cell biochemistry and function Cell Biochem Funct (2014) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cbf.3079
Non-coding RNAs: biological functions and applications Baby Santosh, Akhil Varshney and Pramod Kumar Yadava* School of Life Sciences, Jawaharlal Nehru University, New Delhi, Delhi India
Analyses of the international human genome sequencing results in 2004 converged to a consensual number of ~20 000 protein-coding genes, spanning over 90% of the human genome is likely to be transcribed yielding a complex network of overlapping transcripts that include tens of thousands of long RNAs with little or no protein forming capacity; they are collectively called non-coding RNA. This review highlights the fundamental concepts of biological roles of non-coding RNA and their importance in regulation of cellular physiology under disease conditions like cancer. Copyright © 2014 John Wiley & Sons, Ltd. key words—non-coding RNA; regulatory role; telomere biology; chromatin dynamics; gene modulation; structural organization list of abbreviations—ncRNA, non-coding RNA; lncRNA, long non-coding RNA; snpcRNA, small non-protein coding RNA; siRNA, small interfering RNA; snoRNA, small nucleolar RNA; miRNA, microRNA; snRNA, small nuclear RNA; piRNA
NON-CODING RNAS Transcripts of genomic sequences that are not meant to be translated are often called non-translated or non-coding RNA (ncRNA) molecules.1 A vast number of ncRNAs are encoded by human genome. Among them, most of the ncRNAs have been widely implicated in regulating cellular homeostasis.2 Some of these ncRNAs are directly involved in epigenetic changes and/or modifications in cells. Total cellular RNAs are classified on the basis of their functions as presented in Figure 1. A subset of ncRNAs is processed in to functionally important RNAs such as transfer RNA (tRNA), ribosomal RNA (rRNA), small nucleoar RNA (snoRNAs), microRNAs, small interfering RNAs (siRNAs), PIWI-interacting RNAs (piRNAs) and the long non-coding RNAs (lncRNAs). Two widely studied lncRNA are Xinactivation specific transcript (xist) and HOX antisense intergenic RNA (HOTAIR).60,29 The total number of ncRNAs encoded within the human genome is not completely known; however, recent transcriptomic and bioinformatic studies suggest the existence of thousands of ncRNAs with their functional importance. Because many of the newly identified ncRNAs have not been validated for
*Correspondence to: Pramod Kumar Yadava, Jawaharlal Nehru University School of Life Sciences Lab no. 111, SLS, JNu Room no. 111, SLS, JNu New Delhi India 110067.E-mail: [email protected]
Copyright © 2014 John Wiley & Sons, Ltd.
their function, it is possible that many are non-functional. However, examples of the function and role of ncRNAs are still emerging. ncRNAs are divided on the basis of the length of RNA formed post-transcriptionally, i.e. short non-coding RNA (sncRNAs < 30 nt) and long non-coding RNA (lncRNAs > 200 nt). lncRNAs are generally considered as non-protein coding transcripts, longer than 200 nt. This limit is because of practical considerations including the separation of RNAs in common experimental protocols. Additionally, this limit distinguishes lncRNAs from small regulatory RNAs such as miRNAs, siRNAs, piRNAs, snoRNAs (Table 1). miRNAs, a class of short ncRNA, are 18–24 nt long and involved in skin fibrosis.3 The broad functional repertoire of lncRNAs includes role in higher order chromosomal dynamics, telomere biology and in subcellular structural organization.4,5 A new class of ncRNAs, T-UCR is transcribed from ultraconserved region.6 T-UCRs are subset of DNA segments longer that 200 bp and completely conserved between species (Humans, Rat and Mouse genome).6,7 In addition to this, ncRNAs are further subdivided on the basis of nature of genes involved in oncogenesis and/or tumour suppression. For example, steroid receptor RNA activator (SRA), is an oncogenic ncRNA that enhances adipogenesis in response to insulin and inhibits the expression of inflammatory genes associated with adipocytes.8 Maternally expressed gene 3 (MEG3), ncRNA encoding gene is located at chromosome Received 25 August 2014 Revised 13 October 2014 Accepted 31 October 2014
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Figure 1. A classification of RNA types showing the whole new world of RNAs playing important roles in diverse biological functions by modulating gene expression via antisense and miRNA-mediated controls
Table 1. S. no.
Examples of functional non-coding RNAs in mammals Name
Characteristics
Functions
References
Class (a) small non-coding RNAs 1
Small interfering RNAs (siRNAs)
2
MicroRNAs (miRNAs)
3
MicroRNA-offset RNAs (moRNAs) Small nucleolar RNAs (snoRNAs) PIWI-interacting RNAs (piRNAs)
4 6 7
Promoter-associated RNAs (PARs)
~21 to 22 nt long RNAs; produced by dicer cleavage of complementary dsRNA duplexes ~22 nt long; produced by dicer cleavage of imperfect RNA hairpins encoded in long primary transcripts or short introns Small ~20 nt long RNAs derived from regions adjacent to pre-miRNAs Control over rRNA methylation and pseudouridylation Small RNA ~24–30 nt, restricted to the germline and germline bordering somatic cells Long and short RNAs including promoter associated RNAs
Gene regulation, transposon control and viral defence
2.2 kb, transcribed from the HOXC locus
57,66
Post-transcriptional gene regulation
45
Gene regulation
63
Evidence of gene regulatory roles
72
Associated with PIWI-clade Argonaute proteins, regulate transposon mobility and chromatin state Regulate gene expression
73
Epigenetically silences gene expression at the HOX D locus PRC2 recruitment on the inactive X-chromosome Role in X-chromosome inactivation
71
miRNA-like translational regulators
72
Translational repression
51
Regulatory function
12
Involved in telomere maintenance
22
Involved in chromatin modifications
57
Binds and inhibits glucocorticoid receptor
10
74
Class (b) long non-coding RNAs 8 9
HOTAIR (HOX antisense intergenic RNA) Rep A1
10
X-inactivation RNAs (xiRNAs)
11
Sno-derived RNAs (sdRNAs) tRNA-derived RNAs
12 13
MSY2-associated RNAs (MSY-RNAs)
14
Telomere small RNAs (tel-sRNAs) Centrosome-associated RNA (crasi-RNAs) GAS5 (growth-arrest specific 5)
15 16
Copyright © 2014 John Wiley & Sons, Ltd.
1.6 kb, internal to Xist Dicer-dependent small RNAs processed from two lncRNAs duplexes, Xist and Tsix Dicer-dependent small RNAs processed from snoRNAs tRNAs processed into small RNA species by a conserved RNase (angiogenin) ~26–30 nt long, associated with the germ cell-specific DNA/RNA binding protein MSY2 and restricted to the germline ~24 nt RNAs principally derived from the G-rich strand of telomeric repeats A class of ~34–42 nt small RNAs, derived from centrosomes 600 nt long RNA
28 29
Cell Biochem Funct (2014)
NON-CODING RNAS: POWER AND PROMISES 14q32.3 on DLK1-MEG3 locus in humans, and its inactivation causes expression of angiogenesis promoting genes and formation of microvessels in brain.9 GAS5 ncRNA acts as decoy by competing with DNA-glucocorticoid response element (DNA-GRE) at DNA binding domain of glucocorticoid receptor and modulates growth arrest during starvation.10
originating from a region upstream of the major DHFR promoter represses expression of the downstream proteincoding gene19,20 both in cis and trans by forming an RNA–DNA triplex structure with the DHFR promoter and directly interacting with TFIIB, which results in disruption of the pre-initiation complex at the DHFR promoter.20
WIDESPREAD FUNCTIONS OF LNCRNAS
Non-coding RNAs in telomeres
It was presumed previously that lncRNAs are just the transcriptional noise resulting from low RNA polymerase fidelity.11 However, the expression of many lncRNA is restricted to particular developmental stages, and they have tissuespecific expressions.12 According to a recent study, only one fifth of transcription across the human genome is associated with functional protein-coding genes indicating at least four times more long non-coding than coding RNA sequences.13 However, it is large-scale complementary DNA (cDNA) sequencing projects such as functional annotation of mammalian cDNA (FANTOM) that reveal the complexity of this transcription.14 Numerous non-coding transcripts, for example, tRNAs, rRNAs and spliceosomal RNAs, are critical components of cellular machinery. NcRNAs regulate the cellular gene functionality through base pairing; stabilization of target gene translation is promoted by extended base pairing whereas partial base pairing leads to inhibition of target messenger RNA (mRNA) translation while absence of complementarity results in suppression of precursor mRNA by acting as decoy.15 NcRNAs play key regulatory and functional roles in gene expression program of the cell (Figures 2 and 3). One of the crucial functions of ncRNA is to act as ribozymes.16 The group I and II introns, RNase P and hammerhead are naturally occurring ribozymes. A structural class of self-splicing RNAs (group I and II introns) are 200–600 nt long and catalyzes RNA splicing. Although the functions of only limited number of lncRNA transcripts have been identified in different species, numerous paradigms are beginning to be explored (Figure 2). For example, flowering in plants is controlled by flowering locus C (FLC) gene, and a lncRNA cold assisted intronic non-coding RNA (COLDAIR) regulates vernalization, i.e. seasonal time flowering mediated by environmentally induced epigenetic changes, by silencing FLC.17 NcRNAs regulate a remarkable variety of biological functions in transcriptional interference, telomere maintenance, epigenetic changes, imprinting, post-transcriptional, translational control, structural organization, cell differentiation and development.18
Telomeres are functional nucleoprotein complexes that protect the terminus of the eukaryotic chromosomes from degradation and activation of DNA damage responses that would otherwise follow telomeres being recognized as DNA double-strand breaks. It maintains the genomic stability, senescence and aging.21 Telomeres have been long considered transcriptionally silent DNA-protein complexes because of their presence in heterochromatin state. It was recently shown that telomeres are transcribed in several organisms (e.g. Homo sapiens, Mus musculus, Danio rerio, yeast, plants) and give rise to lncRNA called telomere repeat containing RNA (TERRA) or TelRNAs.22,23 TERRA molecules are involved in telomere homeostasis through regulating telomere length, telomerase activity and formation of heterochromatin at the end of the chromosomes. Altered expression of these molecules may lead to chromosomal instability and can promote cellular senescence.5 TERRA transcript originates from the subtelomeric region of telomeres and proceeds towards the chromosome end by RNA polymerase II. The length of TERRA varies from 100 nt to 9 kb in mammalian cells and consists of Grich telomeric repeats.23 The association of these molecules with chromatin is negatively regulated by suppressors with morphogenetic defects in genitalia (SMG) proteins. Thus, SMG proteins play connecting link between TERRA (lncRNA) and telomere-specific heterochromatin modifications.23 These studies strongly suggest for an involvement of telomeric ncRNAs in telomere biology. Transcripts arising from telomere transcription are known in several eukaryotes. TERRA is part of the heterochromatic region of the telomere. TERRA may have a role in replication of telomeres and in heterochromatinization.24 Telomerase seems to elongate actively transcribing telomeres in cancer cells.25
Transcriptional interference: modulation of gene expression through ncRNA transcript NcRNAs transcript from an upstream promoter can either negatively or positively affect the expression of the downstream gene by inhibiting RNA polymerase II recruitment or inducing chromatin remodelling.1 For example, an lnc transcript of human dihydrofolate reductase (DHFR) locus Copyright © 2014 John Wiley & Sons, Ltd.
Epigenetic modifications It has been known previously that subsets of ncRNAs have key role in maintaining the active or inactive state of chromosomes by recruiting polycomb group (PcG) and trithorax group (TrxG) proteins on target site. PcG proteins bind and silence the expression of more than a thousand mammalian genes26,27, and TrxG protein counteracts the action of PcG proteins to maintain active transcription states, and, interestingly, both PcG and TrxG are recruited to their target loci by lncRNA.28 For example, a histone methyl transferase and PRC-2 (member of polycomb-repressive complex 2 or polycomb chromatin remodeling complex-2) were found to directly interact with 1.6 kb lncRNA (RepA) transcribed Cell Biochem Funct (2014)
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Figure 2. A schematic representation of biological functions of non-coding RNAs showing their roles in alternative splicing, epigenetic regulation of gene expression, mRNA turnover, translational repression, activation and generation of siRNA
from xist locus. RepA recruits PRC-2 to silence one Xchromosome, whereas PRC-2 is inactivated on the other Xchromosome by the antisense transcript Tsix, but one study explains the alternative mechanism that Xist and Tsix anneal to form an RNA duplex that is processed by dicer to generate siRNA that is required for the repressive chromatin modification on the inactive X-chromosome.29 Post-transcriptional modulation through ncRNAs As a result of cellular heat shock response, heat shock RNA1 (HSR1) ncRNA forms a complex with heat shock tran-
scription factor 1 activating the transcription factor to induce the expression of heat shock proteins.30 ncRNA derived from small interspersed elements binds to RNA polymerase II during heat shock to inhibit transcription of other mRNA such as actin.31 Some isoforms of the ncRNA, SRA function as transcriptional co-activator of steroid receptor.8,32 It has been found that ncRNAs also modulate the activity of proteins by regulating their subcellular localization. The transcription factor nuclear factor of activated T cells (NFAT) localizes to the cytoplasm and is imported back into the nucleus by Ca2+ dependent signals and activates transcription of target genes.33 ncRNA non-coding repressor of NFAT
Figure 3. Paradigms for non-coding RNAs including epigenetic control of gene expression, generation of si/miRNAs, interference with transcription, acting as precursors of small RNAs and regulation of chromatin organization Copyright © 2014 John Wiley & Sons, Ltd.
Cell Biochem Funct (2014)
NON-CODING RNAS: POWER AND PROMISES (NRON) has been found to regulate nuclear trafficking of the transcription factor NFAT. NRON specifically inhibits the nuclear accumulation of NFAT by binding to the members of the nucleocytoplasmic trafficking machinery but not the other transcription factors such as p53 that also translocate from the cytoplasm to the nucleus.34 Translational controls of other protein by ncRNAs Several reports suggest that RNA transcripts derived from pseudogenes are able to hybridize to their corresponding spliced mRNA, resulting in the formation of doublestranded RNA that are cleaved by dicer to generate endogenous siRNA (endo-siRNA). Thus, coding mRNA is consumed in generating endo-siRNA that may direct RNA-induced silencing complex (RISC) to cleave additional copies of the mRNA transcript, resulting in further downregulation of the protein-coding gene.35 It can be concluded that pseudogenes are not just a non-functional element but are the key regulators of gene expression when transcribed as lncRNAs. Pre-mRNAs after alternative splicing provide a major source of transcriptome and proteome diversity in cells.36 lncRNA controls mRNA translation by acting upon miRNA production. lncRNA H19 produces miR-675 in placenta in dicer-dependent manner. Ultimately, miR-675 suppresses the growth and proliferation of embryonic stem cells, trophoblastic stem cells and mouse embryo fibroblasts while reducing the translation of Igf1r (insulin growth factor receptor1). Production of miR-675 is regulated by HuR (an RNA binding protein), and thus, H19 limits the growth of placenta in mammals.37 Regulation of structural organizations by ncRNAs lncRNAs may, in some cases, serve as key structural component.4 There are numerous RNA-binding proteins within a nucleus, including paraspeckles protein component-1 (PSP1α), NONO and 68kd subunit of cleavage factor Im, as well as a nuclear retained mRNA (CTNRNA) localized to paraspeckles.38,39 Paraspeckles are newly identified ribonucleoproteins present in the interchromatin space of mammalian cell nuclei. These proteins, along with the lncRNA nuclear-enriched autosomal transcript 1 (NEAT1), associate to form paraspeckles and maintain their integrity. Nuclear speckles (interchromatin granule clusters), about 20–30 per nucleus are highly dynamic subnuclear domains enriched with pre-mRNA splicing factor.40 It has been proposed that speckles are sites from where splicing factors are recruited to active sites of transcription.40 From the whole transcriptome analysis, paraspeckles may be a new area for a class of subnuclear bodies formed around lncRNA. The paraspeckles have been suggested to function as storage sites for nuclear-retained RNAs.39 RNAs may be a critical component of these nuclear structures because of three reasons: Firstly, RNase A treatment disrupts the structural integrity of paraspeckles, second, paraspeckle proteins contain RNA-binding motifs Copyright © 2014 John Wiley & Sons, Ltd.
and third, PSP1α requires its RNA binding domain for paraspeckle targeting.41 Disease condition associated with ncRNAs Microsatellite expansion in non-coding region of genes can induce disease through an RNA gain of function mechanism.42 For example, the cause of myotonic dystrophy-1 is a CTG expansion in the 3′UTR region of dystrophia myotonica protein kinase gene.42 Further, it affects the activity of splicing factors by accumulation of RNA repeats. Another, gain of RNA function is reported in case of fragile X-associated tremor ataxia syndrome in which, CGG repeats occur in the fragile X mental retardation gene-1 and causes dysregulation in alternative splicing by recruiting a set of splicing regulators in to nuclear inclusions.43 The human genome share several hundred miRNAs and those ncRNAs seem to regulate 30% of human genome either by translation inhibition or its degradation.7 It has been reported that expression profiles of miRNAs differ in normal and cancerous tissue types, and high-throughput experimental results proved the perturbed expression of proteome as a result of the binding of miRNAs across the transcriptome at different sites.44,45 In addition to this, miRNA expression pattern were found to be altered in cardiac dysfunction.46 For example, miR-200 family are responsible for regulation of epithelial to mesenchymal transition and most of the Bcell chronic lymphocytic leukaemia (CLL) involved dysregulation of miR-15 and miR-16 RNA cluster.7,6,47 miRNAs encoded by miR-15a/b, miR-16-1/2 act as tumour suppressor, and deletion or inhibition of these two miRNAs enhances E2F1-induced G1/S transition.48 miR-15 and miR-16 are direct transcriptional targets of E2F that limits induction of E2F proliferation through cyclin E.48 miR-21 is classified as oncomiR on the basis of their deregulation almost in all types of cancer including cardiac and pulmonary fibrosis as well as myocardial infarction.49 Till date, most of the ncRNAs dysfunctioning are associated with diseased condition (Table 2). Online databases are available for miRNA (Human MicroRNA Disease Database) with miRNA names, disease associations, dysfunction evidences and PubMed ID of related papers (http://202.38.126.151/hmdd/mirna/md/). Non-coding RNAs act as small RNA precursors It is proposed that mature long transcript (both proteincoding mRNAs and lncRNAs) may be post-transcriptionally processed to yield small RNAs that are further modified by the addition of a cap structure.50 Each mature transcript can display a distinct subcellular localization and have a unique function. A nascent transcript can be processed to yield two ncRNAs (3′MALAT1 and 5′masc RNA) that localize to distinct subcellular compartments.51 Metastasisassociated lung adenocarcinoma transcript 1 (MALAT1) also known as nuclear-enriched autosomal transcript 252 is a highly conserved lncRNA (~7 kb), dysregulated in many cancers.53,54 It is retained specifically in the nuclear speckle domains that are thought to be involved in the assembly, Cell Biochem Funct (2014)
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An illustrative list of ncRNAs reported in cancer and diseases
Name miR-9 miR-15 and miR-16 miR-21 miR-34b and miR-34c miR-106b-25 miR-128 miR-129-2 miR-137 miR-141 miR-148a miR-151 miR-196b miR-200c miR-205 miR-517c and miR-520 g miR-675 and H19 mir-675 Uc.283 + A Uc.160+ Uc.346+ Uc.21 U50 Uc.72 Uc.159
Class
Consequence of deregulation
Disease association
References
miRNA miRNA
Metastasis BCL2 over-expression
Colon, melanoma, head and neck cancer Haematological cancer
miRNA miRNA
Malignant phenotype Metastasis
Carcinoma and cardiovascular disease Many different tumour types
miRNA miRNA miRNA miRNA miRNA miRNA miRNA miRNA miRNA miRNA miRNA
p21 and BIM depletion Tumourigenesis and cancer development SOX2 overexpression CDC42 overexpression EMT Metastasis Metastasis Unknown EMT EMT Wnt upregulation
Oesophageal adenocarcinoma and prostate cancer Carcinoma Colon, endometrial and gastric carcinoma Colon, head, stomach and neck carcinoma Colon, breast and lung carcinoma Colon, melanoma and breast carcinoma Hepatocellular carcinoma Gastric and Crohn disease Colon, breast and lung carcinoma Bladder carcinoma Neuroectodermal brain tumours
miRNA and lncRNA miRNA T-UCR T-UCR T-UCR T-UCR snoRNA T-UCR T-UCR
Mutation (epigenetic changes), modulation of expression of TGFB1 Modulation of tumour suppressor RUNX-1 Cell survival, mitosis CpG island hypermethylation-associated silencing CpG island hypermethylation-associated silencing Unknown Increase growth Unknown Unknown
Silver–Russell syndrome, prostate cancer Human gastric cancer cell proliferation Colon, breast and lung carcinoma Colon, breast and lung carcinoma Colon, breast and lung carcinoma Epithelial tumours and leukaemia Breast cancer Epithelial tumours and leukaemia Epithelial tumours and leukaemia
7 47 49 45 75,76 77 78 62 79 62 79 80 79 81 82 37,83,84 85 86 6 6 87 7,87 87 87
BCL2, B-cell CLL/lymphoma 2; BIM, also known as BCL2L11; CDC42, cell division cycle 42; CDK6, cyclin-dependent kinase 6; EMT, epithelial-to-mesenchymal transition; RUNX1, Runt domain transcription factor 1; T-UCR, transcribed-ultraconserved region.
modification and/or storage of the pre-mRNA processing machinery.40 In contrast to mature long MALAT1 transcript, small MALAT1-associated small cytolasmic RNA (masc RNA) is localized exclusively to the cytoplasm and is generated by the processing of the MALAT1 nascent transcript51 by RNase P that recognizes the tRNA-like structure in the nascent RNA polymerase II transcript and then cleaves to simultaneously generate 3′ end of the mature MALAT1 transcript and 5′ end of the mascRNA. Regulatory roles of the lncRNA MALAT1 It is assumed that MALAT1 could act as ‘molecular sponge’ by interacting with serine/arginine-rich (SR) proteins in the nuclear speckles thereby modulating the concentration of SR proteins.55 SR proteins are a family of splicing factors involved in both constitutive and alternative splicing.54 Although SR proteins are abundantly and ubiquitously expressed in cells and tissues, their cellular expression levels are tightly regulated. They all share Nterminal RNA recognition motifs (RRM) and an RS (arginine-serine) domain. RS domain is target for extensive phosphorylation and is important for the activity of SR proteins in splicing. Computational analysis identified SR protein binding motifs in MALAT1 RNA. SRSF1 (member of SR protein family) showed specific binding to MALAT1 through RRM.55 So, MALAT1 was required for proper localization of SSF-1 as well as several other specific factors Copyright © 2014 John Wiley & Sons, Ltd.
to the nuclear speckles. Both studies confirm that depletion of MALAT1 leads to changes in alternative splicing of a subset of transcripts.55,56 Diagnostic and therapeutic applications In eukaryotic cells, a large number of lncRNAs play vital role in regulating gene expression.1,57 The increasing functional diversity among lncRNAs observed in the mammalian genome provides the complex networks needed to regulate cell function and could be responsible for the genome paradox.1,57 In brief, lncRNAs serve as backbone of cells by directly or indirectly recruiting other factors in various crucial steps such as pre-mRNA splicing, mRNA decay, translation, whereas sncRNAs serve as critical component of the backbone by directly acting upon the target genes. It is proved that lncRNAs have very important role in cancer, by maintaining cellular homeostasis and can be used as diagnostic tool in combination with proteincoding genes.7 Increased awareness of ncRNA biology provides a new paradigm of regulatory role of lncRNAs in tumourigenesis.58 In some cancer prognosis like in colon, lung and breast cancer, there is strong association with miRNA expression signifying the diagnostic application of ncRNAs in combination with therapeutic targets.59,60 A signature of miR-200 family may be sufficient for cancer categorization.61 The diagnostic use of lncRNA is advantageous over protein-coding RNA because RNA itself is the effector Cell Biochem Funct (2014)
NON-CODING RNAS: POWER AND PROMISES molecule and its expression levels may be a better indicator of tumour. By using miRNA expression profiling, it is easy to accurately identify the origin of poorly differentiated tumours and carcinomas, revealing the developmental lineage and differential state of the tumours.62 It seems possible to deal with early detection of colon and other cancers by profiling miRNAs from serum, saliva and tissues of individuals.63 Confidence in RNA molecules being employed as therapeutic agents has grown. There is increasing use of RNAs as therapeutics like miRNA activity in human cancers.59,64 RNA-based and RNA-targeted therapies were encouraged by early successes using siRNAs in human in vitro culture systems,65 and in targeting HIV-1 and human BCL2 with siRNA-like molecules.66 siRNAs effectively modulate alternative splicing.67 miRNAs expression in vivo may be a powerful therapeutic mechanism adding new dimensions to phenotypic diversity. It is found that over-expression of a single miRNA (miR-302) is capable of inducing stemness.68
biomarkers for specific disease states. In brief, from this evidence, it is clear that lncRNAs are molecules that keep in perfect tune the balance of gene expression networks, and discordance in their function results homeostatic imbalance, often leading to cellular transformation. CONFLICT OF INTEREST The authors have declared that there is no conflict of interest. ACKNOWLEDGEMENT BS is an INSPIRE Fellow of the Department of Science and Technology. Financial support received from UGC, DST, DBT and ICMR are gratefully acknowledged.
REFERENCES FUTURE PERSPECTIVES A major current challenge is to understand the effect of molecular function and mechanism of action of lncRNAs. lncRNAs are shedding new light on cancer pathways and may represent a ‘missing link’ in cancer. For example, ncRNAs had a role in the formation of photoreceptors in the developing retina69 as well as in regulation of cell survival and cell cycle progression during mammary gland development.70 The mechanisms by which these lncRNA transcripts may involve in tumourigenesis are being better understood. According to a recent study, over-expression of HOTAIR level in primary breast tumours is a powerful predictor of metastasis and death.71 This phenotype is an outcome of PRC-2-dependent gene repression that is induced by HOTAIR. It is reported that enforced expression of HOTAIR results in altered pattern of H3K27 methylation and increased invasiveness, whereas the depletion of HOTAIR causes the opposite cellular phenotype.71 These studies clearly demonstrate that the oncogenic link of RNAs can hijack the epigenetic machinery to reshape the epigenetic landscape leading to cancer. An intriguing common theme is emergence of large ncRNAs forming ribonucleoprotein complexes that impart key regulatory functions in cellular circuits. It is clear that HOTAIR gene, MALAT1 among others, share a common functionality of forming RNA-protein complexes with chromatin regulatory factors. So, lncRNA MALAT1 is used as a tumour marker that is over-expressed in many different tumour types.53 However, it is unclear if MALAT1 acts exclusively through inhibition of the tumour-suppressor PSF. Therefore, a better understanding of cancer will require a comprehensive identification of lncRNAs with their associated protein complexes misregulated across a spectrum of cancer types. We shall need to identify lncRNAs that could potentially serve as Copyright © 2014 John Wiley & Sons, Ltd.
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b. santosh b. santosh 19. Blume SW, Meng Z, Shrestha K, Snyder RC, Emanuel PD. The 5’untranslated RNA of the human dhfr minor transcript alters transcription pre-initiation complex assembly at the major (core) promoter. J Cell Biochem 2003; 88: 165–180. 20. Martianov I, Ramadass A, Serra Barros A, Chow N, Akoulitchev A. Repression of the human dihydrofolate reductase gene by a noncoding interfering transcript. Nature 2007; 445: 666–670. 21. Blasco MA. Telomeres and human disease: ageing, cancer and beyond. Nat Rev Genet 2005; 6: 611–622. 22. Azzalin CM, Reichenbach P, Khoriauli L, Giulotto E, Lingner J. Telomeric repeat containing RNA and RNA surveillance factors at mammalian chromosome ends. Science (New York, NY) 2007; 318: 798–801. 23. Schoeftner S, Blasco MA. Developmentally regulated transcription of mammalian telomeres by DNA-dependent RNA polymerase II. Nat Cell Biol 2008; 10: 228–236. 24. Feuerhahn S, Iglesias N, Panza A, Porro A, Lingner J. TERRA biogenesis, turnover and implications for function. FEBS Lett 2010; 584: 3812–3818. 25. Farnung BO, Brun CM, Arora R, Lorenzi LE, Azzalin CM. Telomerase efficiently elongates highly transcribing telomeres in human cancer cells. PLoS One 2012; 7: e35714. 26. Boyer LA, Plath K, Zeitlinger J, Brambrink T, Medeiros LA, Lee TI, et al. Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 2006; 441: 349–353. 27. Bracken AP, Dietrich N, Pasini D, Hansen KH, Helin K. Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions. Genes Dev 2006; 20: 1123–1136. 28. Sanchez-Elsner T, Gou D, Kremmer E, Sauer F. Noncoding RNAs of trithorax response elements recruit Drosophila Ash1 to Ultrabithorax. Science 2006; 311: 1118–1123. 29. Ogawa Y, Sun BK, Lee JT. Intersection of the RNA Interference and X-Inactivation Pathways. Science 2008; 320: 1336–1341. 30. Shamovsky I, Ivannikov M, Kandel ES, Gershon D, Nudler E. RNAmediated response to heat shock in mammalian cells. Nature 2006; 440: 556–560. 31. Allen TA, Von Kaenel S, Goodrich JA, Kugel JF. The SINE-encoded mouse B2 RNA represses mRNA transcription in response to heat shock. Nat Struct Mol Biol 2004; 11: 816–821. 32. Lanz RB, McKenna NJ, Onate SA, Albrecht U, Wong J, Tsai SY, et al. A steroid receptor coactivator, SRA, functions as an RNA and is present in an SRC-1 complex. Cell 1999; 97: 17–27. 33. Hogan PG, Chen L, Nardone J, Rao A. Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev 2003; 17: 2205–2232. 34. Willingham AT, Orth AP, Batalov S, Peters EC, Wen BG, Aza-Blanc P, et al. A strategy for probing the function of noncoding RNAs finds a repressor of NFAT. Science (New York, NY) 2005; 309: 1570–1573. 35. Tam OH, Aravin AA, Stein P, Girard A, Murchison EP, Cheloufi S, et al. Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes. Nature 2008; 453: 534–538. 36. Pan Q, Shai O, Lee LJ, Frey BJ, Blencowe BJ. Deep surveying of alternative splicing complexity in the human transcriptome by highthroughput sequencing. Nat Genet 2008; 40: 1413–1415. 37. Keniry A, Oxley D, Monnier P, Kyba M, Dandolo L, Smits G, et al. The H19 lincRNA is a developmental reservoir of miR-675 that suppresses growth and Igf1r. Nat Cell Biol 2012; 14: 659–665. 38. Fox AH, Lam YW, Leung AKL, Lyon CE, Andersen J, Mann M, et al. Paraspeckles: a novel nuclear domain. Curr Bio: CB 2002; 12: 13–25. 39. Prasanth KV, Prasanth SG, Xuan Z, Hearn S, Freier SM, Bennett CF, et al. Regulating gene expression through RNA nuclear retention. Cell 2005; 123: 249–263. 40. Lamond AI, Spector DL. Nuclear speckles: a model for nuclear organelles. Nat Rev Mol Cell Biol 2003; 4: 605–612. 41. Fox AH, Bond CS, Lamond AI. P54nrb Forms a Heterodimer with PSP1 That Localizes to Paraspeckles in an RNA-dependent Manner. Mol Biol Cell 2005; 16: 5304–5315. 42. Ranum LPW, Cooper TA. RNA-mediated neuromuscular disorders. Annu Rev Neurosci 2006; 29: 259–277. 43. Sellier C, Rau F, Liu Y, Tassone F, Hukema RK, Gattoni R, et al. Sam68 sequestration and partial loss of function are associated
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NON-CODING RNAS: POWER AND PROMISES 67. Alló M, Buggiano V, Fededa JP, Petrillo E, Schor I, de la Mata M, et al. Control of alternative splicing through siRNA-mediated transcriptional gene silencing. Nat Struct Mol Biol 2009; 16: 717–724. 68. Kuo CH, Deng JH, Deng Q, Ying SY. A novel role of miR-302/367 in reprogramming. Biochem Biophys Res Commun 2012; 417: 11–16. 69. Young TL, Matsuda T, Cepko CL. The noncoding RNA taurine upregulated gene 1 is required for differentiation of the murine retina. Curr Biol: CB 2005; 15: 501–512. 70. Ginger MR, Shore AN, Contreras A, Rijnkels M, Miller J, GonzalezRimbau MF, et al. A noncoding RNA is a potential marker of cell fate during mammary gland development. Proc Natl Acad Sci U S A 2006; 103: 5781–5786. 71. Gupta RA, Shah N, Wang KC, Kim J, Horlings HM, Wong DJ, et al. Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature 2010; 464: 1071–1076. 72. King TH, Liu B, McCully RR, Fournier MJ. Ribosome structure and activity are altered in cells lacking snoRNPs that form pseudouridines in the peptidyl transferase center. Mol Cell 2003; 11: 425–435. 73. Aravin AA, Sachidanandam R, Girard A, Fejes-Toth K, Hannon GJ. Developmentally regulated piRNA clusters implicate MILI in transposon control. Science 2007; 316: 744–747. 74. Mattick JS. The Genetic Signatures of Noncoding RNAs. PLoS Genet 2009; 5: e1000459. 75. Hudson RS, Yi M, Esposito D, Glynn SA, Starks AM, Yang Y, et al. MicroRNA-106b-25 cluster expression is associated with early disease recurrence and targets caspase-7 and focal adhesion in human prostate cancer. Oncogene 2013; 32: 4139–4147. 76. Kan T, Sato F, Ito T, Matsumura N, David S, Cheng Y, et al. The miR106b-25 polycistron, activated by genomic amplification, functions as an oncogene by suppressing p21 and Bim. Gastroenterology 2009; 136: 1689–1700. 77. Li M, Fu W, Wo L, Shu X, Liu F, Li C. miR-128 and its target genes in tumorigenesis and metastasis. Exp Cell Res 2013; 319: 3059–3064.
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78. Huang YW, Liu JC, Deatherage DE, Luo J, Mutch DG, Goodfellow PJ, et al. Epigenetic repression of microRNA-129-2 leads to overexpression of SOX4 oncogene in endometrial cancer. Cancer Res 2009; 69: 9038–9046. 79. Nana-Sinkam SP, Croce CM. Non-coding RNAs in cancer initia tion and progression and as novel biomarkers. Mol Oncol 2011; 5: 483–491. 80. Tsai KW, Hu LY, Wu CW, Li SC, Lai CH, Kao HW, et al. Epigenetic regulation of miR-196b expression in gastric cancer. Genes Chromosomes Cancer 2010; 49: 969–980. 81. Qin AY, Zhang XW, Liu L, Yu JP, Li H, Wang SZ, et al. MiR-205 in cancer: an angel or a devil? Eur J Cell Biol 2013; 92: 54–60. 82. Li M, Lee KF, Lu Y, Clarke I, Shih D, Eberhart C, et al. Frequent amplification of a chr19q13.41 microRNA polycistron in aggressive primitive neuroectodermal brain tumors. Cancer Cell 2009; 16: 533–546. 83. Bartholdi D, Krajewska-Walasek M, Ounap K, Gaspar H, Chrzanowska KH, Ilyana H, et al. Epigenetic mutations of the imprinted IGF2-H19 domain in Silver-Russell syndrome (SRS): results from a large cohort of patients with SRS and SRS-like phenotypes. J Med Genet 2009; 46: 192–197. 84. Zhu M, Chen Q, Liu X, Sun Q, Zhao X, Deng R, et al. lncRNA H19/miR-675 axis represses prostate cancer metastasis by targeting TGFBI. FEBS J 2014; 281: 3766–3775. 85. Zhuang M, Gao W, Xu J, Wang P, Shu Y. The long non-coding RNA H19-derived miR-675 modulates human gastric cancer cell proliferation by targeting tumor suppressor RUNX1. Biochem Biophys Res Commun 2014; 448: 315–322. 86. Plosky BS. An Ultraconserved lnc to miRNA Processing. Mol Cell 2014; 55: 3–4. 87. Calin GA, Liu CG, Ferracin M, Hyslop T, Spizzo R, Sevignani C, et al. Ultraconserved regions encoding ncRNAs are altered in human leukemias and carcinomas. Cancer Cell 2007; 12: 215–229.
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Non-coding RNAs: biological functions and applications Baby Santosh, Akhil Varshney and Pramod Kumar Yadava* School of Life Sciences, Jawaharlal Nehru University, New Delhi, Delhi India
Analyses of the international human genome sequencing results in 2004 converged to a consensual number of ~20 000 protein-coding genes, spanning over 90% of the human genome is likely to be transcribed yielding a complex network of overlapping transcripts that include tens of thousands of long RNAs with little or no protein forming capacity; they are collectively called non-coding RNA. This review highlights the fundamental concepts of biological roles of non-coding RNA and their importance in regulation of cellular physiology under disease conditions like cancer. Copyright © 2014 John Wiley & Sons, Ltd. key words—non-coding RNA; regulatory role; telomere biology; chromatin dynamics; gene modulation; structural organization list of abbreviations—ncRNA, non-coding RNA; lncRNA, long non-coding RNA; snpcRNA, small non-protein coding RNA; siRNA, small interfering RNA; snoRNA, small nucleolar RNA; miRNA, microRNA; snRNA, small nuclear RNA; piRNA
NON-CODING RNAS Transcripts of genomic sequences that are not meant to be translated are often called non-translated or non-coding RNA (ncRNA) molecules.1 A vast number of ncRNAs are encoded by human genome. Among them, most of the ncRNAs have been widely implicated in regulating cellular homeostasis.2 Some of these ncRNAs are directly involved in epigenetic changes and/or modifications in cells. Total cellular RNAs are classified on the basis of their functions as presented in Figure 1. A subset of ncRNAs is processed in to functionally important RNAs such as transfer RNA (tRNA), ribosomal RNA (rRNA), small nucleoar RNA (snoRNAs), microRNAs, small interfering RNAs (siRNAs), PIWI-interacting RNAs (piRNAs) and the long non-coding RNAs (lncRNAs). Two widely studied lncRNA are Xinactivation specific transcript (xist) and HOX antisense intergenic RNA (HOTAIR).60,29 The total number of ncRNAs encoded within the human genome is not completely known; however, recent transcriptomic and bioinformatic studies suggest the existence of thousands of ncRNAs with their functional importance. Because many of the newly identified ncRNAs have not been validated for
*Correspondence to: Pramod Kumar Yadava, Jawaharlal Nehru University School of Life Sciences Lab no. 111, SLS, JNu Room no. 111, SLS, JNu New Delhi India 110067.E-mail: [email protected]
Copyright © 2014 John Wiley & Sons, Ltd.
their function, it is possible that many are non-functional. However, examples of the function and role of ncRNAs are still emerging. ncRNAs are divided on the basis of the length of RNA formed post-transcriptionally, i.e. short non-coding RNA (sncRNAs < 30 nt) and long non-coding RNA (lncRNAs > 200 nt). lncRNAs are generally considered as non-protein coding transcripts, longer than 200 nt. This limit is because of practical considerations including the separation of RNAs in common experimental protocols. Additionally, this limit distinguishes lncRNAs from small regulatory RNAs such as miRNAs, siRNAs, piRNAs, snoRNAs (Table 1). miRNAs, a class of short ncRNA, are 18–24 nt long and involved in skin fibrosis.3 The broad functional repertoire of lncRNAs includes role in higher order chromosomal dynamics, telomere biology and in subcellular structural organization.4,5 A new class of ncRNAs, T-UCR is transcribed from ultraconserved region.6 T-UCRs are subset of DNA segments longer that 200 bp and completely conserved between species (Humans, Rat and Mouse genome).6,7 In addition to this, ncRNAs are further subdivided on the basis of nature of genes involved in oncogenesis and/or tumour suppression. For example, steroid receptor RNA activator (SRA), is an oncogenic ncRNA that enhances adipogenesis in response to insulin and inhibits the expression of inflammatory genes associated with adipocytes.8 Maternally expressed gene 3 (MEG3), ncRNA encoding gene is located at chromosome Received 25 August 2014 Revised 13 October 2014 Accepted 31 October 2014
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Figure 1. A classification of RNA types showing the whole new world of RNAs playing important roles in diverse biological functions by modulating gene expression via antisense and miRNA-mediated controls
Table 1. S. no.
Examples of functional non-coding RNAs in mammals Name
Characteristics
Functions
References
Class (a) small non-coding RNAs 1
Small interfering RNAs (siRNAs)
2
MicroRNAs (miRNAs)
3
MicroRNA-offset RNAs (moRNAs) Small nucleolar RNAs (snoRNAs) PIWI-interacting RNAs (piRNAs)
4 6 7
Promoter-associated RNAs (PARs)
~21 to 22 nt long RNAs; produced by dicer cleavage of complementary dsRNA duplexes ~22 nt long; produced by dicer cleavage of imperfect RNA hairpins encoded in long primary transcripts or short introns Small ~20 nt long RNAs derived from regions adjacent to pre-miRNAs Control over rRNA methylation and pseudouridylation Small RNA ~24–30 nt, restricted to the germline and germline bordering somatic cells Long and short RNAs including promoter associated RNAs
Gene regulation, transposon control and viral defence
2.2 kb, transcribed from the HOXC locus
57,66
Post-transcriptional gene regulation
45
Gene regulation
63
Evidence of gene regulatory roles
72
Associated with PIWI-clade Argonaute proteins, regulate transposon mobility and chromatin state Regulate gene expression
73
Epigenetically silences gene expression at the HOX D locus PRC2 recruitment on the inactive X-chromosome Role in X-chromosome inactivation
71
miRNA-like translational regulators
72
Translational repression
51
Regulatory function
12
Involved in telomere maintenance
22
Involved in chromatin modifications
57
Binds and inhibits glucocorticoid receptor
10
74
Class (b) long non-coding RNAs 8 9
HOTAIR (HOX antisense intergenic RNA) Rep A1
10
X-inactivation RNAs (xiRNAs)
11
Sno-derived RNAs (sdRNAs) tRNA-derived RNAs
12 13
MSY2-associated RNAs (MSY-RNAs)
14
Telomere small RNAs (tel-sRNAs) Centrosome-associated RNA (crasi-RNAs) GAS5 (growth-arrest specific 5)
15 16
Copyright © 2014 John Wiley & Sons, Ltd.
1.6 kb, internal to Xist Dicer-dependent small RNAs processed from two lncRNAs duplexes, Xist and Tsix Dicer-dependent small RNAs processed from snoRNAs tRNAs processed into small RNA species by a conserved RNase (angiogenin) ~26–30 nt long, associated with the germ cell-specific DNA/RNA binding protein MSY2 and restricted to the germline ~24 nt RNAs principally derived from the G-rich strand of telomeric repeats A class of ~34–42 nt small RNAs, derived from centrosomes 600 nt long RNA
28 29
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NON-CODING RNAS: POWER AND PROMISES 14q32.3 on DLK1-MEG3 locus in humans, and its inactivation causes expression of angiogenesis promoting genes and formation of microvessels in brain.9 GAS5 ncRNA acts as decoy by competing with DNA-glucocorticoid response element (DNA-GRE) at DNA binding domain of glucocorticoid receptor and modulates growth arrest during starvation.10
originating from a region upstream of the major DHFR promoter represses expression of the downstream proteincoding gene19,20 both in cis and trans by forming an RNA–DNA triplex structure with the DHFR promoter and directly interacting with TFIIB, which results in disruption of the pre-initiation complex at the DHFR promoter.20
WIDESPREAD FUNCTIONS OF LNCRNAS
Non-coding RNAs in telomeres
It was presumed previously that lncRNAs are just the transcriptional noise resulting from low RNA polymerase fidelity.11 However, the expression of many lncRNA is restricted to particular developmental stages, and they have tissuespecific expressions.12 According to a recent study, only one fifth of transcription across the human genome is associated with functional protein-coding genes indicating at least four times more long non-coding than coding RNA sequences.13 However, it is large-scale complementary DNA (cDNA) sequencing projects such as functional annotation of mammalian cDNA (FANTOM) that reveal the complexity of this transcription.14 Numerous non-coding transcripts, for example, tRNAs, rRNAs and spliceosomal RNAs, are critical components of cellular machinery. NcRNAs regulate the cellular gene functionality through base pairing; stabilization of target gene translation is promoted by extended base pairing whereas partial base pairing leads to inhibition of target messenger RNA (mRNA) translation while absence of complementarity results in suppression of precursor mRNA by acting as decoy.15 NcRNAs play key regulatory and functional roles in gene expression program of the cell (Figures 2 and 3). One of the crucial functions of ncRNA is to act as ribozymes.16 The group I and II introns, RNase P and hammerhead are naturally occurring ribozymes. A structural class of self-splicing RNAs (group I and II introns) are 200–600 nt long and catalyzes RNA splicing. Although the functions of only limited number of lncRNA transcripts have been identified in different species, numerous paradigms are beginning to be explored (Figure 2). For example, flowering in plants is controlled by flowering locus C (FLC) gene, and a lncRNA cold assisted intronic non-coding RNA (COLDAIR) regulates vernalization, i.e. seasonal time flowering mediated by environmentally induced epigenetic changes, by silencing FLC.17 NcRNAs regulate a remarkable variety of biological functions in transcriptional interference, telomere maintenance, epigenetic changes, imprinting, post-transcriptional, translational control, structural organization, cell differentiation and development.18
Telomeres are functional nucleoprotein complexes that protect the terminus of the eukaryotic chromosomes from degradation and activation of DNA damage responses that would otherwise follow telomeres being recognized as DNA double-strand breaks. It maintains the genomic stability, senescence and aging.21 Telomeres have been long considered transcriptionally silent DNA-protein complexes because of their presence in heterochromatin state. It was recently shown that telomeres are transcribed in several organisms (e.g. Homo sapiens, Mus musculus, Danio rerio, yeast, plants) and give rise to lncRNA called telomere repeat containing RNA (TERRA) or TelRNAs.22,23 TERRA molecules are involved in telomere homeostasis through regulating telomere length, telomerase activity and formation of heterochromatin at the end of the chromosomes. Altered expression of these molecules may lead to chromosomal instability and can promote cellular senescence.5 TERRA transcript originates from the subtelomeric region of telomeres and proceeds towards the chromosome end by RNA polymerase II. The length of TERRA varies from 100 nt to 9 kb in mammalian cells and consists of Grich telomeric repeats.23 The association of these molecules with chromatin is negatively regulated by suppressors with morphogenetic defects in genitalia (SMG) proteins. Thus, SMG proteins play connecting link between TERRA (lncRNA) and telomere-specific heterochromatin modifications.23 These studies strongly suggest for an involvement of telomeric ncRNAs in telomere biology. Transcripts arising from telomere transcription are known in several eukaryotes. TERRA is part of the heterochromatic region of the telomere. TERRA may have a role in replication of telomeres and in heterochromatinization.24 Telomerase seems to elongate actively transcribing telomeres in cancer cells.25
Transcriptional interference: modulation of gene expression through ncRNA transcript NcRNAs transcript from an upstream promoter can either negatively or positively affect the expression of the downstream gene by inhibiting RNA polymerase II recruitment or inducing chromatin remodelling.1 For example, an lnc transcript of human dihydrofolate reductase (DHFR) locus Copyright © 2014 John Wiley & Sons, Ltd.
Epigenetic modifications It has been known previously that subsets of ncRNAs have key role in maintaining the active or inactive state of chromosomes by recruiting polycomb group (PcG) and trithorax group (TrxG) proteins on target site. PcG proteins bind and silence the expression of more than a thousand mammalian genes26,27, and TrxG protein counteracts the action of PcG proteins to maintain active transcription states, and, interestingly, both PcG and TrxG are recruited to their target loci by lncRNA.28 For example, a histone methyl transferase and PRC-2 (member of polycomb-repressive complex 2 or polycomb chromatin remodeling complex-2) were found to directly interact with 1.6 kb lncRNA (RepA) transcribed Cell Biochem Funct (2014)
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Figure 2. A schematic representation of biological functions of non-coding RNAs showing their roles in alternative splicing, epigenetic regulation of gene expression, mRNA turnover, translational repression, activation and generation of siRNA
from xist locus. RepA recruits PRC-2 to silence one Xchromosome, whereas PRC-2 is inactivated on the other Xchromosome by the antisense transcript Tsix, but one study explains the alternative mechanism that Xist and Tsix anneal to form an RNA duplex that is processed by dicer to generate siRNA that is required for the repressive chromatin modification on the inactive X-chromosome.29 Post-transcriptional modulation through ncRNAs As a result of cellular heat shock response, heat shock RNA1 (HSR1) ncRNA forms a complex with heat shock tran-
scription factor 1 activating the transcription factor to induce the expression of heat shock proteins.30 ncRNA derived from small interspersed elements binds to RNA polymerase II during heat shock to inhibit transcription of other mRNA such as actin.31 Some isoforms of the ncRNA, SRA function as transcriptional co-activator of steroid receptor.8,32 It has been found that ncRNAs also modulate the activity of proteins by regulating their subcellular localization. The transcription factor nuclear factor of activated T cells (NFAT) localizes to the cytoplasm and is imported back into the nucleus by Ca2+ dependent signals and activates transcription of target genes.33 ncRNA non-coding repressor of NFAT
Figure 3. Paradigms for non-coding RNAs including epigenetic control of gene expression, generation of si/miRNAs, interference with transcription, acting as precursors of small RNAs and regulation of chromatin organization Copyright © 2014 John Wiley & Sons, Ltd.
Cell Biochem Funct (2014)
NON-CODING RNAS: POWER AND PROMISES (NRON) has been found to regulate nuclear trafficking of the transcription factor NFAT. NRON specifically inhibits the nuclear accumulation of NFAT by binding to the members of the nucleocytoplasmic trafficking machinery but not the other transcription factors such as p53 that also translocate from the cytoplasm to the nucleus.34 Translational controls of other protein by ncRNAs Several reports suggest that RNA transcripts derived from pseudogenes are able to hybridize to their corresponding spliced mRNA, resulting in the formation of doublestranded RNA that are cleaved by dicer to generate endogenous siRNA (endo-siRNA). Thus, coding mRNA is consumed in generating endo-siRNA that may direct RNA-induced silencing complex (RISC) to cleave additional copies of the mRNA transcript, resulting in further downregulation of the protein-coding gene.35 It can be concluded that pseudogenes are not just a non-functional element but are the key regulators of gene expression when transcribed as lncRNAs. Pre-mRNAs after alternative splicing provide a major source of transcriptome and proteome diversity in cells.36 lncRNA controls mRNA translation by acting upon miRNA production. lncRNA H19 produces miR-675 in placenta in dicer-dependent manner. Ultimately, miR-675 suppresses the growth and proliferation of embryonic stem cells, trophoblastic stem cells and mouse embryo fibroblasts while reducing the translation of Igf1r (insulin growth factor receptor1). Production of miR-675 is regulated by HuR (an RNA binding protein), and thus, H19 limits the growth of placenta in mammals.37 Regulation of structural organizations by ncRNAs lncRNAs may, in some cases, serve as key structural component.4 There are numerous RNA-binding proteins within a nucleus, including paraspeckles protein component-1 (PSP1α), NONO and 68kd subunit of cleavage factor Im, as well as a nuclear retained mRNA (CTNRNA) localized to paraspeckles.38,39 Paraspeckles are newly identified ribonucleoproteins present in the interchromatin space of mammalian cell nuclei. These proteins, along with the lncRNA nuclear-enriched autosomal transcript 1 (NEAT1), associate to form paraspeckles and maintain their integrity. Nuclear speckles (interchromatin granule clusters), about 20–30 per nucleus are highly dynamic subnuclear domains enriched with pre-mRNA splicing factor.40 It has been proposed that speckles are sites from where splicing factors are recruited to active sites of transcription.40 From the whole transcriptome analysis, paraspeckles may be a new area for a class of subnuclear bodies formed around lncRNA. The paraspeckles have been suggested to function as storage sites for nuclear-retained RNAs.39 RNAs may be a critical component of these nuclear structures because of three reasons: Firstly, RNase A treatment disrupts the structural integrity of paraspeckles, second, paraspeckle proteins contain RNA-binding motifs Copyright © 2014 John Wiley & Sons, Ltd.
and third, PSP1α requires its RNA binding domain for paraspeckle targeting.41 Disease condition associated with ncRNAs Microsatellite expansion in non-coding region of genes can induce disease through an RNA gain of function mechanism.42 For example, the cause of myotonic dystrophy-1 is a CTG expansion in the 3′UTR region of dystrophia myotonica protein kinase gene.42 Further, it affects the activity of splicing factors by accumulation of RNA repeats. Another, gain of RNA function is reported in case of fragile X-associated tremor ataxia syndrome in which, CGG repeats occur in the fragile X mental retardation gene-1 and causes dysregulation in alternative splicing by recruiting a set of splicing regulators in to nuclear inclusions.43 The human genome share several hundred miRNAs and those ncRNAs seem to regulate 30% of human genome either by translation inhibition or its degradation.7 It has been reported that expression profiles of miRNAs differ in normal and cancerous tissue types, and high-throughput experimental results proved the perturbed expression of proteome as a result of the binding of miRNAs across the transcriptome at different sites.44,45 In addition to this, miRNA expression pattern were found to be altered in cardiac dysfunction.46 For example, miR-200 family are responsible for regulation of epithelial to mesenchymal transition and most of the Bcell chronic lymphocytic leukaemia (CLL) involved dysregulation of miR-15 and miR-16 RNA cluster.7,6,47 miRNAs encoded by miR-15a/b, miR-16-1/2 act as tumour suppressor, and deletion or inhibition of these two miRNAs enhances E2F1-induced G1/S transition.48 miR-15 and miR-16 are direct transcriptional targets of E2F that limits induction of E2F proliferation through cyclin E.48 miR-21 is classified as oncomiR on the basis of their deregulation almost in all types of cancer including cardiac and pulmonary fibrosis as well as myocardial infarction.49 Till date, most of the ncRNAs dysfunctioning are associated with diseased condition (Table 2). Online databases are available for miRNA (Human MicroRNA Disease Database) with miRNA names, disease associations, dysfunction evidences and PubMed ID of related papers (http://202.38.126.151/hmdd/mirna/md/). Non-coding RNAs act as small RNA precursors It is proposed that mature long transcript (both proteincoding mRNAs and lncRNAs) may be post-transcriptionally processed to yield small RNAs that are further modified by the addition of a cap structure.50 Each mature transcript can display a distinct subcellular localization and have a unique function. A nascent transcript can be processed to yield two ncRNAs (3′MALAT1 and 5′masc RNA) that localize to distinct subcellular compartments.51 Metastasisassociated lung adenocarcinoma transcript 1 (MALAT1) also known as nuclear-enriched autosomal transcript 252 is a highly conserved lncRNA (~7 kb), dysregulated in many cancers.53,54 It is retained specifically in the nuclear speckle domains that are thought to be involved in the assembly, Cell Biochem Funct (2014)
b. santosh b. santosh Table 2.
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An illustrative list of ncRNAs reported in cancer and diseases
Name miR-9 miR-15 and miR-16 miR-21 miR-34b and miR-34c miR-106b-25 miR-128 miR-129-2 miR-137 miR-141 miR-148a miR-151 miR-196b miR-200c miR-205 miR-517c and miR-520 g miR-675 and H19 mir-675 Uc.283 + A Uc.160+ Uc.346+ Uc.21 U50 Uc.72 Uc.159
Class
Consequence of deregulation
Disease association
References
miRNA miRNA
Metastasis BCL2 over-expression
Colon, melanoma, head and neck cancer Haematological cancer
miRNA miRNA
Malignant phenotype Metastasis
Carcinoma and cardiovascular disease Many different tumour types
miRNA miRNA miRNA miRNA miRNA miRNA miRNA miRNA miRNA miRNA miRNA
p21 and BIM depletion Tumourigenesis and cancer development SOX2 overexpression CDC42 overexpression EMT Metastasis Metastasis Unknown EMT EMT Wnt upregulation
Oesophageal adenocarcinoma and prostate cancer Carcinoma Colon, endometrial and gastric carcinoma Colon, head, stomach and neck carcinoma Colon, breast and lung carcinoma Colon, melanoma and breast carcinoma Hepatocellular carcinoma Gastric and Crohn disease Colon, breast and lung carcinoma Bladder carcinoma Neuroectodermal brain tumours
miRNA and lncRNA miRNA T-UCR T-UCR T-UCR T-UCR snoRNA T-UCR T-UCR
Mutation (epigenetic changes), modulation of expression of TGFB1 Modulation of tumour suppressor RUNX-1 Cell survival, mitosis CpG island hypermethylation-associated silencing CpG island hypermethylation-associated silencing Unknown Increase growth Unknown Unknown
Silver–Russell syndrome, prostate cancer Human gastric cancer cell proliferation Colon, breast and lung carcinoma Colon, breast and lung carcinoma Colon, breast and lung carcinoma Epithelial tumours and leukaemia Breast cancer Epithelial tumours and leukaemia Epithelial tumours and leukaemia
7 47 49 45 75,76 77 78 62 79 62 79 80 79 81 82 37,83,84 85 86 6 6 87 7,87 87 87
BCL2, B-cell CLL/lymphoma 2; BIM, also known as BCL2L11; CDC42, cell division cycle 42; CDK6, cyclin-dependent kinase 6; EMT, epithelial-to-mesenchymal transition; RUNX1, Runt domain transcription factor 1; T-UCR, transcribed-ultraconserved region.
modification and/or storage of the pre-mRNA processing machinery.40 In contrast to mature long MALAT1 transcript, small MALAT1-associated small cytolasmic RNA (masc RNA) is localized exclusively to the cytoplasm and is generated by the processing of the MALAT1 nascent transcript51 by RNase P that recognizes the tRNA-like structure in the nascent RNA polymerase II transcript and then cleaves to simultaneously generate 3′ end of the mature MALAT1 transcript and 5′ end of the mascRNA. Regulatory roles of the lncRNA MALAT1 It is assumed that MALAT1 could act as ‘molecular sponge’ by interacting with serine/arginine-rich (SR) proteins in the nuclear speckles thereby modulating the concentration of SR proteins.55 SR proteins are a family of splicing factors involved in both constitutive and alternative splicing.54 Although SR proteins are abundantly and ubiquitously expressed in cells and tissues, their cellular expression levels are tightly regulated. They all share Nterminal RNA recognition motifs (RRM) and an RS (arginine-serine) domain. RS domain is target for extensive phosphorylation and is important for the activity of SR proteins in splicing. Computational analysis identified SR protein binding motifs in MALAT1 RNA. SRSF1 (member of SR protein family) showed specific binding to MALAT1 through RRM.55 So, MALAT1 was required for proper localization of SSF-1 as well as several other specific factors Copyright © 2014 John Wiley & Sons, Ltd.
to the nuclear speckles. Both studies confirm that depletion of MALAT1 leads to changes in alternative splicing of a subset of transcripts.55,56 Diagnostic and therapeutic applications In eukaryotic cells, a large number of lncRNAs play vital role in regulating gene expression.1,57 The increasing functional diversity among lncRNAs observed in the mammalian genome provides the complex networks needed to regulate cell function and could be responsible for the genome paradox.1,57 In brief, lncRNAs serve as backbone of cells by directly or indirectly recruiting other factors in various crucial steps such as pre-mRNA splicing, mRNA decay, translation, whereas sncRNAs serve as critical component of the backbone by directly acting upon the target genes. It is proved that lncRNAs have very important role in cancer, by maintaining cellular homeostasis and can be used as diagnostic tool in combination with proteincoding genes.7 Increased awareness of ncRNA biology provides a new paradigm of regulatory role of lncRNAs in tumourigenesis.58 In some cancer prognosis like in colon, lung and breast cancer, there is strong association with miRNA expression signifying the diagnostic application of ncRNAs in combination with therapeutic targets.59,60 A signature of miR-200 family may be sufficient for cancer categorization.61 The diagnostic use of lncRNA is advantageous over protein-coding RNA because RNA itself is the effector Cell Biochem Funct (2014)
NON-CODING RNAS: POWER AND PROMISES molecule and its expression levels may be a better indicator of tumour. By using miRNA expression profiling, it is easy to accurately identify the origin of poorly differentiated tumours and carcinomas, revealing the developmental lineage and differential state of the tumours.62 It seems possible to deal with early detection of colon and other cancers by profiling miRNAs from serum, saliva and tissues of individuals.63 Confidence in RNA molecules being employed as therapeutic agents has grown. There is increasing use of RNAs as therapeutics like miRNA activity in human cancers.59,64 RNA-based and RNA-targeted therapies were encouraged by early successes using siRNAs in human in vitro culture systems,65 and in targeting HIV-1 and human BCL2 with siRNA-like molecules.66 siRNAs effectively modulate alternative splicing.67 miRNAs expression in vivo may be a powerful therapeutic mechanism adding new dimensions to phenotypic diversity. It is found that over-expression of a single miRNA (miR-302) is capable of inducing stemness.68
biomarkers for specific disease states. In brief, from this evidence, it is clear that lncRNAs are molecules that keep in perfect tune the balance of gene expression networks, and discordance in their function results homeostatic imbalance, often leading to cellular transformation. CONFLICT OF INTEREST The authors have declared that there is no conflict of interest. ACKNOWLEDGEMENT BS is an INSPIRE Fellow of the Department of Science and Technology. Financial support received from UGC, DST, DBT and ICMR are gratefully acknowledged.
REFERENCES FUTURE PERSPECTIVES A major current challenge is to understand the effect of molecular function and mechanism of action of lncRNAs. lncRNAs are shedding new light on cancer pathways and may represent a ‘missing link’ in cancer. For example, ncRNAs had a role in the formation of photoreceptors in the developing retina69 as well as in regulation of cell survival and cell cycle progression during mammary gland development.70 The mechanisms by which these lncRNA transcripts may involve in tumourigenesis are being better understood. According to a recent study, over-expression of HOTAIR level in primary breast tumours is a powerful predictor of metastasis and death.71 This phenotype is an outcome of PRC-2-dependent gene repression that is induced by HOTAIR. It is reported that enforced expression of HOTAIR results in altered pattern of H3K27 methylation and increased invasiveness, whereas the depletion of HOTAIR causes the opposite cellular phenotype.71 These studies clearly demonstrate that the oncogenic link of RNAs can hijack the epigenetic machinery to reshape the epigenetic landscape leading to cancer. An intriguing common theme is emergence of large ncRNAs forming ribonucleoprotein complexes that impart key regulatory functions in cellular circuits. It is clear that HOTAIR gene, MALAT1 among others, share a common functionality of forming RNA-protein complexes with chromatin regulatory factors. So, lncRNA MALAT1 is used as a tumour marker that is over-expressed in many different tumour types.53 However, it is unclear if MALAT1 acts exclusively through inhibition of the tumour-suppressor PSF. Therefore, a better understanding of cancer will require a comprehensive identification of lncRNAs with their associated protein complexes misregulated across a spectrum of cancer types. We shall need to identify lncRNAs that could potentially serve as Copyright © 2014 John Wiley & Sons, Ltd.
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