RNA Sequencing Identifies New RNase III Cleavage Sites in Escherichia coli and Reveals Increased Regulation of mRNA

Abstract

Ribonucleases facilitate rapid turnover of RNA, providing cells with another mechanism to adjust transcript and protein levels in response to environmental conditions. While many examples have been documented, a comprehensive list of RNase targets is not available. To address this knowledge gap, we compared levels of RNA sequencing coverage of Escherichia coli and a corresponding RNase III mutant to expand the list of known RNase III targets. RNase III is a widespread endoribonuclease that binds and cleaves double-stranded RNA in many critical transcripts. RNase III cleavage at novel sites found in aceEF, proP, tnaC, dctA, pheM, sdhC, yhhQ, glpT, aceK, and gluQ accelerated RNA decay, consistent with previously described targets wherein RNase III cleavage initiates rapid degradation of secondary messages by other RNases. In contrast, cleavage at three novel sites in the ahpF, pflB, and yajQ transcripts led to stabilized secondary transcripts. Two other novel sites in hisL and pheM overlapped with transcriptional attenuators that likely serve to ensure turnover of these highly structured RNAs. Many of the new RNase III target sites are located on transcripts encoding metabolic enzymes. For instance, two novel RNase III sites are located within transcripts encoding enzymes near a key metabolic node connecting glycolysis and the tricarboxylic acid (TCA) cycle. Pyruvate dehydrogenase activity was increased in an rnc deletion mutant compared to the wild-type (WT) strain in early stationary phase, confirming the novel link between RNA turnover and regulation of pathway activity. Identification of these novel sites suggests that mRNA turnover may be an underappreciated mode of regulating metabolism.

Transcript degradation and codon usage regulate gene expression in a lytic phage

“…Bacteria use transcript degradation extensively as a strategy for gene regulation [27][28][29][30][31][32][37][38][39]. Degradation helps to tightly couple transcription and translation, such that protein abundances closely match those of transcripts and bacteria can more quickly respond to changes in their environment.…”

“…Degradation helps to tightly couple transcription and translation, such that protein abundances closely match those of transcripts and bacteria can more quickly respond to changes in their environment. In many cases, however, specific transcript properties drive differential degradation patterns [30]. In E. coli operons, for example, secondary structure creates patterns of differential degradation within single polycistronic transcripts [39].…”

“…Pinetree employs a directional model of transcript degradation modeled after observations from E. coli [27][28][29]. In E. coli, degradation occurs either from the 5'-end of a newly-synthesized transcript or from the 5'-end of a transcript recently cleaved by an RNase III ribnuclease [30]. A combination of endo-and exonucleases degrade transcripts, but the net effect is that transcript degradation is directional-the 5' end of a transcript has a shorter lifespan than the 3' end [31].…”

Enzymes Involved in Posttranscriptional RNA Metabolism in Gram-Negative Bacteria

“…However, besides RNase E, there are a significant number of other endonucleases that initiate the decay of mRNAs as minor players. For example, RNase III, which will be discussed in more detail later in the context of rRNA processing, has been shown to affect the steady-state levels of up to 10% of the mRNAs in exponentially growing cells of E. coli resulting in either destabilization or stabilization of the transcripts (24, 25). RNase P, which is a ribozyme that contains an RNA catalytic subunit (26), specifically cleaves a small number of polycistronic mRNAs in intercistronic regions (27).…”

Tombusvirusp19 captures RNase III-cleaved double-stranded RNAs formed by overlapping sense and antisense transcripts inE. coli

“…asRNA and RNase III regulate 12 gene expression of plasmids and toxins. The E. coli ColE1 plasmid replication origin encodes 13 two non-coding RNAs -RNA II, which serves as a DNA replication primer, and RNA I, which 14 is shorter and fully complementary to the 5´ portion of RNA II (7)(8)(9)(10). RNA I inhibits plasmid 15 replication by binding to the RNA II plasmid replication primer.…”