Roles of RNA helicases in small non-coding RNA functions in Escherichia coli

Abstract

E. coli is a good model with which to study the functions of DEAD-box proteins, due to the reactions in which they participate, i.e., ribosome assembly and mRNA degradation. E. coli possesses five such proteins, DbpA, DeaD, RhlB, RhlE, and SrmB, in which the helicase core is flanked with C-terminal extensions of various lengths. In E. coli, only four DEAD-box proteins are involved in ribosome biogenesis. Of the five E. coli DEAD-box proteins, RhlB is part of the bacterial degradosome, a multi-protein complex involved in the RNA degradosome. In many model organisms, several DEAD-box proteins have also been found to be essential. In contrast, none of the five E. coli DEAD-box proteins is required for rapid growth at $37^\circ C$. From the results of in vivo and in vitro experiments, I identified the interconversion of DEAD-box proteins. An analysis of the function of RNA helicases is difficult because these features and the precise substrates and sites of action remain unknown for most of them. I expect that RNA helicases interact with sRNA and participate in RNA metabolism. To verify this assumption, I applied a gene-silencing system to an analysis of the roles of RNA helicases. The abundance of artificial small RNA (afsRNA) ARlacZ1 with a SibC-derived scaffold was increased by only rhlB deletion in contrast to dSL6 with its double-stem-loop (dSL) scaffold. An increase in the ARlacZ1 level was induced by blocking the degradation by RNase III / E. The increased ARlacZ1 also underwent base-pairing with other target mRNAs, after which the base-paired mRNAs were degraded by RNase. These results demonstrated that RhlB recognizes the target sRNA and helps RNase E to degrade sRNA, mRNA, or both. Another experimental approach was the construction of a DEAD-box gene multiple-deletion strain and a search for the target sRNA of RNA Helicases at a low temperature using these mutation strains. DeaD and SrmB are essential proteins at low temperatures. The removal of rhlE did not alter the growth process at either $37^\circ C$ or$ 25^\circ C$, but the $\Delta deaD$ and $\Delta srmB$ strains showed the opposite effect, with rhlE deletion during the growth process. I used this feature to find the target sRNAs of RNA helicase proteins. The abundance and stability of sRNAs individually changed in DEAD-box gene multiple-deletion strains at low temperatures. A comparison of DsrA and GlmZ as induced by a cold shock showed the opposite propensity. An intracellular level change was found in only DsrA among RprA, ArcZ, and DsrA known to be activators of rpoS expression. The results of this study distinctly indicate that RNA helicases specifically target certain sRNAs and participate in sRNA processing through the regulation of the stability and abundance of sRNA.