Small RNA Modules Confer Different Stabilities and Interact Differently with Multiple Targets

Bacterial Hfq-associated small regulatory RNAs (sRNAs) parallel animal microRNAs in their ability to control multiple target mRNAs. The small non-coding MicA RNA represses the expression of several genes, including major outer membrane proteins such as ompA, tsx and ecnB. In this study, we have characterised the RNA determinants involved in the stability of MicA and analysed how they influence the expression of its targets. Site-directed mutagenesis was used to construct MicA mutated forms. The 5′linear domain, the structured region with two stem-loops, the A/U-rich sequence or the 3′ poly(U) tail were altered without affecting the overall secondary structure of MicA. The stability and the target regulation abilities of the wild-type and the different mutated forms of MicA were then compared. The 5′ domain impacted MicA stability through an RNase III-mediated pathway. The two stem-loops showed different roles and disruption of stem-loop 2 was the one that mostly affected MicA stability and abundance. Moreover, STEM2 was found to be more important for the in vivo repression of both ompA and ecnB mRNAs while STEM1 was critical for regulation of tsx mRNA levels. The A/U-rich linear sequence is not the only Hfq-binding site present in MicA and the 3′ poly(U) sequence was critical for sRNA stability. PNPase was shown to be an important exoribonuclease involved in sRNA degradation. In addition to the 5′ domain of MicA, the stem-loops and the 3′ poly(U) tail are also shown to affect target-binding. Disruption of the 3′U-rich sequence greatly affects all targets analysed. In conclusion, our results have shown that it is important to understand the “sRNA anatomy” in order to modulate its stability. Furthermore, we have demonstrated that MicA RNA can use different modules to regulate its targets. This knowledge can allow for the engineering of non-coding RNAs that interact differently with multiple targets.