Mapping RNA–RNA interactome and RNA structure in vivo by MARIO

Abstract

The pervasive transcription of our genome presents a possibility of revealing new genomic functions by investigating RNA interactions. Current methods for mapping RNA–RNA interactions have to rely on an ‘anchor' protein or RNA and often require molecular perturbations. Here we present the MARIO (Mapping RNA interactome in vivo) technology to massively reveal RNA–RNA interactions from unperturbed cells. We mapped tens of thousands of endogenous RNA–RNA interactions from mouse embryonic stem cells and brain. We validated seven interactions by RNA antisense purification and one interaction using single-molecule RNA–FISH. The experimentally derived RNA interactome is a scale-free network, which is not expected from currently perceived promiscuity in RNA–RNA interactions. Base pairing is observed at the interacting regions between long RNAs, including transposon transcripts, suggesting a class of regulatory sequences acting in trans. In addition, MARIO data reveal thousands of intra-molecule interactions, providing in vivo data on high-order RNA structures.

Advances and challenges towards the study of RNA-RNA interactions in a transcriptome-wide scale

“…However, psoralen and AMT can only crosslink pyrimidines, and with very limited efficiency [93,94]. For instance, the cross-linking efficiency is less than 6% in most related techniques [41,43,45,46]. In addition, the efficiency of proximity ligation is also low and influenced by the secondary and tertiary structures of target RNAs, which may result in deteriorated performance on small ncRNAs [41,45].…”

“…RNA followed by high-throughput sequencing) [45] ( Figure 3C), and MARIO (mapping RNA interactome in vivo) ( Figure 3D) [46]. PARIS employs a psoralenderivative 4′-aminomethyltrioxsalen (AMT) as the nucleic acid cross-linker to cross-link RNA base pairs in living cells [41].…”

“…Studies have been carried out to collect the data and analyze them from a perspective of systems biology. It is found that, very similar to protein-protein interaction (PPI) networks [89], RRI networks are also scale-free and modular in human [88], mouse [46], and yeast [90]. One characteristic of a scale-free network is the existence of a set of nodes with relatively high interaction degrees [89,91].…”

“…The recent breakthrough in transcriptome-wide methods for RRI discovery depends on the crosslinking of interacting duplexes by small molecules including psoralen and its derivates [41,43,45,46]. However, psoralen and AMT can only crosslink pyrimidines, and with very limited efficiency [93,94].…”

“…For instance, RNA duplexes usually migrate slower in electrophoresis gel, allowing them to be separated from non-interacting RNA fragments [22,23]. Recently, with the advent of next generation sequencing, new techniques based on deep sequencing have been developed to reveal the full network of RRIs, i.e., the interactome, for all transcripts in a cell [41,43,45,46]. They can also be used to study all interactions concerning a certain RNA, usually combined with RNA pull-down with a set of target-specific antisense probes [38,39].…”