Clp protease and antisense RNA jointly regulate the global regulator CarD to mediate mycobacterial starvation response

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    Evaluation Summary:

    CarD is an RNA polymerase interacting protein that is essential for mycobacterial viability, the levels of which are important for controlling gene expression in mycobacteria during various stress conditions. This study reports two mechanisms that regulate levels of CarD under stress conditions, including starvation. The authors report that CarD levels are tightly regulated and that there was a dramatic decrease in the levels of CarD when cells switched from the nutrient-rich to the starvation condition. They discovered two synergistic mechanisms that led to this dramatic decrease in CarD. The first is SigF-dependent induction of antisense RNA of CarD (AscarD), which inhibits CarD translation and a second mechanism involving Clp protease-mediated degradation of intracellular CarD. The work will be of interest to researchers studying non-coding RNAs, microbial gene expression, physiology and stress response.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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Abstract

Under starvation conditions, bacteria tend to slow down their translation rate by reducing rRNA synthesis, but the way they accomplish that may vary in different bacteria. In Mycobacterium species, transcription of rRNA is activated by the RNA polymerase (RNAP) accessory transcription factor CarD, which interacts directly with RNAP to stabilize the RNAP-promoter open complex formed on rRNA genes. The functions of CarD have been extensively studied, but the mechanisms that control its expression remain obscure. Here, we report that the level of CarD was tightly regulated when mycobacterial cells switched from nutrient-rich to nutrient-deprived conditions. At the translational level, an antisense RNA of carD (AscarD) was induced in a SigF-dependent manner to bind with carD mRNA and inhibit CarD translation, while at the post-translational level, the residual intracellular CarD was quickly degraded by the Clp protease. AscarD thus worked synergistically with Clp protease to decrease the CarD level to help mycobacterial cells cope with the nutritional stress. Altogether, our work elucidates the regulation mode of CarD and delineates a new mechanism for the mycobacterial starvation response, which is important for the adaptation and persistence of mycobacterial pathogens in the host environment.

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  1. Evaluation Summary:

    CarD is an RNA polymerase interacting protein that is essential for mycobacterial viability, the levels of which are important for controlling gene expression in mycobacteria during various stress conditions. This study reports two mechanisms that regulate levels of CarD under stress conditions, including starvation. The authors report that CarD levels are tightly regulated and that there was a dramatic decrease in the levels of CarD when cells switched from the nutrient-rich to the starvation condition. They discovered two synergistic mechanisms that led to this dramatic decrease in CarD. The first is SigF-dependent induction of antisense RNA of CarD (AscarD), which inhibits CarD translation and a second mechanism involving Clp protease-mediated degradation of intracellular CarD. The work will be of interest to …

  2. Reviewer #1 (Public Review):

    Manuscript by Li et al. entitled "Clp protease and antisense RNA jointly regulate the global regulator CarD to mediate mycobacterial starvation response" reports two mechanisms that regulate levels of CarD under stress conditions, including starvation. They report that level of CarD was tightly regulated, and there was a dramatic decrease in the levels of CarD when cells switched from the nutrient-rich to the starvation condition. They discovered two synergistic mechanisms that led to this dramatic decrease in the levels of CarD: 1) SigF-dependent induction of antisense RNA of CarD (AscarD), which inhibits CarD translation, and 2) Clp protease-mediated degradation of intracellular CarD.

    CarD is an essential global transcription regulator which activates transcription and modulates the expression of about …

  3. Reviewer #2 (Public Review):

    Our understanding of bacterial mechanisms of transcriptional regulation has been pushed forward by studies in a few model organisms that were initially amenable to biochemical experiments. More recently, however, it has become clear that many regulatory pathways are distinct from these model organisms. This fact, coupled with continued appearance of drug-resistant strains of pathogenic bacteria motivates the serious study of important pathways directly in the pathogenic bacteria themselves.

    The mycobacterial transcription factor CarD has been studied via many approaches and the biophysical mechanisms underlying its effect on transcription initiation are well-described. Curiously though, the biological role of CarD is far less well-understood. The authors have approached this question by uncovering two modes …

  4. Reviewer #3 (Public Review):

    This is a study of the regulation of CarD expression by proteolysis and anti sense RNA. CarD is a well-studied RNAP interacting protein with complex roles on RNAP promoter dynamics, as the authors note, but the full mechanisms controlling its cellular levels under various stress conditions are not well defined. One prior report indicated that CarD is subject to proteolysis by the ClpP system. This study demonstrates that CarD protein levels are subject to ClpP proteolysis during stationary phase and that conditional expression of ClpP2 reverses this effect. The authors also identify an antisense RNA at the CarD locus that also negatively regulates CarD protein levels and overexpression of this antisense RNA confers sensitivity to various stresses, phenocopying prior data with CarD depletion.

    Overall, the …