Decoding LncRNAs

Abstract

Non-coding RNAs (ncRNAs) have been considered as unimportant additions to the transcriptome. Yet, in light of numerous studies, it has become clear that ncRNAs play important roles in development, health and disease. Long-ignored, long non-coding RNAs (lncRNAs), ncRNAs made of more than 200 nucleotides have gained attention due to their involvement as drivers or suppressors of a myriad of tumours. The detailed understanding of some of their functions, structures and interactomes has been the result of interdisciplinary efforts, as in many cases, new methods need to be created or adapted to characterise these molecules. Unlike most reviews on lncRNAs, we summarize the achievements on lncRNA studies by taking into consideration the approaches for identification of lncRNA functions, interactomes, and structural arrangements. We also provide information about the recent data on the involvement of lncRNAs in diseases and present applications of these molecules, especially in medicine.

Keywords: biomarkers; gene regulation; gene therapy; lncRNA; miRNA sponge.

Figure 1

Chemical/enzymatic methods for lnRNAs structure analysis. (1) Probing lnRNAs by chemical probes which form covalent adducts, or by enzymes; (2) reverse transcription of modified or fragmented lnRNAs; (a) ‘stop’ event (blue dot) occurs when RT (reverse transcriptase) stalls or dissociates before adduct, resulting in a truncated fragment; (b) ‘mutation’ event occurs when RT proceeds through the adduct introducing a mutation; (3) detection using mutation or fractionation readout approaches. This is an original figure created in BioRender.com (accessed on 25 May 2020).

Figure 2

Methods for detection of RNA-RNA interactions. (A) Crosslinking RNAs directly, (B) crosslinking RNAs in the presence of RNA binding protein (RBP). PARIS, SPLASH, LIGR-seq include crosslinking RNAs with psoralen, digestion with RNase, proximity ligation and sequencing. In hiCLIP after RNA digestion, adjacent RNAs ends are ligated using a linker. In MARIO, RNA binding protein (RBP) is biotinylated, enabling immobilisation of RNA–RBP followed by digestion of RNAs ends and their ligation with added biotinylated linker; the tagged RNA–protein complexes are then purified and sequenced. This is an original figure.

Figure 3

Reporter systems used to study lncRNA functioning as (A) transcriptional activator, (B) complex transcriptional repressor, (C) direct transcriptional repressor, (D) miRNA sponges. Abbreviations: UAS: upstream activation sequence, min P: minimal promoter, P: promoter, RBP: RNA binding protein, DBP: DNA binding protein, LUC: luciferase, IFN-β: interferon beta, PGK: phosphoglycerate kinase, promoter. This is an original figure.

Figure 4

Targeting lncRNAs by RNAi, ASOs, PNAs, LNAs to knockdown and/or map lncRNAs functional domains. (A) RNA interference (RNAi) recruits the multiprotein RNAi-induced silencing complex (RISC) containing a siRNA (small interfering RNA), shRNA (short hairpin RNA) or esiRNA (endoribonuclease-prepared RNA) to specifically degrade the targeted RNA. (B) Antisense oligonucleotides (ASOs) bind to their targeted RNA, triggering endogenous RNase H1 to cleave the RNA/DNA heteroduplex. (C) Peptide Nucleic Acids (PNAs) and (D) Locked Nucleic Acids (LNAs), hybridize with complementary RNA sequences and affect activity of targeted lncRNAs. This is an original figure.

Figure 5

Schematic representation of Tiling CRISPR. Several guide RNAs target Cas9 endonuclease to sequences mapping wild-type (WT) lncRNA. As a consequence, insertions/deletions are introduced to lncRNA. This is an original figure.

Figure 6

Schematic of anti-tumour gene therapy approaches involving lncRNA. The altered expression of lncRNA is widely observed in tumour tissues. The initial step for gene therapy includes collecting and growing tumour cells that are obtained from the patient (or from commercial cell line collections). The next step is testing the function of lncRNA of interest in tumour, which might be done with cell-editing assays using targeting lncRNA by RNAi, ASOs, PNAs or CRISPR/Cas9. Resulted cell lines with altered expression of lncRNA are characterized in line with unmodified tumour cells. The cell-based experiments include analysis of cell viability, proliferation, migratory potential, response to therapeutic compounds. The obtained results need to be confirmed in vivo for example after injection of lncRNA expressing cells/lncRNA-silenced cells to immunodeficient mouse (xenograft models). The tumour growing in xenografts mimics the patient’s tumour. The characteristics of tumour mass, growth rate and specific aspects of its behavior, such as assessing metastatic growth, is needed to validate the therapeutic potential of lncRNA. Additionally, xenografts might be subjected to anti-tumour therapy to test response to drugs. The verified therapeutic lncRNA gene might be then encapsuled with the non-immunogenic vectors like viruses and injected to the patient. Ultimately, stem cell lines such as MCSs (mesenchymal stem cells), HCSs (haematopoietic stem cells) or iPCSs (induced pluripotent stem cells) might be transfected with the lncRNA to obtain cells expressing lncRNA. The injection of these modified cells to the patient increases the ability to generate healthy cells. Several lncRNAs tested for gene therapy are described within the text. This is an original figure.