In the study published in the journal Nature Communications on December 7, entitled “In vivo RNA interactome profiling reveals 3'UTR-processed small RNA targeting a central regulatory hub”, Prof. CHAO Yanjie’s group at the Institute of Shanghai Institute of Immunity and Infection of the Chinese Academy of Sciences reported a novel RNA interactome profiling technology (iRIL-seq), which identifies RNA-RNA molecular interactions in live microbial cells. Using iRIL-seq, the researchers for the first time mapped the RNA-RNA interactome networks of Salmonella enterica at different growth stages, and discovered many novel non-coding RNAs and important mRNA regulatory hubs. 

Direct RNA-RNA interaction is a main mechanism by which noncoding RNAs exert their functions. In prokaryotic cells, non-coding small RNAs recognize target mRNAs through very short and imperfect basepairing interactions, which are difficult to be predicted and identified using conventional methods. Bacterial non-coding RNAs have important regulatory functions, controlling the expression of many target genes at the post-transcriptional level, and playing crucial regulatory roles in many biological processes, such as bacterial infection, antibiotic resistance, central metabolism, stress response, quorum sensing, and biofilm formation, etc. The analysis of the RNA-RNA interaction network is of great significance to the study of microbial physiology and the functions of non-coding RNAs.

However, the existing RNA-RNA interactome profiling techniques are complicated, which require fixed cells and numerous in vitro steps such as RNA digestion, repair and ligation, yielding insufficient RNA interactions with low resolution and accuracy. 

In this study, the researchers developed a novel in vivo RNA proximity-ligation approach, iRIL-seq (intracellular RNA interaction by ligation and sequencing). In this technology, the researchers induced the expression of T4 RNA ligase in living microbial cells, which leads to the proximity-ligation between interacting RNA molecules in vivo; next enriched the non-coding small RNAs and their interacting transcripts by immunoprecipitation of the major small RNA chaperone Hfq; and finally determined the identity and sequence of the interacting RNA molecules using high-throughput RNA sequencing. The main experimental process can be completed within a single day, thanks to the rapid ligation in vivo and streamlined workflow.

This new technology not only illustrates the atlas of microbial RNA-RNA interaction network, but also identifies RNA-RNA basepairing interactions at the single-nucleotide resolution. Therefore, the iRIL-seq technology provides a simple, fast, accurate and universal technology for analyzing the RNA-RNA interactions in microorganisms, and also proposes a new way to study RNA-RNA interactions in eukaryotic cells.