Linking RNA biology to lncRNAs

Abstract

The regulatory potential of RNA has never ceased to amaze: from RNA catalysis, to RNA-mediated splicing, to RNA-based silencing of an entire chromosome during dosage compensation. More recently, thousands of long noncoding RNA (lncRNA) transcripts have been identified, the majority with unknown function. Thus, it is tempting to think that these lncRNAs represent a cadre of new factors that function through ribonucleic mechanisms. Some evidence points to several lncRNAs with tantalizing physiological contributions and thought-provoking molecular modalities. However, dissecting the RNA biology of lncRNAs has been difficult, and distinguishing the independent contributions of functional RNAs from underlying DNA elements, or the local act of transcription, is challenging. Here, we aim to survey the existing literature and highlight future approaches that will be needed to link the RNA-based biology and mechanisms of lncRNAs in vitro and in vivo.

Figure 1.

Experimental approaches to manipulate the expression, perturb the activity, or evaluate the functions of long noncoding RNAs. Most commonly used are transient expression of exogenous oligonucleotides designed to exploit the endogenous RNAi machinery or RNase H activity to degrade an RNA (A) or occlude putative functional regions (B). Recently, strategies have used the flexible CRISPR/Cas9 system to positively or negatively affect the transcription of a lncRNA gene (C) or the incorporation of an auto-catalytic regulatory RNA element to destabilize the nascent lncRNA transcript (D). An alternative to manipulating lncRNA expression levels involves the recruitment of the RNA of interest to a particular genomic locus or reporter gene through fusion of DNA-binding proteins with RNA-binding elements such as MS2 stem–loops (E) or covalent tethering of a lncRNA transcript to the CRISPR/Cas9 guide RNA (F).

Figure 2.

Genetic approaches to evaluate lncRNA functional contributions. (A) Genetic ablation of a lncRNA gene or a lncRNA promoter is a powerful technique that can be used to confirm any regulatory activity arising from a particular locus. The optional incorporation of a reporter gene at the deleted locus can be used to visualize the biological contexts in which the lncRNA gene is expressed and evaluate any contribution of the local act of transcription to an observed phenotype. (B) An alternative strategy is to introduce a premature transcriptional terminator sequence, which will prevent transcription of the full-length lncRNA transcript. With this approach, however, it is not possible to assess the contribution of the act of transcription. (C) With either approach, the rescue of any observed phenotype by transgene expression is currently still considered the gold standard to confirm a functional lncRNA molecule; however, a rescue may not be possible if the RNA works exclusively in cis.

Figure 3.

Functionally equivalent RNA and DNA domains. Despite distinct mechanisms by which they achieve their equivalence, RNA domains are capable of affecting many biological activities that have traditionally been ascribed exclusively to proteins. Much work remains, however, in identifying common RNA primary, secondary, and tertiary structures that comprise orthologous functional domains and can mediate these and other subcellular activities.