Biochemical characterization and functional analysis for noncoding RNAs in Escherichia coli

Abstract

Noncoding RNAs have been extensively found in many different organisms, from bacteria to mammals. In Escherichia coli, about 100 of noncoding RNAs (50~400 nucleotides in length) have been identified and they have been recognized as important regulators of a number of cellular processes. However, the biological functions and regulatory mechanisms of most noncoding RNAs remained elusive. In this thesis, biochemical analyses were performed to investigate the regulatory mechanism of Sib RNA, the tight control of M1 RNA biogenesis. In addition, biogenesis and functions of SraH were investigated. SibC is a small noncoding RNA of about 140 nucleotides. Recently, it has been reported that SibC functions as an antitoxin RNA to repress the synthesis of the toxin protein IbsC. Four additional Sib RNAs (SibA, B, D, and E) have been found in the E. coli genome, which are known as an antitoxin Sib RNA family. Although the sequences of five Sib RNAs are highly homologous one another, they uniquely regulate their own targets (ibs mRNAs) without cross-regulation. Therefore, the biochemical characterization was also focused on how Sib RNAs recognize their targets. The sequences responsible for RNA-RNA interactions between SibC and ibsC mRNA were defined with various SibC RNA derivatives by using co-transformation assays and the interactions were biochemically probed. It was found that SLII of SibC is important for its target recognition. The SLII region recognizes SLIV of ibsC mRNA. A specific single strand region (ss) of SibC, which recognizes ss2 of ibsC mRNA, also serves for the target recognition. Indeed, if either one of these two sites of SibC was replaced by those of SibD, the SibC variants were able to suppress the toxicity of not only ibsC but also ibsD expression. When both of two sites were replaced by the counterpart of SibD, no more ibsC repression was observed due to the loss of specificity. These results suggest that the sequences in these two regions of Sib RN...