of ncRNAs [2]. On the basis of molecular size ncRNAs can be classified into two groups. One group includes short RNAs that are less than 200 nucleotides in length such as microRNAs (20–25 nucleotides Length), piwi-interacting RNAs etc. The other group includes Long-non coding RNAs of around 200 nucleotides or more in length. Among the ncRNAs, long non coding RNAs represent a greater portion. Regulatory non coding RNAs with length ≥ 200 nucleotides belong to long non coding RNAs (lncRNAs). Due to their low expression levels, lncRNAs in the beginning were considered to be transcription noise. According to HUMAN GENCODE statistics, there are around 16,000 lncRNA genes, although some estimates put the number at over 100,000 [3, 4]. RNA polymerase II is primarily responsible for lncRNA transcription, but other RNA polymerases are also involved. The resultant lncRNAs are frequently capped at their 5′ ends with 7-methyl guanosine (m7G), polyadenylated at their 3′ ends, and spliced similarly to mRNAs. lncRNAs, in contrast, have a median turnover of 3.5 h, while it is 5.1 h for mRNAs. After transcription, the widely held lncRNAs stay in the nucleus [5]. LncRNAs regulate gene expression at different domains including the ones at epigenetic, transcriptional, post-transcriptional, translational, and post-translational levels by interacting principally with DNA, mRNA, protein, and miRNA [5]. LncRNAs regulate gene transcription by promoting or inhibiting the formation of transcription loops and recruiting or blocking regulators. LncRNAs also operate as precursors to other ncRNAs, such as microRNAs (miRNAs), and influence mRNA splicing [6]. They are involved in RNA processing, transcriptional interference, chromatin remodelling, transcriptional activation, and mRNA translation, among other biological processes. Besides these roles they also act as oncogenes or tumour suppressors by regulating different signalling cascades [7]. Based on their functions lncRNAs are classified into three types: non-functional lncRNAs, these are products of transcriptional noise; lncRNAs for which the act of transcription is adequate for their activity but the transcript itself is not required and functional lncRNAs that act in both cis and trans ways [8]. In recent years, lncRNAs which account for the immense majority of non-coding RNAs (ncRNAs), have become a hot topic in disease diagnostics and target therapeutics in recent years.
2. Biogenesis of lncRNAs
Understanding the biosynthesis of these lncRNAs is not only crucial but also ineluctable in order to decipher their functional value, relevance, and differentiation from other forms of RNAs. LncRNA biogenesis is both cell type and stage specific, regulated by stage and cell specific stimuli [9]. Enhancers, promoters, and intergenic regions are among the DNA components in eukaryotic genomes that transcribe diverse types of lncRNAs [10]. LncRNA biogenesis involve ribonuclease P for cleavage to create mature ends, the production of protein (snoRNP) complex caps at their ends, small nucleolar RNA, and the development of circular structures [11, 12]. During specific lncRNAs biogenesis, “paraspeckles” (sub-nuclear structures) have been discovered [13]. The identity of 4 paraspeckle proteins (PSPs) required for paraspeckle formation was made possible by RNAi analyses of 40 paraspeckle proteins [14]. Overall, the biology dealing with regulation, synthesis of various lncRNAs is still unknown.
3. Origin of LncRNAs
Compared to protein-coding genes, very less information regarding origins and evolution of lncRNAs is available. They reveal that among mammals, sequence conservation is poor and evolution is fast [15]. For the origin of lncRNAs, various evolutionary assumptions have been considered. The first idea is that the protein-coding gene undergoes transformation as a result of a gene-duplication process [16, 17]. Throughout evolution, one copy of a protein-coding gene acquires mutations and loses its capacity to code for proteins. Then, among other coding fractions, a new functional lncRNA gene is generated with polyadenylation sequences, splicing signals, regulatory elements and exon sequences [16, 18].
X inactive-specific transcript (XIST), meant for dosage compensation in mammals is assumed to have arisen from the chicken protein-coding Lnx3 gene [16, 17, 19], was classified as a pseudo gene [18]. The 5-UTR of Lnx3 exons 1 and 2 were used to create the XIST promoter region. Lnx3 exons 4 and 11 were also used to create exons 4 and 5 of the XIST gene of humans [20]. Other lncRNA genes, such as short and long non-coding RNAs, can duplicate segments or whole genes to generate lncRNAs. According to genomic research large homologous protein-coding gene families, protein coding gene duplication is ubiquitous; yet apart from protein coding genes, there is minimal proof for the entire duplication of lncRNAs. This might be owing to the fast sequence divergence of lncRNAs. The duplicated lncRNA mouse nuclear-enriched abundant transcript 2 is paralogous to non exonic sequences in the mouse genome [18]. The generation of lncRNAs appears to be aided by segmental gene duplication within antisense non-coding RNAs (ancRNAs).
De novo creation is another option for lncRNA origins. Examples include genome changes viz. chromosomal rearrangement, creation of (proto-) splice sites and (proto-) promoters transformed non-functional genomic stretches into functional lncRNAs [16]. Genesis of lncRNAs via insertion of transposable elements is the most recent and final hypothesis [17, 18, 21]. The bulk of human lncRNAs have TE segments as related to other genes such as pseudo genes, tiny lncRNAs, and protein-coding genes. Internal exons, transcription start sites, polyadenylation (polyA) sites, or a mix of these components can all include TEs. According to this research at least 75% of human lncRNAs contain at least one exon containing partial TE origin.
4. Mechanism of action of LncRNAs
4.1 LncRNAs as chromatin regulators
The figure of lncRNAs with known roles is gradually increasing, and the majority of the studies focus on their controlling potential. Histone modifications, DNA methylation, and chromatin remodelling are all functional steps where lncRNAs regulate chromatin structure.
LncRNAs are frequently used as important regulators (modulators) of protein coding gene expression by acting in cis and trans ways [22]. Binding of lncRNAs to histone modification complexes like PRC1 and PRC2 [in-specific] causes methylation of lysine 27 on histone 3, a histone signature connected to suppressed transcriptional state. Xist, a lncRNA abundantly generated from inactive X chromosomes in females (Xi), enhances PRC2 recruitment to the Xi to mute gene expression, according to studies on mammalian X chromosome inactivation [23, 24]. Other example includes HOTAIR lncRNA, synthesised from the HoxC gene cluster but targets histone H3K4me1/2 demethylase LSD1 and PRC2 complex to induce transcriptional gene suppression at the HoxD locus in trans [25]. Some well-studied lncRNAs, including ANRIL and KCNQ1OT1, bring epigenetic modifiers to particular loci, allowing for chromatin remodelling. For example, KCNQ1OT1, binds to PRC2 as well as the methyl-transferase G9A, whereas ANRIL binds to both polycomb repressive complexes [26]. Many different lncRNAs act as scaffolds and work by leading restrictive histone modifying complexes to particular loci [26]. By forming complexes with the trithorax group (TrxG) and the polycomb repressive complex 2, lncRNA steroid receptor RNA activator also plays a role in transcriptional control [27].
Examples of lncRNA that interact with PRC1 complex include FAL1 RNA (focally amplified lncRNA on chromosome 1). BMI1, a PRC1 subunit, interacts with FAL1. FAL1 controls not only the stability of BMI1, but also its connection with target promoter regions, altering target gene expression [28].
4.2 Transcriptional regulation
At gene promoters transcription is thought to be regulated by the interaction of transcription factors and chromatin modifying factors. LncRNAs regulate gene expression by interacting with other molecules such as Proteins, RNA and DNA near their target genes promoters or enhancers. LncRNAs have a number of ways for controlling transcription.
Enhancer RNAs: Enhancer RNAs (eRNAs) are a sort of long noncoding RNA (lncRNA) derived from gene enhancer regions that act along with DNA to upregulate gene transcription via two mechanisms: transcriptional machinery tracking and enhancer-promoter looping [29]. Kim et al. found a 2 kb eRNA transcribed bidirectional from active enhancers. The function of enhancer region was correlated with the expression of this eRNA, suggesting that eRNAs play a task in enhancer function and influence gene transcription [30, 31].
Activating ncRNAs: Class of lncRNAs that acts as a transcriptional activator and is produced from independent loci instead of enhancers. In an RNA-dependent way, activating ncRNAs need the activation of the coding gene promoter and exclusively trigger the transcription of neighbouring coding genes. The mediator complex has been connected to a variety of activating ncRNAs, and attenuation of such complex prevents looping between the activating ncRNAs locus and its target gene.
By recruitment of chromatin modifiers: LncRNAs can modulate target genes by triggering epigenetic alteration viz. DNA methylation, histone modification, and by bringing chromatin remodelling complexes to specific genomic loci, typically promoter regions. LncRNAs might have two purposes. Firstly, by attaching to a protein or protein complex, lncRNAs act as a bridge scaffold for chromatin conformational changes [32]. Second, lncRNAs lead chromatin modifying enzymes to particular DNA patterns by acting as a tethered scaffold. For instance, the lncRNA HOTAIR serves as an epigenetic protein scaffold with several binding domains for various proteins. HOTAIR aids in the demethylation of H3K4 by interacting with LSD1 (lysine-specific histone demethylase 1A), REST (restriction element 1-silencing transcription) factor & REST corepressor1 at the 3′ end. HOTAIR (At the 5′ end) is a transcriptional gene silencing factor derived from the HOXC locus that leads to transcriptional gene silencing in trans across 40 kb of the HOXD locus by inducing a repressive chromatin state, recruiting PRC2, & reinforcing H3K27 methylation [33].
Long non-coding RNAs also function as cofactors, influencing the activity of transcription factors. For example, the ncRNA Evf2 is produced from a conserved distal enhancer and attracts DlX2 (TF) to the same enhancer, causing neighbouring protein-coding genes to be expressed.
4.3 Regulation after transcription
Because ncRNAs can recognise complementary sequences, they can affect how mRNAs are processed after transcription, including capping, splicing, editing, transport, translation, destruction, and stability. LncRNAs compete with microRNAs to impact mRNA levels by changing mRNA stability, degradation, and translation [34].
Regulation of mRNA splicing by LncRNAs : Alternative splicing can be aided by lncRNAs in a number of ways. LncRNAs interact with splicing factors most of the time to regulate gene splicing. MALAT1, a notable lncRNA is involved in pre-mRNA splicing. MALAT1 is needed for the precise localization of SRSF1 and numerous other splicing factors to nuclear speckles. Deletion of MALAT1 is associated with the change in the alternative splicing of a group of transcripts [35]. Other lncRNA like Gomafu/MIAT, restricted to a nuclear domain and expresses neuronally, may influence mRNA splicing and obstruct spliceosome formation by sequestering splicing factor1 [36]. Chromatin-mediated splicing control is the other way through these ncRNAs can modulate alternative splicing. For instance, nuclear antisense lncRNA generated from FGFR2 locus promotes FGFR2 epithelial-specific alternative splicing. The lncRNA establishes a chromatin environment that precludes the binding of a restrictive chromatin-splicing adaptor complex required for mesenchymal-specific splicing by attracting Polycomb-group proteins and KDM2a (Histone demethylase) [37].Regulation of mRNA stability : Because of interactions between their 3′-UTR and RBPs (RNA Binding Proteins), many genes have a limited mRNA half-life. Because lncRNAs can interact with RBPs, this type of interaction is expected to alter not just the number of RBPs in the pool, but also the functioning of lncRNAs that share binding sites with other genes, both coding and noncoding. By this way, mRNA molecules are either stabilised or destabilised. RBPs of different kinds may have a function in mRNA stability and, as a result, mRNA level. HuR is destabilised when the lncRNA OCC-1 binds with it and recruits the ubiquitin E3 ligase-TrCP1 to it [38]. HuR works as a stabilising factor for a wide number of mRNAs, causing HuR-targeted mRNAs to be down regulated. Linc-RoR interacts with hnRNP I (stabilising factor) and AUF1 (destabilising factor) in the opposite way that it interacts with c-Myc mRNA, according to findings [39].Regulation of protein stability: Many studies have discovered that lncRNAs can influence protein stability through ubiquitination or phosphorylation. DINO, a damage-induced noncoding RNA (lncRNA) that is p53-dependent, affects p53 stability [40]. DINO participates in p53-mediated phenotypes such as Apoptosis and cell cycle arrest with respect to DNA damage. Eminently, DINO binds to and stabilises p53, allowing induced p53 to favourably regulate downstream targets like DINO. Because of its capacity to stabilise the p53 protein, DINO is an important component of the DNA damage-p53 regulation network. lncRNA GUARDIN is another paragon, which is likewise a p53-responsive lncRNA [41].
4.4 LncRNA-mediated regulation of protein activity
Several studies have discovered that lncRNAs can influence protein stability through ubiquitination or phosphorylation. DINO (lncRNA) has a function in phenotypes regulated by p53, such lncRNAs also regulate protein activity in mechanisms other than transcription. The
Despite the fact that lncRNAs are distinct from mRNAs exported to cytoplasm for protein synthesis [47, 48], few lncRNAs are confined in sub-nuclear compartments, suggesting possible activities in these compartments. The schematic figure that represents few mechanisms employed by lncRNAs in gene expression regulation (Figure 1).
Figure 1.
Schematic representation of few mechanisms employed by lncRNAs in regulation gene expression. (A) Regulation by chromatin remodelling (B) Regulation through signalling (C) lncRNA sponging mRNA (D) By acting as scaffolds.
5. Roles and functions of lncRNAs in different biological processes
An emerging view of lncRNAs is that they are key players in many biological processes. These lncRNAs have been discovered to be master regulators in different biological and pathological processes and their dysregulations leads to many life-threatening diseases including different cancers. Some of the pivotal roles and functions carried out by different lncRNAs are well documented.
5.1 Long non-coding RNAs in genomic imprinting: regulation of allelic expression
In layman’s terms genomic imprinting represents a condition in which one of the alleles in an inherited paternal pair is active while the other one remains inactive. It is the parent of origin which will determines the differential expression of inherited parental alleles; in some cases, a genes allele is paternally imprinted, while in others, it is maternally imprinted.
Dna Methylation and Genomic imprinting: The process of adding a methyl (CH3) group to a cytosine known as DNA methylation is commonly located at CpG dinucleotides in mammals and is believed to regulate gene expression. CpG sites are very rare in the genome with the exceptions of CpG islands (which have high CpG amount/density). Generally found near or around promoter region these islands are usually unmethylated. Outside of CpG islands, CpG sites are often methylated, resulting in a bimodal methylation pattern across the genome. A family of DNA methyltransferases catalyses the acquisition of DNA methylation. DNMT1 is a DNA methyltransferase homologue that has an affinity for hemi-methylated DNA and is responsible for sustaining methylation following DNA replication. DNMT3A and DNMT3B catalyse de novo DNA methylation, while DNMT3L is a cofactor with no methyltransferase activity [49]. DNA methylation leads to epigenetic mechanisms and epigenetics leads to allow the transcriptional machinery of the cell to distinguish the two parental chromosomes at imprinted loci.
Imprinting is typically accomplished by altering the histone and/or DNA of a given locus, however lncRNAs have recently been identified to have a role in these phenomena [50]. Imprinted genes must be regulated by cis-regulatory mechanisms since expressed and repressed alleles share the same nucleus. The breakthrough in genomic imprinting was established with the discovery of gametic differentially methylated regions (these are actually control elements) that contain the “imprinting mark” acquired in oocytes and sperm. These markers are then transmitted down through the generations, guiding parental-specific allelic expression in children and embryos.
Control of imprinted lncRNAs is achieved by shared regulatory mechanisms such as parent-of-origin-dependent differentially methylated regions (DMRs) & lncRNAs within a single imprinted cluster [51]. Allele-specific DNA methylation occurs in a separate ICR in the germline in well-studied imprinted clusters, termed as principal DMRs or germline-derived DMRs and persists beyond fertilisation. Epigenetic changes (parent-of-origin-specific) such as DNA methylation, influence parentally inherited allele expression patterns in ICRs in imprinted clusters [52]. There are approximately 35 imprinted gDMRs in the human & 24 in Mouse genome (Monk et al., 2018). For embryonic development to govern imprinted gene expression, gDMRs must be established on paternal or maternal alleles [53]. In early primordial germ cells epigenetic markers including DNA methylation and histone modifications are substantially erased genome-wide. Depending on the parent-of-origin, DNA methylation of ICRs is reinstated in germline cells in gametes. After fertilisation, gDMRs are impervious to subsequent global epigenetic reprogramming. The data on DNA methylation at the ICRs of imprinted areas is retained. Imprinted loci gDMRs establish themselves strongly throughout germline development and are hence resistant to genomic reprogramming after fertilisation. Imprinting markings, on the other hand, are passed down from one generation to the next [54]. The allele-specific methylation statuses of gDMRs are generally identified by transcription regulators in maintaining parent-of-origin specific regulation of imprinted genes for example ZFP57 protein. The development of monoallelic gene expression requires differential methylation statuses of gDMRs on parental alleles. In early embryonic and adult lineages, imprinting control regions regulate DNA methylation and chromatin organisation, resulting in the survival of imprinting patterns through generations and their perpetuation in adult tissues [55].
To explain the control of gene regulation within an imprinted cluster, two fundamental methods have been proposed [56]. The lncRNA model is the earliest and arguably most frequent model. According to this concept, imprinted lncRNAs control imprinted gene expression. In this model, imprinted lncRNAs are closely linked to ICRs. Imprinted lncRNAs are defined by their ability to suppress imprinted genes in the same cluster [57]. As shown by the imprinted cluster Kcnq1/Kcnq1ot1. On paternal allele, the constantly expressed imprinted lncRNA Kcnq1ot1 can repress multiple imprinted genes bidirectionally throughout their gene area. The insulator paradigm, wherein parental allele-specific epigenetic changes at ICRs contribute to topological variations of imprinted gene areas, resulting in gene silence or activation of certain alleles, has been observed in additional imprinted regions. The insulin-like growth factor 2/H19 locus controls imprinted genes mechanistically, according to this concept. The following are some well-known lncRNAs and their roles in genomic imprinting:
Airn lncRNA: 108-kb nuclear-localised transcript that is synthesised in the opposite direction from 3.7-kb ICE of Igf type-2 receptor gene [58]. This lncRNA have been shown to influence the regulation of three parent of origin-specific genes viz. Igf2r, Slc22a2 & Slc22a3. Mice with an ICE deletion show biallelic expression of 3 genes (including Igf2r) and are smaller at birth [59].Kcnq1ot1: Another lncRNA with a well-known involvement in imprinting is Kcnq1ot1. It is a 91-kilobyte RNA with many protein-coding genes that comes from the 1-megabyte Kcnq1/Cdkn1c locus on chromosome 7 [36, 60]. Because of CpG methylation, this lncRNA is suppressed on maternal chromosome and assumes paternal specific expression. The paternal chromosome specific expression is associated with inhibition of some 8–10 protein-coding genes that span many megabases. Kcnq1ot1, like Airn works in cis on the paternal chromosome to mute Phlda2, Kcnq1, Slc22a18, & Cdkn1c genes in all tissues (ubiquitously imprinted loci), Imprinting is a phenomenon that happens in a 600-kb region of mouse chromosome 7 called the imprinting cluster. H19 & Igf2, two genes in this cluster, are expressed maternally and paternally, respectively, and correlate to the human locus 11p15.5 [61, 62]. The H19 gene regulates expression of imprinted loci, forming the imprinted gene network, which is important for embryo development. H19 is a 2.3 kb lncRNA that has been demonstrated to generate an overgrowth phenotype and alleviate imprinting on Igf2 and other IGN genes when knocked out [63].DLK1-DIO3: Human and mouse chromosome 14 & 12 respectvely both have a big imprinted cluster [imprinting status, in prototherians, metatherians and eutherians have also been determined] [64]. The paternally expressed genes DLK1 and DIO3 flank a 1 Mb area bordered by maternally expressed noncoding RNAs including the lncRNA MEG3 [65].SNURF-SNRPN: The SNURF-SNRPN area on chromosome 15q11-13, which spans over 2 Mb and has been related to the neurodevelopment diseases like Angel man Syndrome (AS) and Prader-Willi Syndrome (PWS) [66], is the biggest known imprinted cluster.
5.2 X-chromosome inactivation
Sex in most animals including humans is generally determined by X and Y chromosome system with men carrying XY and females XX chromosomes. In order to have the balance of products of sex-linked and autosomal genes, dosage compensation is required. X-chromosome inactivation (XCI) is a special kind of dosage compensation present in mammalian females involving random selection and transcriptional repression in one among the two X -chromosomes during the early stages of embryonic development [67, 68]. However, humans lack imprinted XCI and instead have XCD (X chromosome dampening) [69, 70, 71]. Most of the research with respect to this has been carried on mouse model and regulated by interactions of many lncRNAs [72]. At the heart of XCI/XCR (X-chromosome reactivation) regulation is a region of X chromosome namely Xic X-inactivation centre (containing many noncoding RNA genes whose expression is regulated by pluripotency factors) [73]. Developmental phase of mice includes two forms of XCI; one being imprinted and another random. Imprinted XCI, in which the paternally inherited X (Xp) is always inactivated, occurs during preimplantation development in the early embryo, when Xist becomes expressed on the Xp from the 2-cell stage onwards and is maintained in the placenta’s extraembryonic tissues [74].
Xist lncRNA regulates all three stages (initiation, establishment, and maintenance) of XCI [75]. The X-linked minimum genetic region (XIC) contains many elements and genes like Tsix & Xist which are meant for XCI initiation [76]. Among the lncRNA loci reported in a 100–500 kb region of the mouse X chromosome and a 2.3 Mb syntenic region of the human X chromosome are RepA Jpx, Xist, Ftx, Tsix, & Xite [75, 76]. XIC contains the lncRNA Xist, and is located 15 kb downstream of Tsix antisense [77].
In first phase, complex factors Xite, OCT4, Tsix & CTCF among others bind Xi and Xa independently to promote X chromosome pairing and counting in the embryo following fertilisation [78]. After counting and pairing, Tsix, Xist, and other genes are elevated, which is regulated by a network of genetic interactions including OCT4, Jpx, Rnf12, and RepA Tsix, Sox2 & PRDM14 [79]. They use alternative transcription fates when full initiation of XCI occurs, with one becoming the Xa and the other Xi chromosome [72]. RNF12, Tsix, and RepA, as well as pluripotency factors (NANOG, OCT4, SOX2) impact lncRNA Xist activation and expression in Xi. Xist binds to Polycomb repressive complex 2 via Repeat A, producing the Xist-PRC2 complex, while YY1 tethers the PRC2-Xist complex to the Xi nucleation centre via Repeat C, where RNA polymerase II gets the lncRNA Xist-PRC2 complex [80]. Upon completion of initiation phase lncRNA Xist recruits different protein complex factors (including heterogeneous nuclear protein U, meant for lncRNA Xist localization, heterogeneous nuclear ribonucleoprotein K for Xist-mediated chromatin modifications) and gene-silencing factor Spen which binds to C, B, F, and A repeats at the 5′ end of the lncRNA Xist and causes the lncRNA Xist topologically associated domains meant for (epigenetic modification and chromatin compaction) to spread along the Xi chromosome at the established phase [76, 81]. ATRX directs PRC1 and PRC2 that cause epigenetic silencing by acetylation of histone H3 and H4 and methylation of CpG islands [56]. The protein complexes SHARP, HDAC3, LBR, Airn and Kcnq1ot1, U1 snRNP, Rsx, and CdK8 are all implicated in the lncRNA Xist spreading process. LncRNA Xist attracts restrictive complexes (like H3K27me3, H2AK119Ub, & the CpG island), which cause immediate histone modifications and DNA methylation, and coats the Xi to generate Xi [82]. Continuous synthesis of lncRNA Xist RNA in an inactive state has been used to establish and maintain the Xi.
6. Role of LncRNAs in cancer
The deadliest disease on the planet is linked to abnormal gene expression. Non-coding areas of the genome have been related to the majority of malignancies. Cancer genomic mutations are found in areas that do not code for proteins [83]. These areas, however, are frequently translated into long non-coding RNAs (lncRNAs). Anomalies in these lncRNAs is thought to have the tumour suppressor or carcinogenic effects and thus play a vital role in tumour establishment. Since lncRNAs have expression that are unique to a tissue, expressed in regulated manner and in association with other gene sets impact cell cycle regulations, immunological responses, survival etc. all of which affect cancer cell transformation [84]. The role of lncRNA is associated with their specific subcellular location. LncRNAs regulate gene expression by different interactions both in the nucleus as well as in the cytoplasm. In nucleus regulate expression at both epigenetic and transcriptional levels including histone modifications [24, 85], DNA methylation regulation [86], chromatin remodelling [87, 88], chromatin modification complexes [89, 90], transcription regulators [91] and proteins present in nucleus [92] while in the cytoplasm these lncRNAs regulate gene expression at both transcriptional and translational levels including interplay with proteins present in the cytoplasm [93], Control of mRNA metabolism [94], as endogenous competitive RNA (ceRNA) interacts with microRNA [95]. As a result, lncRNA play a significant role in cancer cell proliferation, transition, invasion, and treatment resistance [96, 97].
LncRNAs like LUCAT1, KCNQ1OT, HOTAIR, ANRIL, MALAT1(metastasis-associated lung adenocarcinoma transcript 1), Taurine-upregulated gene 1, LINC00152, RP11-385 J1.2 and TUBA4B bring different epigenetic modifiers at their respective loci and modify the chromatin shape and their dysregulation is strongly associated with establishment of different tumours including LUCAT1: digestive system tumours, ANRIL: prostate cancer, MALAT1: breast, liver, and colon cancer [98], HOTAIR: breast cancer KCNQ1OT1: colorectal cancer etc.
Some of known lncRNAs with links to cancer is represented below in a table:
| Symbol/emblem | Cancer phenotype | Cancer association | Mechanism | Reference |
|---|---|---|---|---|
| Promote metastasis | Upregulated in many cancers including those of liver, breast, lung and pancreas | By acting a platform for the PRC2 and LSD1 (chromatin repressors). By turning off | [99] | |
| Promotes both cell metastasis and proliferation | Overexpressed in non-small cell lung cancer, pancreatic, Colon, prostate, breast and hepatocellular carcinomas | Similar to alternative splicing and active transcription. Unique tRNA-like sequence at the 3′ end cleaved off and transformed to produce a short tRNA-like ncRNA (mascRNA) | [35, 98, 100] | |
| Impede cell amplification, relocation and carcinoma establishment | Locus preferentially deleted in sporadic colon cancer, prostate and other different carcinomas | Inveigle for microRNAs that attack | [101] | |
| Impede cell amplification and stimulates Apoptosis | Suppressed in colorectal cancer | Upregulates the transcription of p53 | [102] | |
| Controls cellular events, programmed cell death and resistance to cisplatin in NSCLC cells by attacking IGF-2 | Overexpressed in clear cell renal cell carcinoma ccRCC tissues | Engage in control of amplification, relocation, invasion and impedance to drugs in multiple tumours | [103] |
One of the first lncRNAs to be discovered as having a functional role in cancer formation was the HOTAIR. HOTAIR lncRNAs enhance cancer spread by triggering epigenetic alterations in the chromatin status of tumour cells [104]. Interestingly, various lncRNAs have been reported to originate from the HOX locus, implying that it plays a global regulatory role [21]. In tumours, lncRNAs are also implicated in the control of the epithelial-mesenchymal transition (EMT) [99]. TGF-activated lncRNA (lncRNA-ATB) stimulated the EMT cascade by upregulating the levels of zinc finger E-box-binding homeobox (ZEB1 and ZEB2) [105]. Recent research has discovered that the transcription factor PNUTS has a corresponding lncRNA-PNUTS, which plays a role in breast cancer metastasis by influencing the EMT process [106]. lncRNA human ortholog RNA of Dreh (hDREH), inhibitor of EMT is seen downregulated in many types of cancers. Some tumour suppressor lncRNAs, on the other hand, are expressed at low levels in tumours [107]. It is now possible to increase the expression of these lncRNAs in order to treat cancer. lncRNAs have been implicated in stemness-related signalling pathways in a number of studies. The tumour suppressor long noncoding RNA (TSLNC8), for example, inhibit the STAT3 signalling pathway and thus acted as a tumour suppressor [108]. lncRNA MST1/2-antagonising for YAP activation (MAYA) participate in the Hippo-YAP signalling pathway. These lncRNAs might be directly regulating cell stemness [109]. Thus, these lncRNAs act as potential targets for cancer treatment.
7. Role of LncRNAs in immunology
The study of lncRNA biology is still in its early stages, but it has already revealed key roles in the formation and functions of immune cells. However, because the functions of most lncRNAs are unknown, the field must fill a significant gap in order to comprehend the breadth of their roles in immunity. The function of non-long coding RNAs in the immunology of humans even though in its infancy stage has become hot topic in recent research. In immune system, lncRNAs have a role in immune cell formation, survival, cell fate determination, differentiation, amplification, and activation. Although the functions of most lncRNAs are unknown, novel protein-protein interactions or partnering with RNA or DNA have been discovered to influence innate and adaptive immune responses transcriptionally or post-transcriptionally. According to the recent research lncRNAs have been found in many immune cells including T cells and B cells. Regulation of these lncRNAs including expression levels is considered to be linked to immune cell development, differentiation, and activation [110].
To carry out their functions these IncRNAs can associate with transcription regulators and signalling molecules (NF-B, STAT3) [111, 112], RNA binding proteins including (hnRNP, HuR) [113, 114], and chromatin remodelling components (PRC2, WDR5).
7.1 Long non-coding RNA in T cells
LncRNAs, such as TMEVPG1 (also known as LincR-Ifng-3′AS), have been identified in both mouse and human CD8+ T cells and demonstrated to be positioned inside a cluster of cytokines genes, and have been shown to control Theiler’s virus load in CNS infection [115]. T-bet/Stat, a transcription factor exclusive to Th1 cells that collaborates with TMEVPG1, promotes interferon gamma expression [116]. Interaction of lncRNA called NeST with WDR5, significant component of MLL H3K4 methyltransferases, leads to increase in methylation state of histones at Ifng gene in CD8+/Th1 cells [117]. Hundreds of lncRNAs were found in both CD8+ T cells of animal and human spleens after a genome-wide expression examination utilising a proprietary array, indicating that lncRNAs are important for lymphocyte differentiation and stimulation [118]. To get clearer picture of the role of lncRNAs in T cell growth and differentiation, Hu et al. used RNA-Seq to recognise 1524 genomic regions that produce lincRNAs in 42 subsets of mature peripheral T cells and thymocytes; specific transcription regulators including T-bet, STAT4 for CD8+ descendants, & GATA-3 and STAT6 for CD4+ lineages seemed to be substantially responsible for lineage-specific expression of T cell lincRNAs (LincR-Ccr2-5′AS) [119].
7.2 Long non-coding RNA in B cells
B lymphocytes after being activated by antigen interaction, the major roles are to make antigen-specific antibodies, operate as APCs, and develop into memory cells. In compared to T cells, less is known regarding lncRNA roles in B cells. Bolland et al. [120] have documented how lncRNAs play their part in the chromatin remodelling that occurs during V/D/J gene recombination, which is required for the production of epitopes (Ig or TCR). Further study discovered that the synthesis of such antisense and sense lncRNAs is related to VH portions looping next to DJH areas during pro-B cell recombination, and as a result, it has been dubbed the Igh locus whole transcriptome of sense and antisense transcripts [121]. SAS-ZFAT gene expression which is restricted to CD19+ B cells of peripheral blood lymphocytes may be important for B cell function and AITD development [122].
7.3 Long non-coding RNA in macrophages
Macrophages are white blood cells that surround and destroy pathogens, destroy dead cells and stimulate other immune system cells. Macrophages as APCs also contribute to the optimal operation of both intrinsic and adaptive immune responses. LncRNA acknowledgement and purposes in monocytes/macrophages have received little attention. Macrophages have capital baseline level of ptprj/CD148 (a tyrosine phosphatase with tumour inhibitor-like role), as expected; its level is modulated by LPS, TLR, and CSF-1 treatments in various animals. Dave et al. investigated ptprj-as1, a 1006-nt lncRNA species that is produced antisense to ptprj. Transcripted ptprj-as1 is highly regulated in tissues augmented with macrophages that has been temporarily activated by TLR ligands, similar to ptprj [123]. Thus, the ptprj coding transcript may guide to inflammatory alteration that is promptly connected to macrophages. When myeloid and CD11c + dendritic cells are stimulated by lipopolysaccharide, a TLR4 signalling activator via NFkB, they express lncRNAs-COX2 (Ptgs2) [84]. Li et al. investigated how lincRNA expression changed when innate immune signalling was activated in THP1 macrophages and discovered that an unannotated LincRNA called THRIL was a critical factor in TNF- control and that its activity was clearly lower during the acute period of Kawasaki disease [114]. LncRNA has been observed to regulate healthy and pathological inflammatory immune responses through an RNA-protein complex with hnRNPL [124]. Many lncRNAs discovered as unique binding ally for lincRNA-cox2 in the nucleus and cytoplasm of macrophages, are key switch of immune genes [113].
7.4 Long non-coding RNA in natural-killer cells
Wright et al. uncovered an intron 2 promoter in many KIR genes that generate spliced antisense transcripts. KIR antisense lncRNA is found in progenitor cell lines, and its abundance in NK cells results in the under expression of KIR protein coding genes. KIR’s antisense lncRNA corresponds to exons 1 and 2 of the KIR gene, and also the proximal promoter upstream of KIR. Myeloid zinc finger one (MZF-1) appears to impact KIR antisense lncRNA transcription, resulting in KIR silence via an unknown mechanism [125].
7.5 Functions of immune related lncRNAs
The following table represents the functions of some immune related lncRNAs:
| S. no. | lncRNA | Functions | Reference |
|---|---|---|---|
| 1 | NEAT1 | In response to viral infection, activates IL-8 transcription | [126] |
| 2 | Lnc-DC | Plays a role in transformation of monocytes of humans into dendritic cells | [112] |
| 3 | LincRNA-COX-2 | increases the production of proinflammatory genes (IL-6) & suppresses the synthesis of anti-inflammation genes in non-stimulated cells | [113] |
| 4 | NeST | controls IFN-encoding chromatin epigenetic tagging, IFN-expression, and susceptibility to viral and bacterial pathogens | [117] |
| 5 | Hotair | Through epigenetic changes in the chromatin state, promotes cancer spread and progression | [127] |
| 6 | LincRCcr2–5΄AS | In mouse CD4+ TH2 cells, it controls the synthesis of many chemokine receptor genes via STAT-6 pathway | [119] |
| 7 | LincRNA (THRIL) | Promotes TNF Transcription | [114] |
| 8 | IL1β | Modulation of chromatin | [128] |
| 9 | IL1β-RBT46 | In monocytes regulates IL-1 homeostasis | [128] |
| 10 | IL1b-eRNA | Expression of CXCL8 and IL-1β (proinflammatory mediators) | [128] |
| 11 | Lethe | Activated during inflammation | [111] |
| 12 | NRAV | Transcriptional regulation of numerous interferon-stimulated genes (ISGs), including MxA and IFITM3 | [129] |
| 13 | Morrbid | Control the longevity of myeloid cells with brief lives (neutrophils, eosinophils, and monocytes) | [130] |
| 14 | FAS AS1 | Increase programmed cell death (Fas-driven) in B cell Carcinomas | [131] |
| 15 | lincRNA-EPS | Restriction of inflammation | [132] |
| 16 | TH2-LCR | Th2 cytokine gene transcription regulation | [133] |
| 17 | linc-MAF-4 | Th1 cell differentiation | [134] |
| 18 | lnc-EGFR | Treg differentiation stimulation | [135] |
8. LncRNAs in auto-immune diseases
Auto-immune diseases are conditions in which our immune system wrongly assaults our bodies, assumed to be the result of a complex interplay of genetic, immunological and environmental variables.
8.1 Systemic lupus erythematosus (SLE)
TLR4 signalling dysregulation has been connected to the progression of SLE. A lncRNA has been associated to SLE and is controlled by TLR4 signalling. Nuclear enriched abundant transcript 1 (NEAT1), a lncRNA, has been connected to the pathogenesis of SLE [136]. In a study NEAT1 levels were found to be elevated in PBMCs of SLE patients in comparison with healthy individual. In LPS-stimulated human monocytic cell lines, silencing NEAT1 with siRNA results in lower production of Interleukin-6, CCL2, and CXCL10, all of which have been related to SLE pathogenesis. NEAT1 regulates the stimulation of late mitogen-activated protein kinase signalling pathways, which play a role in the TLR4-driven inflammatory response. SLE patients have lower Gas5 expression in plasma, and in both CD4+ T cells and B cells than in healthy people [137].
8.2 Rheumatoid arthritis (RA)
Is an autoimmune illness characterised by inflammation and proliferation of synovial joint resulting in significant joint damage. Several recent investigations have found that dysregulated lncRNAs play a significant part in aetiology of RA. In 2015, Song et al. discovered that PBMCs and serum exosomes of RA patients have higher expression of HOTAIR than in the healthy controls. Furthermore, increasing HOTAIR increases active macrophage migration, whereas decreased HOTAIR suppresses the synthesis of matrix metalloproteinase (MMP)-2 and MMP-13 in developed osteoclasts and rheumatoid synoviocytes. These results suggest that abnormal HOTAIR expression could perform a part in the development of RA [138]. Low levels of lincRNA-p21 have been linked to increased NF-B activity in persons with Arthritis. Spurlock et al. observed that amount of phosphorylated p65, a hallmark of NF-B activation, was greater in the whole blood of patients suffering from RA however the expression of lincRNA-p21 was found to be downregulated. In comparison with MTX-treated RA patients, untreated counter parts had reduced levels of lincRNA-p21 activity and upregulated levels of p65 [139]. Lu et al. observed that T cells of patients suffering from RA have upregulated lncRNAs LOC100652951 and LOC100506036 in comparison with healthy individuals [140].
8.3 Sjogren’s Syndrome (SS)
Chronic systemic auto-immune illness characterised by symptoms of driest mouth and eyes caused by gland inflammation (exocrine). The lncRNA Tmevpg1, which modulates Th1 responses, may have a role in the aetiology of SS, according to one research. The researchers discovered a connection between SS and the lncRNA Tmevpg1 [141].
8.4 Polymyositis and dermatomyositis (PM/DM)
Muscle inflammatory illness that is persistent. PM/DM has been connected to the expression of the RNA component of signal recognition particles, lncRNA 7SL [142]. Using microarray research, Peng et al. discovered 1198 differently transcribed lncRNAs in diabetes patients muscles compared to normal ones. uc011ihb.2, ENST00000583156.1, ENST00000551761.1, ENST00000541196.1 & linc-DGCR6-1, were among the five lncRNAs confirmed. As per their computational predictions, the linc-DGCR6-1 gene controls USP18, a kind of type 1 interferon-inducible gene found predominantly in the perifascicular portions of muscle fibres of diabetes patients [143].
9. Conclusion
Although there has been immense research going on to understand the role of lncRNAs, still there are many questions that need to be addressed. Efforts are being carried out to elucidate the role of lncRNAs as potential regulators in different biological and pathological processes. The advancement in technologies will further help to pave way in clarifying the mechanisms underlying of lncRNA influence in different diseases. Studying lncRNA role in regulation of host-pathogen interactions can be helpful in identifying different lncRNAs that can serve as potential drug targets and can also serve as novel biomarkers.
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