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 Table of Contents  
REVIEW ARTICLE
Year : 2017  |  Volume : 1  |  Issue : 1  |  Page : 23-29

Biological Functions and Research Methods of Long Noncoding RNAs


1 Department of Biochemistry and Biology, Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, School of Basic Medical Sciences, Fudan University, Shanghai 200030, China
2 Department of Biochemistry and Biology, Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, School of Basic Medical Sciences, Fudan University; Research Center for Birth Defects, Fudan University, Shanghai 200030, China

Date of Web Publication17-Jul-2017

Correspondence Address:
Duan Ma
Department of Biochemistry and Biology, Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, School of Basic Medical Sciences, Fudan University, Shanghai 200030
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2096-2924.210697

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  Abstract 


Long noncoding RNAs (lncRNAs) are functional RNA molecules which are longer than 200 nucleotides in length that do not encode proteins; instead, they regulate target gene expression at transcriptional, posttranscriptional, and epigenetic levels. LncRNAs play important roles in various biological processes such as dosage compensation, genomic imprinting, cell cycle regulation, and cell differentiation. Although their characterizations have been relatively straightforward with recent advances in modern biology, the functions of lncRNAs are largely unknown. Herein, we discuss the biological functions and research methods of lncRNAs.

Keywords: Biological Functions; Long Noncoding RNA; Research Methods


How to cite this article:
Ma J, Chen Q, Ma D. Biological Functions and Research Methods of Long Noncoding RNAs. Reprod Dev Med 2017;1:23-9

How to cite this URL:
Ma J, Chen Q, Ma D. Biological Functions and Research Methods of Long Noncoding RNAs. Reprod Dev Med [serial online] 2017 [cited 2020 Jun 5];1:23-9. Available from: http://www.repdevmed.org/text.asp?2017/1/1/23/210697




  Introduction Top


Whole genome sequencing has found that only 1.5% of the total genome encodes proteins. The majority of the nonprotein coding genome is transcribed into long noncoding RNAs (lncRNAs), indicating that lncRNAs are crucial for various biological processes. LncRNAs are functional RNA molecules longer than 200 nucleotides in length that do not have an open reading frame. They were found in the nucleus or cytoplasm, transcribed by RNA polymerase II, and always capped and polyadenylated.[1] In addition, they have fewer repeat sequences, shorter half-life, single combined site, and multifacet regulation of gene expression levels.

In 2002, Okazaki et al.[2] identified numerous lncRNA transcripts from a large-scale sequencing study of a mouse full-length complementary deoxyribonucleic acid (cDNA) library. And due to unencoding proteins, lncRNA accumulated in the genome during evolution of genome without any biological function, and was named “transcriptional noise”. Ponjavic et al.[3] examined transcriptome data from the Functional Annotation of the Mammalian Genome (FANTOM) study and found that lncRNA primary sequence, promoter sequence, and alternative splicing represented a negative choice with unknown features. In 2007, Rinn et al.[4] reported an interaction between the 2.2 kb lncRNA HOX transcript antisense RNA (HOTAIR) and the polycomb protein complex, which inhibited HOX gene transcription and modified chromatin, thereby regulating the growth and development of organisms. Additional lncRNAs were subsequently identified, such as H19 and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), thus providing a good opportunity to study this growing list of lncRNAs [Table 1].
Table 1: Examples of diverse lncRNAs and their main features

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Based on the relative distance between the lncRNA and the homologous coding gene, lncRNAs can be classified as normal, antisense, bi-directional, intron, promoter, or 3' UTR transcripts.[5],[6] They can also be classified according to their regulatory mechanism as a signaling, decoy, guide, or scaffolding molecule.[7] Functional studies showed that lncRNAs regulated target gene expression at epigenetic, transcriptional, and posttranscriptional levels, and they played important roles in various biological processes such as dosage compensation, genomic imprinting, cell cycle regulation, and cell differentiation.[8],[9] Their in-depth study can help us understand the physiological and pathological mechanism of lncRNAs and higher eukaryotic complex regulatory network. Herein, we discuss the biological functions and the research methods used to study lncRNAs.


  Biological Functions of LncRNAs Top


Epigenetic regulation

Epigenetics refers to the inheritance pattern that alters the functions of genes, resulting in phenotypic changes without changes in the nucleotide sequences of the genes. LncRNAs are also involved in other epigenetic processes such as DNA methylation [Figure 1]a, genomic imprinting [Figure 1]b, chromatin remodeling [Figure 1]c, and histone modification [Figure 1]d.
Figure 1: Major biological functions of lncRNAs in epigenetic, transcriptional, and posttranscriptional regulation. (a) DNA methylation, (b) genomic imprinting, (c) chromatin remodeling, (d) histone modifications, (e) transcriptional regulation, and (f) posttranscriptional regulation.

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DNA methylation and demethylation

CEBPA (CCAAT/enhangcer binding protein alpha) is a well-studied methylation-sensitive gene. A previous study identified a lncRNA transcribed from CEBPA interacting with DNA methyltransferase 1 (DNMT1) that suppressed CEBPA methylation and stabilized its messenger RNA (mRNA) synthesis.[10] In two-cell mouse embryos, on the other hand, bidirectional promoter-associated lncRNAs (pancRNAs) strongly associated with the upregulation of their cognate genes. On downregulation of abundant pancRNAs, mRNA expression was decreased and accompanied by sustained DNA methylation even in the presence of enzymes responsible for DNA demethylation.[11] It was also reported that antisense lncRNA-TARID (TCF21 antisense RNA inducing demethylation) binds to the TCF21 promoter and GADD45A, a regulator of DNA demethylation, thereby activating TCF21 expression by inducing promotor demethylation.[12]

Genomic imprinting

The imprinting control region (ICR) is located between the paternal imprinted gene insulin-like growth factor 2 (IGF2)and the maternal imprinted gene H19. In the maternal allele, ICR is not methylated and not bound to the transcription factor CTCF (CCTC-binding factor), which activates H19 transcription and represses IGF2 transcription. In the paternal allele, by contrast, ICR cannot interact with CTCF because of methylation and it promotes the transcription of IGF2 and inhibits H19.[13] Localized in ICR, lncRNA-Kcnq1ot1 could promote trimethylation of histones H3 at lysine 27 (H3K27) and H3 at lysine 9 by recruiting DNMT1 and euchromatic histone lysine methyltransferase 2 to influence ICR methylation.[14],[15] On the paternal chromosome, the expression of lncRNA-Kcnqlot 1 was increased by methylated ICR and then it in turn cis-silenced neighboring genes. On the maternal chromosome, lncRNA-Kcnq1ot1 silencing enhanced the expression of neighboring genes.[16]

Chromatin remodeling

LncRNA-Evf2 is transcribed from the intergenic region between Dlx5 and Dlx6 genes, which encode homologous domain transcription factors. It can interfere with chromatin remodeling and repress Dlx5 and Dlx6 expression via preventing the combination of chromatin remodeling complex SWI/SNF and the two genes enhancers.[17],[18] Meanwhile, the bond of lncRNA-Evf2 and SWI/SNF chromatin remodeling enzyme brahma-related gene 1 was accompanied by the competition of other lncRNAs with similar lengths, thereby reducing remodeling. It suggested that the binding of lncRNAs to the SWI/SWF complex was complicated. LncRNA-Xist also binds to the SWI/SNF complex to regulate chromatin remodeling.[19] LncRNA-TCF7 is transcribed from 200 kb upstream ofthe TCF7 gene and recruits SWI/SNF to the TCF7 promoter to regulate its expression and activate Wnt signaling pathway in hepatocellular carcinoma cells.[20]

Histone modifications

Within the human HOXC gene cluster, lncRNA-HOTAIR interacts with polycomb repressive complex 2 (PRC2) to regulate PRC2 occupancy and trimethylate histone H3 at lysine 27 (H3K27) of HOXD locus.[4],[21] LncRNA-HOTAIR also binds to the lysine-specific demethylase l complex, which leads to the demethylation of histone H3 at lysine 4 (H3K4) and inhibition of HOXD gene expression.[22] In mouse embryonic stem cells, WDR5 interacts with more than 200 lncRNAs that are important for histone H3K4 trimethylation and important for gene expression and combining with chromatin.[23] Histone H3K4 trimethylation and gene transcription are driven by the direct binding of lncRNA-HOTTIP to WDR5.[24] LncRNA-p21 overexpression significantly repressed H3 and H4 acetylation at the cell surface antigen gene Thy-1 promoter, thus inhibiting Thy-1 expression and possibly causing pulmonary fibrosis in acute respiratory disease syndrome.[25]

Transcriptional and posttranscriptional regulation

Transcriptional regulation

LncRNA affects gene transcription by disrupting transcription or mediating chromatin remodeling and histone modifications [Figure 1]e. A previous study reported that lncRNA, which was transcribed from upstream of the dihydrofolate reductase (DHFR) gene promoter, formed an RNA-DNA triple helix with the DHFR promoter. This structure inhibited DHFR transcription by preventing the transcriptional coregulator TFIID from binding to the promoter.[26] In yeast, lncRNAs derived from PHO84 and GAL1-10 gene clusters inhibit transcription of homologous coding genes by inducing histone modifications.[27] LncRNA may also regulate the activity of transcriptional factors, thus regulating gene transcription. LncRNA that arises from growth specificity inhibitor Gas5 gene imitates glucocorticoid response element (GRE) by RNA secondary folding, which competes with GRE for glucocorticoid receptor (GR)-binding. This lncRNA just suppresses the transcription of downstream genes via inhibiting GRE combination with GR.[28] A previous study reported that lncRNA-PRNCR1 and lncRNA-PCGEM1 were highly overexpressed in aggressive prostate cancer cells and that they could bind with the enhancer of the androgen receptor with an acetylated C-terminus and a methylated N-terminus, thus enhancing the transcriptional activity mediated by ligand-dependent and nonligand-independent androgen receptors.[29]

Posttranscriptional regulation

LncRNAs transcribed from the antisense strand of protein-coding genes can form double-strand RNAs (dsRNAs) with the sense-strand by base pair matching. Their regulation at the posttranscriptional level depends on either preventing the spliceosome from identifying the splice sites and affecting the splicing of the mRNA into several products or producing endogenous small-interfering RNAs (siRNAs) with the ribonuclease Dicer [Figure 1]f. For example, the 5' UTR of zinc finger E-box-binding homeobox 2 (ZEB2) formed dsRNA with its antisense lncRNA, thereby increasing ZEB2 expression by overlapping with the splicing sites.[27] During X-chromosome inactivation, lncRNA-Xist can complementarily pair with its antisense transcript lncRNA-Tsix to form dsRNA, resulting in the production of endogenous siRNA via Dicer and the inhibition of Xist function.[27]

Several lncRNAs serve as precursors of small molecule RNAs, such as microRNAs (miRANs), piRNAs, and small nucleoli RNAs.[30],[31] LncRNAs can also bind to miRNAs in the RNA-induced silencing complex and act as an miRNA sponge as competitive endogenous RNAs (ceRNAs). LncRNAs bind to miRNAs to inhibit the degradation or repression of target mRNAs. The latest study showed that lncRNA-MALAT1 was a ceRNA of miR-206, a tumor suppressor that inhibited ANXA2 and KRAS expression in subjects with gallbladder carcinoma. However, cancer incidence increased when lncRNA-MALAT1 combined with miR-206 to inhibit its role and lead to cancer incidence.[31]

Other functions

LncRNAs have other functions as well. First, lncRNAs affect mRNA stability and translation. For example, lncRNA-TINCR binds to STAU1 to ensure the stable expression of differentiation-related genes, thereby regulating somatic cell differentiation.[32] Second, lncRNAs regulate the activity of specific proteins by binding to interacting proteins. LncRNA-MALAT1 binds to serine/arginine splicing factors to control their phosphorylation and distribution in nuclear speckle domains.[33] Third, lncRNAs mediate interactions between nucleic acids and proteins. For example, lncRNA-NEAT1 is critical for the formation of nuclear structures known as “paraspeckles” upon binding to SPCI, NONO, and CAT2.[34] Fourth, lncRNAs affect the cellular localization of interacting proteins. A previous study showed that nuclear transport factor (NTAT) was sequestered in the cytoplasm by lncRNA-NRON, thus affecting the expression of downstream target genes.[35] Finally, lncRNAs encode short peptides.[36] For example, a muscle-specific lncRNA encoding DWORF, a 34-amino acid peptide localizing to the sarcoplasmic reticulum, enhanced muscle contraction.[37]


  Research Methods to Study LNCRNAs Top


High-throughput analysis and validation of LncRNA expression

High-throughput analysis of lncRNA expression is achieved by microarray and transcriptome sequencing. Microarray allows investigators to screen the entire genome and to identify differences in lncRNA and mRNA expression profiles. By contrast, transcriptome sequencing allows investigators to study the transcriptional activity of any species, including mRNAs, lncRNAs, and other small RNAs. The accuracy of transcriptome sequencing far surpasses that of microarray.

High-throughput data are often validated by various molecular biology methods. For example, Northern blotting and quantitative polymerase chain reaction were used to confirm the identities of eight new lncRNAs.[38] In another study, RNA-fluorescence in situ hybridization was used to confirm the localization of lncRNA-HA117 to the stenotic segment of the intestinal mucosa and the muscularis in patients with congenital megacolon.[39],[40] Finally, lncRNA-gadd7 was silenced by RNA interference and found to regulate endoplasmic reticulum stress.[41]

LncRNA and protein interactions

LncRNA generally mediates interactions not only between RNAs and proteins but also between RNAs and DNAs, RNAs and RNAs. Although these interactions can be identified by various methods [Table 2], further studies are needed to understand the roles of lncRNAs in these interactions.
Table 2: Techniques for identifying lncRNAs according to the interaction with protein, DNA, or RNA

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RNA-binding protein immunoprecipitation

RNA-binding protein immunoprecipitation (RIP) allows investigators to study interactions between RNAs and proteins and to identify lncRNA-binding proteins. For example, a previous study employing RIP showed that PRC2 was coactivated with lncRNA-Xist, lncaRNA-Tsix, and lncRNA-repeat A during X chromosome inactivation.[42] Another study employing the same method also showed that lncRNA-p21 interacted with hnRNP-K to regulate p53-mediated apoptosis.[43] RIP also played a role in the overhaul of microarray and transcriptome sequencing into RIP-Chip and RIP-Seq, which were more conducive to studying lncRNA function. Indeed, more than 9,000 RNA transcripts were found to interact with PRC2 in embryonic stem cells by RIP-Chip and RIP-Seq.[44]

Cross-linking and immunoprecipitation and combined knockdown and localization analysis of noncoding RNAs

Cross-linking and immunoprecipitation (CLIP) allows investigators to study interactions between RNAs and proteins. After cross-linking RNAs and RNA-binding proteins with ultraviolet light, the cells are lysed and proteins are immunoprecipitated using an antibody against the target protein, followed by the isolation and purification of the RNA and RNA-binding protein. The RNA is analyzed by high-throughput sequencing, whereas the RNA-binding protein is examined on a silver-stained polyacrylamide gel and identified by mass spectrometry. By CLIP, several RNA-binding sites were identified within neuron-specific RNA-binding protein NOVA2.[45] On the other hand, combined knockdown and localization analysis of noncoding RNAs (c-KLAN) allows investigators to study the function and cellular localization of a target lncRNA by siRNA and RNA-FISH probes prepared from the cDNA of the target lncRNA. By c-KLAN, lncRNA-Panct1 was found to localize to the nucleus and to function in embryonic stem cell pluripotency.[46]

Chromatin isolation by RNA purification-

Chromatin isolation by RNA purification (ChIRP) is a relatively new method. It can be used to identify the regions of the genome interacting with a target lncRNA such as the coactivation site between the target lncRNA and chromatin. The interacting chromosomal fragments are purified by biotin or streptavidin probes, and the identified RNAs, DNAs, and proteins are then used for downstream analysis. For example, the genomic localization and the binding domain of lncRNA-HOTAIR usually are GA-rich DNA motifs and polycomb proteins aggregation,[46] while Drosophila lncRNA-roX2 is mainly located on the X chromosome and tends to act on the 3' end of all the genes.[46] In another study that through ChIRP, lncRNA-TERC was found to localize to the telomere and to interact with genes of the Wnt signaling pathway.[46] Subsequently, domain-specific ChIRP was developed, which allowed investigators to identify the different functional domains of lncRNAs.[47] For example, the functional domains of lncRNA-roX1 in the Drosophila were revealed this method.[47]

LncRNA secondary structure

Currently, there are several high-throughput methods that can be used to study RNA secondary structures. Selective 2'-hydroxyl acylation analysis by primer extension analysis (SHAPE) allows investigators to directly observe RNA secondary structures in living cells. The target RNA is isolated, labeled, and folded to assemble the truncated products by inhibiting reverse transcriptase. To reconstruct the original structure, the products are then sequenced.[48] SHAPE can also be used to study RNA structural changes.[49] Parallel analysis of RNA structure (PARS) allows investigators to obtain RNA secondary structure information by high-throughput sequencing after digestion with nuclease V1 and S1, which identify and cut dsRNA and single-strand RNA, respectively.[50] Fragmentation sequencing (FRAG-SEG) is comparable to PARS, except that a different nuclease, namely P1, is used for digestion.[51] Finally, ribosome profiling is similar to RNA-Seq with regard to library construction and data analysis. However, ribosome profiling targets only mRNA sequences protected by the ribosome during translation rather than all mRNAs. Thus, it provides useful information on the translation of all mRNAs within the genome.[52]

Bioinformatics

Compared with conventional approaches, bioinformatics tools offer several benefits in predicting lncRNA functions, and it also reduces time and costs and offers experimental blindness. The CatRAPID online algorithm is the best example of how a bioinformatics approach can be used to study lncRNA function. The algorithm predicts the possible interactions between RNAs and proteins on the strength of their secondary structures and the presence of hydrogen bonds and intermolecular forces. CatRAPID algorithm is used to analyze the function of lncRNA in regulating its own regulatory pathway, X chromosome dose compensation, and variable splicing, which is consistent with the previous experimental results.[53]

Several online lncRNA databases are available that can be used to predict the functions of lncRNAs. For example, NCODE,[54] FANTOM,[55] and TCGA [56] databases contain large-scale RNA-Seq data on lncRNAs from different species, including humans. In addition, several investigators maintain databases for this purpose. For example, the lncRNome database [57] is used as a general resource, whereas the lncRNA disease database [58] is used to understand the roles of lncRNAs in disease. Bioinformatic tools can also predict the functions of lncRNAs. The lncRNAtor database [59] is used to identify genes coexpressed with lncRNAs, whereas LNCipedia and lncRNome [57],[60] databases are used to identify similar DNA motifs and to predict RNA structures. The lncRNome database [57] also integrates epigenetic information.

Clustered regularly interspaced short palindromic repeats associated protein 9 and antisense oligonucleotide

The most widely used eukaryotic genome editing technique, clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas9), can modify genes in cells and organisms.[61] It can also silence lncRNA genes, as well as overexpress lncRNA genes, because specific endonuclease Cas9 is unable to digest nucleic acids with transcriptional activators such as the VP64 activation domain.[61],[62],[63] Both properties of CRISPR/Cas9 can be used to study lncRNA function.

Antisense oligonucleotides (ASOs) are short DNA sequences that hybridize with the target RNA to affect its function. Through regulating cellular activities, chemical modification can extend their half-life and reduce their degradation by nucleases.[64] SiRNA and shRNA are also examples of ASOs, except that their efficiency in silencing lncRNAs in the nucleus is low because siRNA and shRNA work in the cytoplasm. Locked nucleic acids are RNA derivatives analogous to ASOs. Although thousands of lncRNAs bind to PRC2 to inhibit gene transcription, the interaction of some specific lncRNA and PRC2 could be disturbed by locked nucleic acids and upregulate target genes expression.[65],[66]


  Conclusion and Future Perspectives Top


The research on lncRNAs is in full swing. The roles of lncRNAs in various biological processes and human diseases are becoming increasingly important. Nevertheless, this is just the tip of iceberg. However, the identities and regulatory mechanisms of several lncRNAs remain unknown, which may be due to the fact that (i) several lncRNAs are weakly expressed, poor sequence conservation, expression easily affected by environment, varied and complex action modes; (ii) high-throughput technologies are not yet fully developed; and (iii) there are very few lncRNA prediction tools and databases. With the advent of new methods, studies on lncRNAs will enter a new stage. New technologies will help us further analyze lncRNA function and their regulatory mechanisms, thus providing answers for unresolved issues that have to do with evolution, development, and disease.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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