The bacterial endoribonuclease RNase E can cleave RNA in the absence of the RNA chaperone Hfq

Abstract

RNase E is a component of the RNA degradosome complex and plays a key role in RNA degradation and maturation in Escherichia coli. RNase E-mediated target RNA degradation typically involves the RNA chaperone Hfq and requires small guide RNAs (sRNAs) acting as a seed by binding to short (7-12-bp) complementary regions in target RNA sequences. Here, using recombinantly expressed and purified proteins, site-directed mutagenesis, and RNA cleavage and protein cross-linking assays, we investigated Hfq-independent RNA decay by RNase E. Exploring its RNA substrate preferences in the absence of Hfq, we observed that RNase E preferentially cleaves AU-rich sites of single-stranded regions of RNA substrates that are annealed to an sRNA that contains a monophosphate at its 5-end. We further found that the quaternary structure of RNase E is also important for complete, Hfq-independent cleavage at sites both proximal and distal to the sRNA-binding site within target RNAs containing monophosphorylated 5-ends. Of note, genetic RNase E variants with unstable quaternary structure exhibited decreased catalytic activity. In summary, our results show that RNase E can degrade its target RNAs in the absence of the RNA chaperone Hfq. We conclude that RNase E-mediated, Hfq-independent RNA decay in E. coli requires a cognate sRNA sequence for annealing to the target RNA, a 5-monophosphate at the RNA 5-end, and a stable RNase E quaternary structure.

Adaptor protein RapZ activates endoribonuclease RNase E by protein–protein interaction to cleave a small regulatory RNA

“…RNase E contributes to sRNA biogenesis, maturation as well as decay (Göpel et al 2013;Miyakoshi et al 2015;Chao et al 2017). Some sRNAs recruit RNase E to degrade the target RNA, while others have protective roles by occlusion of RNase E cleavage sites upon base-pairing (Morita et al 2005;McCullen et al 2010;Fröhlich et al 2013;Baek et al 2019).…”

Regulatory RNAs: A Universal Language for Inter-Domain Communication

“…In Gram-positive bacteria, RNase Y is the key nuclease of the degradosome and is thought to be the primary nuclease of asRNA-mediated mRNA cleavage [ 74 , 75 ]. For many trans -asRNAs, the interaction with RNase E is mediated by the RNA chaperone protein, Hfq ( Figure 1 B) [ 29 , 30 ]. Hfq stabilises the trans -asRNA and facilitates binding between the trans -asRNA and its mRNA target, despite the limited complementarity in RNA sequence [ 72 , 76 ].…”

“…( B ) Newly transcribed bacterial trans -asRNAs can be stabilised by Hfq. Hfq can also facilitate binding between the trans- asRNA and the mRNA target [ 29 , 30 ]. In Gram-negative bacteria, Hfq can facilitate binding of the trans- asRNA and mRNA to RNase E which degrades both the asRNA and mRNA [ 31 ].…”

Kinetic model of small RNA-mediated regulation suggests that a small RNA can regulate co-transcriptionally

“…D/U t=15 in the WT cell was about ~70% of that in the ΔsgrS cell, suggesting that the regulation by SgrS caused reduction in the abundance of the downstream region compared to the upstream region of the SgrS binding site on the target mRNA. The reduced D/U t=15 upon SgrS regulation may be explained by the directionality of RNase E activity, i.e., an enhanced RNase E activity on the downstream fragment with 5' monophosphate (55)(56)(57). In comparison, D/U t=1 in the WT cell was about ~40% of that in the ΔsgrS cell, suggesting that in the nascent-mRNA enriched pool, the regulation by SgrS led to more reduction in the abundance of the downstream region compared to the upstream region, and supporting our prediction that SgrS repressed the generation of the downstream portion co-transcriptionally.…”