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What are lncRNAs?

Advances in RNA sequencing technologies have revealed the complexity of our genome. Non-coding RNAs make up the majority (98%) of the transcriptome, and several different classes of regulatory RNA with important functions are being discovered. Understanding the significance of this RNA world is one of the most important challenges facing biology today, and the non-coding RNAs within it represent a gold mine of potential new biomarkers and drug targets.

lncRNA sequences

Long non-coding RNAs (lncRNAs) are a large and diverse class of transcribed RNA molecules with a length of more than 200 nucleotides that do not encode proteins (or lack > 100 amino acid open reading frame). lncRNAs are thought to encompass nearly 30,000 different transcripts in humans, hence lncRNA transcripts account for the major part of the non-coding transcriptome. lncRNA discovery is still at a preliminary stage. There are many specialized lncRNA databases, which are organized and centralized through RNAcentral.

lncRNAs can be transcribed as whole or partial natural antisense transcripts (NAT) to coding genes, or located between genes or within introns. Some lncRNAs originate from pseudogenes (Milligan & Lipovich, 2015). lncRNAs may be classified into different subtypes (Antisense, Intergenic, Overlapping, Intronic, Bidirectional, and Processed) according to the position and direction of transcription in relation to other genes (Peschansky & Wahlestedt, 2014, Mattick & Rinn, 2015).

lncRNA expression

Gene expression profiling and in situ hybridization studies have revealed that lncRNA expression is developmentally regulated, can be tissue- and cell-type specific, 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 (Derrien et al., 2012).

In general, the expression level of lncRNA is at least one order of magnitude below that of mRNA. Many lncRNAs are located exclusively in the nucleus, but some are cytoplasmic or are located in both nucleus and cytoplasm.

lncRNA functions

To date, very few lncRNAs have been characterized in detail. However, it is clear that lncRNAs are important regulators of gene expression, and lncRNAs are thought to have a wide range of functions in cellular and developmental processes. lncRNAs may carry out both gene inhibition and gene activation through a range of diverse mechanisms, adding yet another layer of complexity to our understanding of genomic regulation. It is estimated that 25 – 40% of coding genes have overlapping antisense transcription, so the impact of lncRNAs on gene regulation is not to be underestimated.

Figure 1

Overview of some of the functions of long non-coding RNA. (Click for a larger image) lncRNAs are involved in gene regulation through a variety of mechanisms. The process of transcription of the lncRNA itself can be a marker of transcription and the resulting lncRNA can function in transcriptional regulation or in chromatin modification (usually via DNA and protein interactions) both in cis and in trans. lncRNAs can bind to complementary RNA and affect RNA processing, turnover or localization. The interaction of lncRNA with proteins can affect protein function and localization as well as facilitate formation of riboprotein complexes. Some lncRNAs are actually precursors for smaller regulatory RNAs such as microRNAs or piwi RNAs. Figure modified from Wilusz et al. Genes Dev. 2009. 23: 1494-1504. PMID: 19571179.

lncRNA mechanisms of gene regulation

lncRNAs are not defined by a common mode of action, and can regulate gene expression and protein synthesis in a number of different ways (Figure 1). Some lncRNAs are relatively highly expressed, and appear to function as scaffolds for specialized subnuclear domains. lncRNA possess secondary structures which facilitate their interactions with DNA, RNA and proteins. lncRNA may also bind to DNA or RNA in a sequence-specific manner. Gene regulation may occur in cis (e.g. in close proximity to the transcribed lncRNA) or in trans (at a distance from the transcription site). In the case of chromatin modulation, the effect of lncRNA is typically gene-specific, exerted at a local level (in cis) however regulation of chromatin can also occur in trans.

A few lncRNAs have had their functions experimentally defined and have been shown to be involved in fundamental processes of gene regulation including:
  • Chromatin modification and structure
  • Direct transcriptional regulation
  • Regulation of RNA processing events such as splicing, editing, localization, translation and turnover/degradation
  • Post-translational regulation of protein activity and localization
  • Facilitation of ribonucleoprotein (RNP) complex formation
  • Modulation of microRNA regulation
  • Gene silencing through production of endogenous siRNA (endo-siRNA)
  • Regulation of genomic imprinting
It has recently been attempted to categorize the various types of molecular mechanisms that may be involved in lncRNA function. lncRNAs may be defined as one or more of the following five archetypes:
  • The Signal archetype: functions as a molecular signal or indicator of transcriptional activity.
  • The Decoy archetype: binds to and titrates away other regulatory RNAs (e.g. microRNAs) or proteins (e.g. transcription factors).
  • The Guide archetype: directs the localization of ribonucleoprotein complexes to specific targets (e.g. chromatin modification enzymes are recruited to DNA).
  • The Scaffold archetype: has a structural role as platform upon which relevant molecular components (proteins and or RNA) can be assembled into a complex or spatial proximity.
  • The Enhancer archetype: controls higher order chromosomal looping in an enhancer-like model.

lncRNA and disease

With such a wide range of functions, it is not surprising that lncRNA play a role in the development and pathophysiology of disease. Interestingly, genome wide association studies have demonstrated that most disease variants are located outside of protein-coding genes.

lncRNAs have been found to be differentially expressed in various types of cancer including leukemia, breast cancer, hepatocellular carcinoma, colon cancer, and prostate cancer. Key oncogenes and tumor suppressors including PTEN and KRAS are now known to be regulated by corresponding lncRNA pseudogenes which also act as competing endogenous RNAs (ceRNAs) or microRNA sponges (Poliseno et al., 2010,Johnsson et al.,). This highlights the important role that lncRNAs play in oncogenesis.

Other diseases where lncRNAs are dysregulated include cardiovascular diseases, neurological disorders and immune-mediated diseases and genetic disorders. One of the first lncRNA to be discovered was the Xist lncRNA which plays an important role in X chromosome inactivation (Penny et al., 1996), an extreme case of genomic imprinting. lncRNAs are present at almost all imprinted loci, arguing for an important role for lncRNAs in this form of epigenetic regulation.

lncRNAs represent a gold mine of potential new biomarkers and drug targets, as well as a step change in the way we understand mechanisms of disease.

The challenges of studying lncRNA

Only a relatively small proportion of lncRNAs have so far been investigated and although we can start to classify different types of lncRNA functions, we are still far from being able to predict the function of new lncRNAs. This is mainly due to the fact that unlike protein-coding genes whose sequence motifs are indicative of their function, lncRNA sequences are not usually conserved and they don’t tend to contain conserved motifs. Other differences between lncRNA and mRNA are summarized in Table 1.

The main challenges of working with lncRNA are the fact that they can be present in very low amounts (typically an order of magnitude lower than mRNA expression levels), can overlap with coding transcripts on both strands and are often restricted to the nucleus.

Exiqon offer a comprehensive range of products and services for all aspects of lncRNA research – from discovery and expression analysis, to elucidation of lncRNA functions. We are committed to developing novel tools and approaches to advance our understanding of the complexity of lncRNA.

Table 1

mRNA

lncRNA

Tissue-specific expression

Tissue-specific expression

Form secondary structure

Form secondary structure

Undergo post-transcriptional processing, i.e. 5' cap, polyadenylation, splicing

Undergo post-transcriptional processing, i.e. 5' cap, polyadenylation, splicing

Important roles in dieases and development

Important roles in diseases and development

Protein coding transcript

Non-protein coding, regulatory functions

Well conserved between species

Poorly conserved between species

Present in both nucleus and cytoplasm

Many predominantly nuclear, others nuclear and/or cytoplasmic

Total 20-24,000 mRNAs

Currently ~30,000 lncRNA transcripts, predicted 3-100 fold of mRNA in number

Expression level: low to high

Expression level: very low to moderate

Similarities (gray) and differences (cyan) between mRNA and lncRNA

lncRNA discovery and profiling using Next Generation Sequencing

Expression profiling is one way to start to uncover the function of lncRNA. Identifying lncRNAs that are differentially expressed during development or in particular situations can shed light on their potential functions. Alternatively, looking for lncRNAs and protein-coding genes whose expression is correlated, can perhaps indicate co-regulation or related functions.

Whole transcriptome RNA sequencing is the method of choice for comprehensive lncRNA expression profiling, including the discovery of novel lncRNAs. Whole transcriptome sequencing enables the characterization of all RNA transcripts, including both the coding mRNA and non-coding RNA larger than 170 nucleotides in length, regardless of whether they are polyadenylated or not.

Exiqon offers a comprehensive whole transcriptome NGS Service including everything from RNA isolation to the final report including advanced data analysis and interpretation.

Advantages of LNA™-enhanced research tools for lncRNA

Exiqon offer a broad range of sensitive and specific tools specifically designed to address the challenges faced when investigating lncRNA expression and function. Exiqon’s tools are based on the Locked nucleic acid (LNA™) technology. LNA™ is a class of high-affinity RNA analogs that exhibit unprecedented thermal stability when hybridized to a complementary DNA or RNA strand. Hence, LNA™ enables superior sensitivity and specificity in any hybridization-based approach. We continue to use the LNA™ technology to develop new and innovative ways to improve our understanding of lncRNAs in this rapidly developing field.

Functional analysis of lncRNAs has been revolutionized by the development of Antisense LNA™ GapmeRs which enable efficient silencing of lncRNA both in vitro and in vivo. Exiqon also offer tools to investigate lncRNA function in other ways, for example using LNA™ oligos to block interactions between lncRNA and DNA, RNA or proteins. ExiLERATE LNA™ qPCR assays have been developed to enable robust detection of even low abundance and challenging lncRNAs by qPCR. Precise subcellular localization of lncRNAs can be studied using LNA™ probes for in situ hybridization.

Silencing lncRNA to disrupt their function

One strategy to study the function of lncRNA is to silence them using specific and potent antisense oligonucleotides (Antisense LNA™ GapmeRs).

The nuclear localization of many lncRNAs has meant that siRNA approaches to knockdown lncRNA, have met with limited success. The double-stranded siRNA duplex has difficulty crossing the nuclear membrane and the passenger strand (non-targeting sequence) of the duplex can often elicit its own effect, confounding interpretation of results.

Antisense LNA™ GapmeRs overcome this challenge by enabling highly efficient RNase H mediated silencing of all lncRNA. RNase H is present both in the cytoplasm and in the nucleus and it has been shown that LNA™ Gapmers offer significantly better knockdown of nuclear targets than siRNA mediated silencing. In addition, the single stranded LNA™ GapmeRs are an advantage for lncRNAs that are transcribed as antisense transcripts to coding genes because there is no second strand that could compromise specificity.

Antisense LNA™ GapmeRs show high potency in a broad range of tissues in vivo when administered systemically without formulation in animal models. This makes LNA™ GapmeRs very promising antisense drugs for lncRNA targets in the future.

Studying lncRNA interactions with DNA, RNA and proteins

The fact that the molecular mechanism of lncRNAs often relies on sequence specific interaction with DNA, RNA or proteins means that it is possible to design highly specific LNA™-oligonucleotides that can be used to inhibit these interactions and thereby reveal the details of how lncRNAs function. Please contact us and our experts can help you with the design of custom LNA™ oligonucleotides for studying lncRNA interactions.

lncRNA analysis by qPCR

Short, high affinity, LNA™-enhanced qPCR primers offer an advantage for the detection of low abundance targets. In addition, the use of LNA™ to adjust primer melting temperature provides greater flexibility in primer design which is important for qPCR analysis of overlapping transcripts. The ExiLERATE LNA™ qPCR System offers a sophisticated primer design tool combined with highly sensitive and specific qPCR assays for any RNA target.

ExiLERATE LNA™ qPCR assays are ideal to monitor the efficiency of LNA™ GapmeR-mediated RNA knockdown. Validated LNA™ qPCR primer sets are available to detect the lncRNA targeted by Antisense LNA™ GapmeR positive controls.

ExiLERATE LNA™ qPCR primer sets also provide a convenient way to validate RNA sequencing data. Our advanced online design algorithm can design LNA™ qPCR assays for novel lncRNA transcripts, isoforms or splice variants. LNA™qPCR assays for multiple lncRNAs can easily be designed using the batch mode function in our online design algorithm.

Subcellular localization of lncRNA expression by in situ hybridization

Understanding the subcellular localization of a lncRNA is important information when starting to hypothesize the potential functions that the lncRNA may be performing. LNA™-enhanced probes for in situ hybridization have increased affinity for their target sequence and offer increased sensitivity and increased signal to noise ratio, which is important for detection of rare targets such as lncRNA.

For details of all the services products available for studying lncRNA, please use the links below.

Exiqon Products and Services for lncRNA research

References

Durham et al. ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature. 2012. 489(7414):57-74. PMID: 22955616

Derrien et al., The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome Res. 2012 Sep;22(9):1775-89. PMID: 22955988

Poliseno et al., A coding-independent function of gene and pseudogene mRNAs regulates tumour biology. Nature. 2010 Jun 24;465(7301):1033-8. PMID: 20577206

Johnsson et al., A pseudogene long-noncoding-RNA network regulates PTEN transcription and translation in human cells. Nat Struct Mol Biol. 2013 Apr;20(4):440-6. PMID: 23435381

Mattick JS, Rinn JL. Discovery and annotation of long noncoding RNAs. Nat Struct Mol Biol. 2015 Jan;22(1):5-7. PubMed PMID:

Peschansky VJ, Wahlestedt C. Non-coding RNAs as direct and indirect modulators of epigenetic regulation. Epigenetics. 2014 Jan;9(1):3-12. PMID: 24739571

Penny et al., Requirement for Xist in X chromosome inactivation. Nature. 1996 Jan 11;379(6561):131-7. PMID: 8538762

Wilusz et al. Long noncoding RNAs: functional surprises from the RNA world, Genes Dev. 2009. 23: 1494-1504. PMID: 19571179

Bhartiya et al. Conceptual approaches for lncRNA drug discovery and future strategies. Expert Opin Drug Discov. 2012. 7: 503-513. PMID: 22559214

Ma et al. Molecular Mechanisms and Function Prediction of Long Noncoding RNA, ScientificWorldJournal. 2012; 2012: 541786. PMID: 23319885

Wang et al. Molecular mechanisms of long noncoding RNAs. Mol Cell. 2011. 43(6):904-14. PMID: 21925379

Bernard et al. A long nuclear-retained non-coding RNA regulates synaptogenesis by modulating gene expression. EMBO J. 2010. 29: 3082-3093. PMID: 20729808

Sarma et al. Locked nucleic acids (LNAs) reveal sequence requirements and kinetics of Xist RNA localization to the X chromosome. Proc Natl Acad Sci U S A. 2010. 107: 22196-22201. PMID: 21135235

Gutschner et al. The Noncoding RNA MALAT1 Is a Critical Regulator of the Metastasis Phenotype of Lung Cancer Cells, Cancer Res 2013;73:1180-1189.

Lee et al. RNase H-mediated degradation of toxic RNA in myotonic dystrophy type 1. Proc Natl Acad Sci U S A. 2012. 109(11):4221-6. PMID: 22371589

 
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