Seed pairing between a small regulatory RNA and target mRNA determines cleavage by a RNase E with a minimal C-terminal scaffold.

Abstract

Small regulatory RNAs (sRNAs) can specifically bind to their target mRNAs to inhibit their expression, either by promoting degradation or by blocking translation. However, the molecular determinants that select which repression mode an sRNA employs remain poorly understood. To investigate this, we used the model target mRNA hdeD, which is repressed by two sRNAs, CyaR and RprA, both of which use distinct modes of repression. Whereas CyaR promotes the target mRNA hdeD degradation, the sRNA RprA specifically blocks hdeD translation initiation, leaving hdeD mRNA intact. We found that mutating two nucleotides in the seed pairing region of RprA with hdeD is sufficient to switch its mode of repression from translation inhibition to degradation by recruiting RNase E. We investigated this further by using various RNase E deletion mutants of the C-terminal domain (CTD). Among these was a RNase E mutant deleted to contain a minimal C-terminal scaffold (rne 701), a form which typically fails to promote sRNA-induced mRNA degradation. Our data indicated that even with a minimal scaffold, the mutated RprA sRNA still promoted hdeD mRNA degradation, whereas the native sRNA CyaR could not. Our data suggested that the mutated RprA sRNA relies primarily on the arginine-rich RNA-binding (ARRBD) domain of RNase E to induce target mRNA hdeD degradation. This suggests that the mutated RprA sRNA requires fewer components of the CTD of RNase E and RNA degradosome to induce target mRNA degradation.  .