Patricia F. Fitzpatrick Dimond Ph.D. Technical Editor of Clinical OMICs President of BioInsight Communications
Because They Are Such Elusive Prey, lncRNAs Have Yet to Emerge as Therapeutic Targets
Advanced high-throughput analyses of mammalian transcriptomes have provided a greatly expanded view of genome complexity. These analyses, most experts agree, confirm that about 50% of the human genome is transcribed to produce noncoding RNAs (ncRNAs) that are numerous and vary in size. Although ncRNAs are not translated into proteins, they are not without function.
Such insights go back at least 10 years. According to mammalian transcriptome annotation studies cited by a 2012 review in EMBO Reports, thousands of RNA transcripts have been identified that do not seem to be derived from known genes.1 Many of these transcripts accumulate to significant levels and resemble messenger RNA in being capped, polyadenylated, and spliced; but they don’t overlap the exons of known protein-encoding genes.
The ncRNAs that are longer than 200 nucleotides and appear to have little or no protein-encoding capacity have been typically termed long noncoding RNAs (lncRNAs). Their relatively large size distinguishes lcRNAs from small regulatory RNAs such as microRNAs (miRNAs), short interfering RNAs (siRNAs), and short hairpin RNAs (shRNAs).
Described by some as the “dark matter of the genome,” lncRNA transcripts have emerged as a “cryptic, but critical” layer in the genetic regulatory code.2 But, no clear picture has yet emerged as to how these transcripts work.
In a 2013 review published in Genetics,3 lncRNA experts wrote that “lncRNAs of all kinds have been implicated in a range of developmental processes and diseases, but knowledge of the mechanisms by which they act is still surprisingly limited, and claims that almost the entirety of the mammalian genome is transcribed into functional noncoding transcripts remain controversial.”
All the same, the reviewers noted, a few well-studied lncRNAs have yielded important information about the biology of these molecules, and a few key functional and mechanistic themes have begun to emerge.
The lncRNAs are highly diverse, are expressed throughout the cell, and include “thousands of different species”—in the sense that genomes of complex organisms are characterized by the pervasive expression of different types of ncRNAs.4 Encoded by a significant proportion of the genome, studies suggest that lncRNAs fulfill a wide variety of regulatory roles at almost every stage of gene expression. These roles, which encompass signal, decoy, scaffold, and guide capacities, derive from folded modular domains in lncRNAs.
Increasing numbers of studies demonstrate that lncRNAs play critical roles in the regulation of protein-coding genes, the maintenance of genomic integrity, dosage compensation, genomic imprinting, mRNA processing, cell differentiation, and development. Misregulation of lncRNAs is associated with a variety of human diseases including cancer, immune disorders, and neurological disorders.
Oxford University scientists Thomas C. Roberts, Ph.D., and Matthew J. A. Wood, Ph.D., have provided a good general functional description of lncRNAs as adaptors between chromatin, proteins, and other RNAs.5 Properties of lncRNA molecules that allow these interactions include their capacity to bind to DNA or other RNA molecules by complementary Watson-Crick base pairing to form hetero- or homo-duplexes, formation of DNA-DNA-RNA triplexes by Hoogstein and reverse-Hoogstein base pairing, or by direct RNA recognition of chromatin surface features.
The inherent flexibility of RNA, these scientists have indicated, permits the formation of complex secondary structures that can function as binding domains for proteins or small molecules. These combined properties enable a much wider range of functions than is possible with miRNAs. Additionally, lncRNAs may also contain multiple binding modules allowing for complex multifunctional interactions.
The expression of lncRNAs is developmentally regulated; can be specific to different tissue and cell types; and can vary spatially, temporally, or in response to stimuli. Many lncRNAs are expressed in a more tissue-specific fashion and with greater variation between tissues compared to protein-coding genes.
The lncRNAs may execute either gene inhibition and activation through a diverse series of mechanisms, adding yet another layer of complexity to understanding of genomic regulation.6 They have been “roughly” classified based on their position relative to protein-coding genes as intergenic (between genes), intragenic/intronic (within genes), and antisense.7 Initial efforts to characterize these molecules have shown that they function in cis, regulating their immediate genomic neighbors. Examples include AIR, XIST, and Kcnq1ot, genes that recruit chromatin-modifying complexes to silence adjacent sites.
Understanding of the scope of lncRNAs in gene regulation grew as more about these enigmatic molecules was discovered. For example, when Rinn et al.8 found that the lncRNA HOTAIR (Hox Transcript Antisense RNA) could exhibit trans as well as cis regulatory capacities, these investigators suggested that the transcription of ncRNA may demarcate chromosomal domains of gene silencing at a distance.
HOTAIR is transcribed at the intersection of opposing chromatin domains in the HOXC locus. This 2.2-kilobase spliced RNA transcript interacts with the polycomb group proteins, including polycomb repressive complex 2 (PRC2), that are responsible for cellular differentiation during development via transcriptional repression. The newly characterized HOTAIR-PRC2 interaction appears to modify chromatin and repress transcription of the human HOX genes, which regulate development. How HOTAIR does this remains unclear.
Khalil et al.9 established that hundreds of large intergenic noncoding (lincRNAs) are physically associated with polycomb and other chromatin-modifying complexes. The researchers added to the catalog of human lincRNAs, bringing the total to about 3,300 by analyzing chromatin-state maps of various human cell types.
Since the well-characterized lincRNA HOTAIR binds PRC2, the researchers tested whether many lincRNAs are physically associated with the complex. The scientists found that about 20% of lincRNAs expressed in various cell types are bound by PRC2, and that additional lincRNAs are bound by other chromatin-modifying complexes.
They further showed that siRNA-mediated depletion of certain lincRNAs associated with PRC2 leads to changes in gene expression and that the upregulated genes are enriched for those normally silenced by PRC2. The authors proposed a model in which some lincRNAs guide chromatin-modifying complexes to specific genomic loci to regulate gene expression.
Dysregulation and Cancer
The lncRNAs have captured great interest among cancer scientists as they appear to regulate complex cellular functions that are commonly deregulated in cancer, including growth, differentiation, and establishment of cellular identity. The growth in the number of publications associating them with cancer attests to this interest, rising from around 1,000 in 2010 to about 4,000 in 2015.
Some have already been linked to poor prognosis in multiple tumor types, some have acquired clinical relevance as biomarkers, and some have been shown to be deregulated in diverse human cancers and associated with disease progression. For example, HOTAIR is highly expressed in breast cancer. It can predict metastasis formation and indicate a poor prognosis.10
Gupta et al., writing in Nature, reported that that the HOTAIR expression level in primary tumors is a predictor of eventual metastasis and death.11 Enforced expression of HOTAIR in epithelial cancer cells induces genome-wide retargeting of PRC2 to an occupancy pattern more resembling embryonic fibroblasts, leading to altered histone H3 lysine 27 methylation, gene expression, and increased cancer invasiveness and metastasis in a manner dependent on PRC2.
The authors further reported that loss of HOTAIR can inhibit cancer invasiveness, particularly in cells that possess excessive PRC2 activity. These findings, the authors said, indicate that lincRNAs have active roles in modulating the cancer epigenome and may be important targets for cancer diagnosis and therapy.
Other groups have found a role for lncRNA in hematopoietic development. For example, Hu et al. found that one lncRNA has an essential role in the maturation of red blood cells, mediating antiapoptotic activity in murine erythroid terminal differentiation.12
Transcriptome profiling on mouse erythroid cells at different developmental stages revealed differential expression of more than 400 lncRNAs during this developmental process. The scientists characterized a lncRNA, lncRNA-EPS, which is required for the maturation of mouse erythroid cells by inhibiting apoptosis.
Markedly induced during the terminal differentiation of mouse erythroid cells, lncRNA-EPS inhibition results in apoptosis and severely compromises differentiation and downstream enucleation from erythroid cells. Conversely, the scientists reported, ectopic expression of lncRNA-EPS can protect erythroid progenitor cells from apoptosis triggered by erythropoietin starvation. Mechanistic studies suggest that lncRNA-EPS regulates apoptosis by repressing expression of several pro-apoptotic proteins.
As for novel drug targeting, in cases where a lncRNA is directly linked to disease pathogenesis, conventional RNAi or antisense oligonucleotides could be used, some propose, to modulate its expression. At present, the number of such situations remains relatively small, although further investigation into the role that lncRNAs play in disease is likely to identify “a plethora” of novel therapeutic targets.
In their paper in Genetics, lncRNA experts Kung et al. caution that given the current state of data in the field, “at present, it may be best to avoid blanket statements about structure, function, and mechanism, as indeed we have barely begun to scratch the surface of the lncRNA world. There is still plenty to learn and much work to be done.”3
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9. Khalil AM, Guttman M, Huarte M, Garber M, Raj A, Rivea Morales D, Thomas K, Presser A, Bernstein BE, van Oudenaarden A, et al. Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc Natl Acad Sci USA 2009; 106: 11667–11672. doi: 10.1073/pnas.0904715106.,
10. Gutschner T, Hämmerle M, Eissmann M, Hsu J, Kim Y, Hung G, Revenko A, Arun G, Stentrup M, Groß M. The noncoding RNA MALAT1 is a critical regulator of the metastasis phenotype of lung cancer cells. Cancer Res. 2013; 73: 1180–1189. doi: 10.1158/0008-5472.CAN-12-2850.,
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12. Hu W, Yuan B, Flygare J, Lodish HF. Long noncoding RNA-mediated anti-apoptotic activity in murine erythroid terminal differentiation. Genes Dev 2011; 25(24): 2573–2578. doi: 10.1101/gad.178780.111.