The coordinated action of RNase III and RNase G controls enolase expression in response to oxygen availability in Escherichia coli

Abstract

Rapid modulation of RNA function by endoribonucleases during physiological responses to environmental changes is known to be an effective bacterial biochemical adaptation. We report a molecular mechanism underlying the regulation of enolase (eno) expression by two endoribonucleases, RNase G and RNase III, the expression levels of which are modulated by oxygen availability in Escherichia coli. Analyses of transcriptional eno-cat fusion constructs strongly suggested the existence of cis-acting elements in the eno 5′ untranslated region that respond to RNase III and RNase G cellular concentrations. Primer extension and S1 nuclease mapping analyses of eno mRNA in vivo identified three eno mRNA transcripts that are generated in a manner dependent on RNase III expression, one of which was found to accumulate in rng-deleted cells. Moreover, our data suggested that RNase III-mediated cleavage of primary eno mRNA transcripts enhanced Eno protein production, a process that involved putative cis-antisense RNA. We found that decreased RNase G protein abundance coincided with enhanced RNase III expression in E. coli grown anaerobically, leading to enhanced eno expression. Thereby, this posttranscriptional up-regulation of eno expression helps E. coli cells adjust their physiological reactions to oxygen-deficient metabolic modes. Our results revealed a molecular network of coordinated endoribonuclease activity that post-transcriptionally modulates the expression of Eno, a key enzyme in glycolysis.

RNase III Participates in the Adaptation to Temperature Shock and Oxidative Stress in Escherichia coli

“…In the case of the precursor mRNA of the T7 phage [28,71], RNase III was shown to proceed to single strand cleavages, releasing individual mRNAs with a stable hairpin in their 3 -ends. Other cases of positive regulation by RNase III have been identified, but the mechanism has not been elucidated, such as eno [26], ahpC, pflB, yajQ [32] and sucA [72] mRNAs. The importance of RNase III in the positive regulation of gene expression is further suggested by two independent transcriptomic analyses showing that 23% (120 out of 511 [30]) and 47% (87 out 187 [29]) of the genes whose expression was altered in an rnc mutant were downregulated.…”

“…As a consequence, inactivation of RNase III results in the overexpression of PNPase [23]. In a small number of cases, RNase III was also shown to be involved in the positive post-transcriptional regulation of a diverse set of genes, including gadE mRNA, encoding a regulator involved in acid resistance [24]; adhE mRNA encoding an alcohol dehydrogenase essential for fermentation [25]; eno mRNA encoding the metabolic enzyme enolase, which is also associated with the degradosome [26], cIII and N mRNAs from the phage λ [15,27] and the pre-mRNA of phage T7 [28]. Thus, in addition to its role in RNA decay, RNase III is also involved in the positive regulation of gene expression.…”

Endoribonuclease-mediated control of hns mRNA stability constitutes a key regulatory pathway for Salmonella Typhimurium pathogenicity island 1 expression

“…RNase G is a bacterial endoribonuclease known to negatively regulate gene expression via mRNA decay [9,[15][16][17]; however, SDS-PAGE and mass spectrometry revealed that some SPI-1 T3SS effectors (SlrP, SopA, SipA, and SipC) were up-regulated in the WT strain compared to that in the Δrng strain, indicating that changes in their mRNA levels were not a direct consequence of mRNA decay by RNase G. To assess this further, we analyzed upstream pathways to identify a SPI-1 T3SS repressor that can be negatively regulated by RNase G. When S. Typhimurium invades host cells or responds to elevated sucrose or salt concentrations, SPI-1 T3SS effector gene expression is activated by the major transcriptional activator HilA, which is generally repressed by H-NS [31,35,52]. To explore the relationships between sipA, sipC, hilA, and hns mRNA expression levels, real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed using total RNA from WT cells cultured under aerobic and anaerobic conditions, since SPI-1 T3SS expression is known to be up-regulated during Salmonella growth under low oxygen conditions and during host cell infection [27,53].…”

“…Both enzymes are single-stranded RNA-specific endoribonucleases with a preference for cleaving AU-rich sequences [11,13,14]. RNase G participates in rRNA maturation and mRNA degradation [9,11,[15][16][17][18][19]. E. coli lacking rne can be made viable by overexpressing RNase G; however, rngcomplementation affects the abundance of a small portion of mRNAs in rne-deficient cells [9], indicating that RNase G and E play distinct physiological roles.…”

The effect of two ribonucleases on the production of Shiga toxin and stx-bearing bacteriophages in Enterohaemorrhagic Escherichia coli

“…Despite some differences in substrate requirements, both enzymes prefer 5’ end-monophosphorylated RNA and attack single-stranded AU-rich sequences 42 – 44 . In E. coli , RNase G cleaves the 5’ end of a 16S rRNA precursor 42 and controls the stability of a limited number of mRNAs 45 – 47 . However, RNase G may have a global role in mRNA stability in some bacteria other than E. coli 48 .…”