The emerging molecular biology toolbox for the study of long noncoding RNA biology

Figures & data

Figure 1.  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.

Table 1.  Summary of the emerging methods used to study long noncoding RNA biology.

Method	Information obtained	Ref.
RNA-Seq	Quantitative reads of the entire transcriptome	[27]
CAGE	Quantitative reads of the transcriptome by capturing the 5′-end of the transcript	[28]
3C (and its derivatives)	Mapping of chromosomal interaction for the identification of spatially proximal genes and genetic elements	[29–31]
ChIP-Seq	Genome wide mapping and quantification of epigenetic marks and protein on DNA	[32]
smFISH (and its derivatives)	Abundance and subcellular localization of transcripts in single cells	[33–39]
Genome and epigenome editing tools	Perturbation of lncRNA transcription and position to determine function	[44,45,50,51,53–55,58,60]
ChIRP	Identification of interacting RNA, DNA and protein partners through the capture of the lncRNA transcript	[61]
Chart	Identification of interacting DNA and protein partners through the capture of the lncRNA transcript	[62]
RAP	Identification of interacting RNA, DNA and protein partners through the capture of the lncRNA transcript	[20]
PAR-CLIP	Identification of lncRNAs that are interacting with a particular protein of interest through the immunoprecipitation of the protein	[63,64]
SHAPE	LncRNA secondary structure at single nucleotide resolution	[65]

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