Reverse-genetics studies of lncRNAs-what we have learnt and paths forward

Abstract

Long non-coding RNAs (lncRNAs) represent a major fraction of the transcriptome in multicellular organisms. Although a handful of well-studied lncRNAs are broadly recognized as biologically meaningful, the fraction of such transcripts out of the entire collection of lncRNAs remains a subject of vigorous debate. Here we review the evidence for and against biological functionalities of lncRNAs and attempt to arrive at potential modes of lncRNA functionality that would reconcile the contradictory conclusions. Finally, we discuss different strategies of phenotypic analyses that could be used to investigate such modes of lncRNA functionality.

Fig. 1

Reverse-genetics approaches for lncRNA functional studies. The illustration shows various methods that target either RNA (based on RNAi, ASOs, or CRISPR/Cas13) or DNA, based on the CRISPR/Cas9 family of methods that can cause deletions and insertions of specific sequences (e.g., polyA cassettes or self-cleaving ribozymes) or bring transcription activators/silencers to promoters depending on specific system employed. Also shown are some of the known problems with these techniques—off-target effects caused by partial sequence matches (1, 4, 6) or non-specific effects such as triggering innate immune response (2), saturation of the endogenous RNAi machinery (3), and interactions with proteins (5), as well inability to discriminate between the targets and other overlapping (7) or shared elements (8) and to target sequences containing repetitive elements (9). More details are in the text

Fig. 2

Emerging strategies for investigating biological functions of lncRNAs. Reverse-genetics methods differ as to their abilities to target transcripts and cause off-target/non-specific effects. As such, unambiguous phenotype-lncRNA assignment, especially using methods that do not exclusively target RNA, requires RNA rescue experiments and combination of multiple approaches. Considering the highly specialized patterns of expression for most lncRNAs, in vivo phenotypes are expected to occur only in the cell types expressing the targeted transcript. In contrast, abnormalities happening in the cells that do not express the lncRNA likely indicate transcript-independent effects. On the other hand, cell-based assays have a number of attractive features and remain the only option for lncRNAs whose in vivo expression is not known or with no known homologs in animal models. In cultured cell systems, a phenotypic analysis can be performed either for a single lncRNA (middle) or in a large-scale high-throughput screen (right). More details are in the text