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.
Highlights
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 cleaves target RNA paired with Small guide RNA (sRNA) in an Hfqindependent manner It has been previously reported that RNase E cleaves singlestranded RNA with 5Ј-monophosphate (i.e. 5Ј-P), but not with 5Ј-hydroxyl (i.e. 5Ј-OH) or triphosphate group [15, 19]
Our experimental results showed that target RNA degradation by RNase E does not necessarily require the activity of the RNA chaperone Hfq (Fig. 1B)
Summary
The bacterial endoribonuclease RNase E can cleave RNA in the absence of the RNA chaperone Hfq. X Yu Mi Baek‡, Kyoung-Jin Jang‡, Hyobeen Lee‡, Soojin Yoon‡, Ahruem Baek‡, Kangseok Lee§, and X Dong-Eun Kim‡1 From the ‡Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Korea and the §Department of Life Science, Chung-Ang University, Seoul 06974, Korea
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have