Long noncoding RNAs (lncRNAs) have been implicated in many biological processes. However, due to the unique nature of lncRNAs and the consequential difficulties associated with their characterization, there is a growing disparity between the rate at which lncRNAs are being discovered and the assignment of biological function to these transcripts. Here researchers from the CSIR South Africa present a molecular biology toolbox equipped to help dissect aspects of lncRNA biology and reveal functionality. They outline an approach that begins with a broad survey of genome-wide, high-throughput datasets to identify potential lncRNA candidates and then narrow the focus on specific methods that are well suited to interrogate the transcripts of interest more closely. This involves the use of imaging-based strategies to validate these candidates and observe the behaviors of these transcripts at single molecule resolution in individual cells. The researchers also describe the use of gene editing tools and interactome capture techniques to interrogate functionality and infer mechanism, respectively. With the emergence of lncRNAs as important molecules in healthy and diseased cellular function, it remains crucial to deepen our understanding of their biology.
An overview of the pipeline and tools used to study lncRNA biology
(A) Publicly available databases provide transcriptomic, conformational and epigenetic information on the entire genome, aiding in the identification of lncRNAs and the generation of broad hypotheses for their function. (B) (left) LncRNAs are able to interface with DNA, RNA and proteins to exert their functions. Many lncRNAs are thought to modify the epigenetic state of chromatin and alter the transcription of genes in cis or trans (Right) lncRNAs can be visually detected by decorating the transcript with fluorescently labelled antisense oligonucleotides for smFISH. The inset shows an example of a lncRNA and a nearby protein coding gene detected by smFISH and fluorescent microscopy. These experiments complement high-throughput biochemical assays and provide useful details on the absolute abundance and the subcellular localization patterns of lncRNA transcripts with single cell resolution. (C)CRISPR/Cas9 tools are able to (i) ablate lncRNA expression by direct mutagenesis to the DNA sequence for loss of function studies (ii) function as a targeting module to recruit activators, inhibitors and chromatin modifiers for locus specific perturbation of lncRNA expression or (iii) target lncRNA transcripts to gene loci to investigate their spatial nature.