Global snapshots of bacterial RNA networks

Abstract

While bacteria were long thought to rely primarily on transcriptional control, it is now well established that they also use numerous small RNAs to regulate mRNA translation and stability. There has recently been a surge in studies, including one by Waters et al (2017) in this issue of The EMBO Journal, that have used clever variations of the RNA‐seq technique to comprehensively map small RNA–target networks.

Figure 1. How CLASH and RIL‐seq work

CLASH: Following in vivo cross‐linking, RNase E is pulled down using a FLAG‐tag, the bound RNA is trimmed, and the complex is purified under denaturing conditions using a His‐tag. Hybrid RNAs are created by ligation of the interacting RNAs with T4 ligase. RIL‐seq: In vivo cross‐linked Hfq is pulled down using a FLAG‐tag, followed by RNA trimming and ligation of the interacting RNAs with T4 ligase. Subsequently, the resulting hybrid RNAs of either protocol are sequenced, allowing in silico analysis of sRNA–target interactions. Bottom left: CLASH reveals new insights into mRNA decay. After binding 3′ of the sRNA–mRNA seed region, RNase E cleaves the mRNA, an oligo(A) stretch is added to the 3′ end, which finally allows decay by 3′ exonucleases. Bottom right: RIL‐seq reveals new sRNAs along with their targets. Bottom middle: Sequencing of RNA hybrids reveals global sRNA interactomes. sRNAs (colored) directly interact with targets (white nodes), which then interact with further targets (gray nodes).