Glutamine synthetase mRNA releases sRNA from its 3′UTR to regulate carbon/nitrogen metabolic balance in Enterobacteriaceae
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eLife assessment
This study revealed the biogenesis of the 3'UTR-derived sRNA GlnZ by RNase E-mediated processing and identified target mRNAs in both E. coli and S. enterica. By introducing point mutations within the predicted seed region of GlnZ and analyzing compensatory mutations in the target mRNAs, the sRNA binding site in those targets could convincingly be mapped. This is an important piece of work and the findings are relevant for researchers within the microbiology and RNA communities and should inspire future studies of 3'derived sRNAs in bacteria. Overall, most of the statements are sufficiently supported by experimental data, but certain amendments to the work are required to fully support the conclusions.
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Abstract
Glutamine synthetase (GS) is the key enzyme of nitrogen assimilation induced under nitrogen limiting conditions. The carbon skeleton of glutamate and glutamine, 2-oxoglutarate, is supplied from the TCA cycle, but how this metabolic flow is controlled in response to nitrogen availability remains unknown. We show that the expression of the E1o component of 2-oxoglutarate dehydrogenase, SucA, is repressed under nitrogen limitation in Salmonella enterica and Escherichia coli . The repression is exerted at the post-transcriptional level by an Hfq-dependent sRNA GlnZ generated from the 3′UTR of the GS-encoding glnA mRNA. Enterobacterial GlnZ variants contain a conserved seed sequence and primarily regulate sucA through base-pairing far upstream of the translation initiation region. During growth on glutamine as the nitrogen source, the glnA 3′UTR deletion mutants expressed SucA at higher levels than the S. enterica and E. coli wild-type strains, respectively. In E. coli , the transcriptional regulator Nac also participates in the repression of sucA . Lastly, this study clarifies that the release of GlnZ from the glnA mRNA by RNase E is essential for the post-transcriptional regulation of sucA . Thus, the mRNA coordinates the two independent functions to balance the supply and demand of the fundamental metabolites.
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eLife assessment
This study revealed the biogenesis of the 3'UTR-derived sRNA GlnZ by RNase E-mediated processing and identified target mRNAs in both E. coli and S. enterica. By introducing point mutations within the predicted seed region of GlnZ and analyzing compensatory mutations in the target mRNAs, the sRNA binding site in those targets could convincingly be mapped. This is an important piece of work and the findings are relevant for researchers within the microbiology and RNA communities and should inspire future studies of 3'derived sRNAs in bacteria. Overall, most of the statements are sufficiently supported by experimental data, but certain amendments to the work are required to fully support the conclusions.
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Reviewer #1 (Public Review):
3' UTR-derived sRNAs are increasingly recognized as post-transcriptional regulators of diverse bacterial processes. While initially assumed to be highly specific regulators with single targets, global RNA interactome approaches challenge this view by suggesting 3'-derived sRNAs can bind and regulate multiple target mRNAs, reminiscent of the regulons of intergenic sRNAs (PMID: 35388892). In Enterobacteriaceae, GlnZ is an sRNA derived from the 3' UTR of the glnA mRNA (PMID: 15718303) that encodes glutamine synthetase. However, the role and function of GlnZ have not previously been determined. In the present work, the authors set out to investigate GlnZ in E. coli and Salmonella enterica. In doing so, they uncover the mechanism of GlnZ biogenesis, namely RNase E-mediated release from the parental glnA mRNA. …
Reviewer #1 (Public Review):
3' UTR-derived sRNAs are increasingly recognized as post-transcriptional regulators of diverse bacterial processes. While initially assumed to be highly specific regulators with single targets, global RNA interactome approaches challenge this view by suggesting 3'-derived sRNAs can bind and regulate multiple target mRNAs, reminiscent of the regulons of intergenic sRNAs (PMID: 35388892). In Enterobacteriaceae, GlnZ is an sRNA derived from the 3' UTR of the glnA mRNA (PMID: 15718303) that encodes glutamine synthetase. However, the role and function of GlnZ have not previously been determined. In the present work, the authors set out to investigate GlnZ in E. coli and Salmonella enterica. In doing so, they uncover the mechanism of GlnZ biogenesis, namely RNase E-mediated release from the parental glnA mRNA. Additionally, they identify several GlnZ targets (involved in carbon/nitrogen metabolism), some of which are conserved between E.coli and Salmonella while others are species-specific. Downstream mutational characterization of sRNA variants and experiments with target reporter constructs allow them to map sRNA-target interaction sites at nucleotide resolution. Together, their findings further support the idea that 3'-derived sRNAs, too, can act as more global post-transcriptional regulators with multiple direct targets.
This is a thoroughly conducted study and both, important and timely. As the corresponding author points out in the cover letter, this is an instance of similar findings being simultaneously reported by more than one group (see preprint from Gisela Storz' lab: https://www.biorxiv.org/content/10.1101/2022.04.01.486790v1). The results of these two, independently conducted studies largely agree and complement one another.
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Reviewer #2 (Public Review):
Miyakoshi et al. investigated the function of the small RNA GlnZ in E. coli and Salmonella. GlnZ originates from the 3'UTR of the glnA mRNA that encodes glutamine synthetase (GS), the central enzyme of nitrogen assimilation in bacteria. It has been confirmed that the processing of glnA and hence GlnZ formation involves Hfq and RNaseE. The authors also reveal that GlnZ regulates the sucA gene encoding the E1o component of 2-oxoglutarate dehydrogenase (OGDH) by complementary base pairing. As 2-OG is part of the TCA cycle as well as a precursor of the GS/GOGAT cycle, GlnZ appears to function as a major element to control carbon flow from the TCA cycle to this nitrogen assimilation pathway. This is an astonishing finding as the glnA gene and many other aspects of nitrogen assimilation via GS are rather …
Reviewer #2 (Public Review):
Miyakoshi et al. investigated the function of the small RNA GlnZ in E. coli and Salmonella. GlnZ originates from the 3'UTR of the glnA mRNA that encodes glutamine synthetase (GS), the central enzyme of nitrogen assimilation in bacteria. It has been confirmed that the processing of glnA and hence GlnZ formation involves Hfq and RNaseE. The authors also reveal that GlnZ regulates the sucA gene encoding the E1o component of 2-oxoglutarate dehydrogenase (OGDH) by complementary base pairing. As 2-OG is part of the TCA cycle as well as a precursor of the GS/GOGAT cycle, GlnZ appears to function as a major element to control carbon flow from the TCA cycle to this nitrogen assimilation pathway. This is an astonishing finding as the glnA gene and many other aspects of nitrogen assimilation via GS are rather well-investigated. Obviously central regulators can still be discovered, even on mRNAs that have been investigated for decades.
The authors present a nice piece of molecular biology work which justifies the major conclusion that GlnZ is formed by processing of glnA and regulates the sucA gene post-transcriptionally. In general, the manuscript is well-written and the data are clearly presented. The figures are great and allow the reader to easily follow the descriptions. Nevertheless, some aspects referring to the actual control of metabolism could be improved. For instance, the authors claim to have proven that "GlnZ represses the expression of SucA TO REDIRECT the carbon flow from the TCA cycle to the nitrogen assimilation pathway". However, the manuscript does not contain data, e.g. of metabolite profiling using glnZ mutants, that really confirm this statement. Even though GlnZ has a significant effect on SucA abundance it is rather weak, especially in E. coli (Fig. 4B). Of course, this is not uncommon for sRNA-dependent expression control and there is no doubt about the importance of the here presented finding. The authors should either include data that indeed show any effect on 2-OG levels and/or metabolic flux through OGDH or at least temper their conclusion and say that their findings only indicate this.
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Reviewer #3 (Public Review):
3' UTRs of mRNAs in bacteria have emerged as a reservoir for trans-acting small RNAs (sRNAs) processed from the full-length transcript by endonucleolytic cleavage. Most sRNAs exert their activity through the formation of imperfect base-pairing interactions with cognate target transcripts, and typically either repress or stimulate translation of bound mRNAs. Best studied in enterobacterial species like E. coli and Salmonella, sRNAs oftentimes rely on the presence of an RNA chaperone, Hfq, which facilitates annealing of complementary RNAs.
In the manuscript, Miyakoshi and co-workers report on the enterobacterial sRNA GlnZ which is released from the 3'UTR of glnA mRNA through RNase E cleavage. Miyakoshi and co-workers demonstrate how GlnZ is induced under nitrogen-limiting conditions. Employing a conserved seed …
Reviewer #3 (Public Review):
3' UTRs of mRNAs in bacteria have emerged as a reservoir for trans-acting small RNAs (sRNAs) processed from the full-length transcript by endonucleolytic cleavage. Most sRNAs exert their activity through the formation of imperfect base-pairing interactions with cognate target transcripts, and typically either repress or stimulate translation of bound mRNAs. Best studied in enterobacterial species like E. coli and Salmonella, sRNAs oftentimes rely on the presence of an RNA chaperone, Hfq, which facilitates annealing of complementary RNAs.
In the manuscript, Miyakoshi and co-workers report on the enterobacterial sRNA GlnZ which is released from the 3'UTR of glnA mRNA through RNase E cleavage. Miyakoshi and co-workers demonstrate how GlnZ is induced under nitrogen-limiting conditions. Employing a conserved seed sequence, GlnZ post-transcriptionally regulates target mRNAs, including sucA and aceE mRNAs. When inhibiting RNase E-mediated processing (through mutation of recognition sites), SucA regulation is abrogated, suggesting that full-length glnA mRNA is inactive as a post-transcriptional regulator.
A characterization of GlnZ, mainly focusing on the E. coli K12 variant, has recently been published elsewhere (Walling et al., 2022, NAR), and it is important to highlight additional findings of this manuscript.
One strength of the manuscript is the comparison of GlnZ-mediated regulation between two different enterobacterial species, Salmonella and E. coli, however this aspect should be assessed more thoroughly. The authors have identified additional targets through a pulse-expression experiment of GlnZ in Salmonella, but the Salmonella-specific targets await validation.
The mechanism by which GlnZ represses its targets sucA and aceE mRNAs through binding far upstream of the ribosome binding site is interesting but not discussed.
The authors speculate on the role of translation regarding the question why GlnZ but not glnA mRNA are able to engage in target regulation. Given the variation in sequence among different enterobacteria it is an open question whether the distance between the translation stop and the sRNA seed influences the regulatory activity.
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