RNA interactomics: recent advances and remaining challenges

Abstract

Tight regulation of cellular processes is key to the development of complex organisms but also vital for simpler ones. During evolution, different regulatory systems have emerged, among them RNA-based regulation that is carried out mainly by intramolecular and intermolecular RNA-RNA interactions. However, methods for the transcriptome-wide detection of these interactions were long unavailable. Recently, three publications described high-throughput methods to directly detect RNA duplexes in living cells. This promises to enable in-depth studies of RNA-based regulation and will narrow the gaps in our understanding of RNA structure and function. In this review, we highlight the benefits of these methods and their commonalities and differences and, in particular, point to methodological shortcomings that hamper their wider application. We conclude by presenting ideas for how to overcome these problems and commenting on the prospects we see in this area of research.

Keywords: RNA; RNA-RNA interactions; Regulation.

Figure 1.. Schematic comparison of Direct Duplex Detection methods.

Overview of the protocols for LIGR-seq , PARIS , and SPLASH . The first column shows the principal steps of the three experimental procedures: crosslinking (violet crosses) was conducted in vivo by 365 nm irradiation and 4′-aminomethyltrioxsalen (AMT) or biotinylated psoralen treatment. Fragmentation was performed enzymatically (LIGR-seq and PARIS) or chemically (SPLASH). Crosslinked RNAs were additionally enriched either by size separation using two-dimensional (2D) gel electrophoresis (crosslinked RNAs above the main diagonal were eluted; PARIS) or by biotin-streptavidin binding to magnetic beads (SPLASH). Proximity ligation was carried out using different ligases. Crosslinks were reverted by 254 nm irradiation. For sequencing, different library preparation strategies were performed. Colors of RNA strands (blue and orange) indicate different RNA molecules. LIGR-seq, ligation of interacting RNA followed by high-throughput sequencing; PARIS, psoralen analysis of RNA interactions and structures; SPLASH, sequencing of psoralen-crosslinked, ligated, and selected hybrids.