Small RNAs (sRNAs) play central roles in post-transcriptional regulation of gene expression, controlling stress responses and adaptive programmes across bacteria and beyond. While their regulatory functions are well characterised, mechanisms for their selective degradation remain elusive. In Escherichia coli, RyhB is a well-established model for sRNA-mediated regulation that modulates iron homeostasis by controlling the stability and translation of multiple mRNA targets through base-pairing. Here, we combine biochemical, structural and functional approaches to characterise how YicC, a widely conserved yet recently discovered ribonuclease, cleaves RyhB.
Using in vitro assays, we mapped a precise cleavage site within RyhB and found that YicC is selective, sparing other regulatory RNAs under identical conditions. Cryo-EM single-particle analysis reveals a novel "clamshell" mechanism: while the ribonuclease adopts an open, inactive conformation in the apo state, it undergoes a large structural rearrangement upon RNA binding. This RNA-induced transition to a closed, catalytically competent state repositions key residues, enabling specific cleavage.
To investigate this mechanism in vivo, we used structure-guided mutagenesis to probe residues involved in the conformational switch. The functional impact of these mutations was assessed using a fluorescent reporter assay, where a fluorescent protein is fused to an mRNA target of RyhB. Loss-of-function variants stabilise the sRNA in vivo, enhancing repression of the fluorescent reporter and providing a live-cell readout of the functional link between conformational dynamics and RNA turnover.
Comparative sequence analysis revealed YicC is widely conserved across diverse bacterial phyla, including species lacking RyhB. This suggests the enzyme targets additional substrates, indicating a broader role in bacterial RNA regulation. To identify its full RNA repertoire in vivo, we are performing RNA-seq analysis on cells following pulse-expression of YicC. Intriguingly, we have also identified homologues in eukaryotes, including rhodophytes (red algae), where they often exist as fusions to a FIG-like (FBPase/IMPase/glpX) phosphatase domain. This conserved fusion suggests a functional link between the ribonuclease and phosphatase activities. We are now characterising these eukaryotic homologues to determine if this unique clamshell mechanism is conserved across kingdoms.
These findings define the structural basis for a new family of ribonucleases, revealing a previously unappreciated mechanism for regulated sRNA turnover.