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Unique features of bacterial small regulatory RNAs in transcriptional networks and translational control – UnifyRNA

The role of small bacterial RNAs in regulatory networks

Unique Features Of Bacterial Small Regulatory RNAs In Transcriptional Networks And Translational Control

Connections between sRNAs and two-component systems in bacteria

Small regulatory RNAs (sRNAs) are now recognized as key regulators in bacteria and are involved in many biological processes. Previous work by many labs has unravelled fundamental aspects of sRNA biology, allowing for instance their identification, as well as that of many of their targets, and the elucidation of their detailed mode of action. Yet several aspects of their physiology remain to be understood. In particular, how sRNAs and the controls that they exert are integrated in larger regulatory networks appears as a crucial question, with the finding that strong connections exist between transcriptional regulatory network and post-transcriptional control by sRNAs. For instance, two-component systems (TCS) are extremely widespread transcriptional regulators in bacteria and synthesis of several of them is known to be post-transcriptionally controlled by sRNAs. However, TCS typically consist of a sensor kinase that phosphorylates a cognate response regulator in response to a specific stimulus, and how these controls by sRNAs affect the level of the phosphorylated form of the response regulator (i.e. the presumably active form) has rarely been addressed. This is really of primary interest given the vast number of TCS that exist in different bacterial genomes (>30 in E. coli for instance) and the fact that many of them are likely to be controlled by sRNAs. <br />The main objectives of the UnifyRNA project are thus (i) to characterize the role of sRNAs in transcriptional networks and the properties of mixed regulatory circuits and (ii) to elucidate non-canonical modes of regulation by sRNAs and TCS.

The main focus of this work is the study of the role of sRNA-mediated feedback loops in gene expression and the definition of biological regulons of sRNAs. Previous studies by several groups have shown that sRNAs commonly target genes encoding transcriptional regulators (TR), in many cases in a feedback-loop (FL) motif (i.e. the sRNA is also regulated by this TR). However, the outcome of such regulations has remained elusive so far. Focusing on the example of the FL that exists between the OmrA and OmrB sRNAs and the OmpR TR, we will study (i) the role of this FL in gene expression and (ii) the regulatory modules of OmrA and OmrB sRNAs, which include the genes indirectly regulated by OmrA/B via changes of the OmpR level. This will involve genomewide analyses of gene expression, combined with molecular genetics and biochemistry, including the analysis of the in vivo phosphorylation of response regulators.
We will also aim at elucidating the mechanisms by which sRNAs can regulate gene expression when pairing to mRNAs outside of the translation initiation region.

So far, we have characterized in details the feedback circuit existing between the EnvZ-OmpR TCS and the OmrA and OmrB sRNAs. In particular, we have shown that, most unexpectedly, transcription of OmrA and OmrB sRNAs was activated by both the phosphorylated and the non-phosphorylated form of the OmpR protein. This is the first time where the non-phosphorylated form of OmpR was reported to have a role in gene expression regulation, despite the fact that EnvZ-OmpR is a paradigm of the TCS in bacteria. Interestingly, OmrA/B decrease the expression of ompR-envZ, which, in agreement with the previously reported robustness of this TCS, does not affect the level of OmpR-P. Thus, OmrA/B have no effect on OmpR-targets such as ompC and ompF, the genes for the major porins in E. coli, that respond only to OmpR-P, but in contrast they can limit their own synthesis by changing the levels of unphosphorylated OmpR. These results are now published (Brosse et al., Nucleic Acids Research, 2016).

sRNAs and two-component systems are both widespread regulators of gene expression in bacteria, acting mostly at the post-transcriptional and the transcriptional level, respectively. Furthermore, two-component systems other than EnvZ-OmpR were reported to be subject to regulation by sRNAs, and it will thus be interesting in the future to determine whether our findings apply to those systems as well.

Publications:

Jagodnik J., Brosse A., Le Lam TN., Chiaruttini C. and Guillier M., 2016, Mechanistic study of base-pairing small regulatory RNAs in bacteria, Methods, in press, doi: 10.1016/j.ymeth.2016.09.012.

Brosse A., Korobeinikova A., Gottesman S. and Guillier M., 2016, Unexpected properties of sRNA promoters allow feedback control via regulation of a two-component system, Nucl. Acids Res., 44(20): 9650-9666.

Jagodnik J., Thieffry D. and Guillier M., 2016, Bacterial small RNAs in mixed regulatory circuits, book chapter in «Stress and Environmental Control of Gene Expression in Bacteria«, edited by Frans de Bruijn (publisher Wiley-Blackwell), in press.


Participation to meetings:

Sept. 2016 81st Harden Conference: RNA and disease, Winchester, UK. - Presenter: M. Guillier (Talk)

August 2016 Molecular Genetics of bacteria and phages meeting, Madison, Wisconsin, USA - Presenter: J. Jagodnik (Poster)

July 2016 Gordon Research Conference “Microbial Stress Response”, Mount Holyoke college, South Hadley, MA, USA. - Presenter: M. Guillier (Poster)

March 2016 10th SifrARN (Structure, Integration, Function and Reactivity of RNA molecules) meeting, Toulouse, France. - Presenters : A. Brosse (Poster), J. Jagodnik (Poster)

Dec. 2015 «Regulating with RNA in Bacteria and Archaea« conference, Cancun, Mexique. - Presenters: M. Guillier (Talk), J. Jagodnik (Poster).

Oct. 2015 EMBL Symposium «New approaches and concepts in microbiology«, EMBL, Heidelberg, Germany. - Presenter: M. Guillier (Poster).

Bacteria are extremely versatile organisms that respond and adapt to a wide variety of environmental changes through their amazing ability to extensively regulate gene expression. The first level of control that has been described is transcription and to date, even though some gaps persist because of its huge complexity, the transcriptional regulatory network of model bacteria such as E. coli has been thoroughly characterized. Two-component systems (TCS), that allow sensing and adaptation to the environment through activation by phosphorylation of a regulator, are among the most widespread transcriptional regulators in bacteria.

However, post-transcriptional control is known to affect gene expression as well. Its importance has been early recognized in bacteria because genes subject to translational control encode proteins whose amount may represent a large portion of the total cellular mass (e.g. genes for ribosomal proteins). Furthermore, the identification and characterization of small RNAs (sRNAs) as widespread post-transcriptional regulators in bacteria has brought a renewal of interest to the field.

sRNAs have been found in many bacteria and are involved in fundamental biological processes, such as quorum-sensing or cell enveloppe homeostasis. A large family of those regulatory molecules act by pairing to target-mRNAs via imperfect and short duplexes, which allows a single sRNA to directly regulate multiple genes. This pairing ultimately leads to changes in translation and/or stability of the target-mRNA according to extremely diverse molecular mechanisms.

Importantly, it has recently become obvious that post-transcriptional control exerted by sRNAs and transcriptional control are intimately connected. Indeed, not only is the synthesis of sRNAs extensively regulated at the transcriptional level, but several of them were also shown to directly regulate the expression of transcriptional regulators (TR), forming a feedback loop in many cases. As a result, biological functions as important as motility or group behaviour are controlled by mixed regulatory circuits, whose properties remain mostly uncharacterized.

The UnifyRNA project will therefore aim at unraveling the role of sRNAs in these mixed circuits by focusing on two examples of choice that we have identified in E. coli. The first one is the feedback loop linking the two OmrA/OmrB apparently redundant sRNAs and the EnvZ-OmpR TCS, different regulators that control cell-surface related processes such as outer membrane composition, adhesion or motility. The other example is the control of the PhoQ-PhoP TCS, involved in magnesium homeostasis and bacterial virulence, by multiple sRNAs such as MicA and GcvB, a system for which we have evidence of surprising complexity. The expected results will not only be of fundamental interest given the central role of PhoQ-PhoP and EnvZ-OmpR in bacterial physiology, but they will also allow to apprehend the functions, the specific properties and the biological roles of mixed regulatory circuits of E. coli and how they integrate into larger global networks. This is especially important given the fact that both TCS and sRNAs are widespread regulators in bacteria and that other examples of sRNAs regulating TCS have been reported. At last, results obtained during these studies should provide important clues in order to apprehend the complete set of targets of OmrA/B sRNAs and, as a result, their biological role.
Using a multidisciplinary approach combining transcriptomics, proteomics, molecular genetics, single cell microscopy and systems biology, the UnifyRNA project will provide an important advance in our understanding of the biology of small regulatory RNAs in bacteria.

Project coordination

Maude Guillier (EXPRESSION GENETIQUE MICROBIENNE)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CNRS FRE 3630 EXPRESSION GENETIQUE MICROBIENNE

Help of the ANR 260,000 euros
Beginning and duration of the scientific project: January 2015 - 36 Months

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