ABSTRACT The success of Mycobacterium tuberculosis as a human pathogen is due in part to its ability to survive stress conditions, such as hypoxia or nutrient deprivation, by entering nongrowing states. In these low-metabolism states, M. tuberculosis can tolerate antibiotics and develop genetically encoded antibiotic resistance, making its metabolic adaptation to stress crucial for survival. Numerous bacteria, including M. tuberculosis , have been shown to reduce their rates of mRNA degradation under growth limitation and stress. While the existence of this response appears to be conserved across species, the underlying bacterial mRNA stabilization mechanisms remain unknown. To better understand the biology of nongrowing mycobacteria, we sought to identify the mechanistic basis of mRNA stabilization in the nonpathogenic model Mycobacterium smegmatis . We found that mRNA half-life was responsive to energy stress, with carbon starvation and hypoxia causing global mRNA stabilization. This global stabilization was rapidly reversed when hypoxia-adapted cultures were reexposed to oxygen, even in the absence of new transcription. The stringent response and RNase levels did not explain mRNA stabilization, nor did transcript abundance. This led us to hypothesize that metabolic changes during growth cessation impact the activities of degradation proteins, increasing mRNA stability. Indeed, bedaquiline and isoniazid, two drugs with opposing effects on cellular energy status, had opposite effects on mRNA half-lives in growth-arrested cells. Taken together, our results indicate that mRNA stability in mycobacteria is not directly regulated by growth status but rather is dependent on the status of energy metabolism. IMPORTANCE The logistics of tuberculosis therapy are difficult, requiring multiple drugs for many months. Mycobacterium tuberculosis survives in part by entering nongrowing states in which it is metabolically less active and thus less susceptible to antibiotics. Basic knowledge on how M. tuberculosis survives during these low-metabolism states is incomplete, and we hypothesize that optimized energy resource management is important. Here, we report that slowed mRNA turnover is a common feature of mycobacteria under energy stress but is not dependent on the mechanisms that have generally been postulated in the literature. Finally, we found that mRNA stability and growth status can be decoupled by a drug that causes growth arrest but increases metabolic activity, indicating that mRNA stability responds to metabolic status rather than to growth rate per se . Our findings suggest a need to reorient studies of global mRNA stabilization to identify novel mechanisms that are presumably responsible.
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The Impact of Leadered and Leaderless Gene Structures on Translation Efficiency, Transcript Stability, and Predicted Transcription Rates in Mycobacterium smegmatis
ABSTRACT Regulation of gene expression is critical for Mycobacterium tuberculosis to tolerate stressors encountered during infection and for nonpathogenic mycobacteria such as Mycobacterium smegmatis to survive environmental stressors. Unlike better-studied models, mycobacteria express ∼14% of their genes as leaderless transcripts. However, the impacts of leaderless transcript structures on mRNA half-life and translation efficiency in mycobacteria have not been directly tested. For leadered transcripts, the contributions of 5′ untranslated regions (UTRs) to mRNA half-life and translation efficiency are similarly unknown. In M. tuberculosis and M. smegmatis , the essential sigma factor, SigA, is encoded by a transcript with a relatively short half-life. We hypothesized that the long 5′ UTR of sigA causes this instability. To test this, we constructed fluorescence reporters and measured protein abundance, mRNA abundance, and mRNA half-life and calculated relative transcript production rates. The sigA 5′ UTR conferred an increased transcript production rate, shorter mRNA half-life, and decreased apparent translation rate compared to a synthetic 5′ UTR commonly used in mycobacterial expression plasmids. Leaderless transcripts appeared to be translated with similar efficiency as those with the sigA 5′ UTR but had lower predicted transcript production rates. A global comparison of M. tuberculosis mRNA and protein abundances failed to reveal systematic differences in protein/mRNA ratios for leadered and leaderless transcripts, suggesting that variability in translation efficiency is largely driven by factors other than leader status. Our data are also discussed in light of an alternative model that leads to different conclusions and suggests leaderless transcripts may indeed be translated less efficiently. IMPORTANCE Tuberculosis, caused by Mycobacterium tuberculosis , is a major public health problem killing 1.5 million people globally each year. During infection, M. tuberculosis must alter its gene expression patterns to adapt to the stress conditions it encounters. Understanding how M. tuberculosis regulates gene expression may provide clues for ways to interfere with the bacterium’s survival. Gene expression encompasses transcription, mRNA degradation, and translation. Here, we used Mycobacterium smegmatis as a model organism to study how 5′ untranslated regions affect these three facets of gene expression in multiple ways. We furthermore provide insight into the expression of leaderless mRNAs, which lack 5′ untranslated regions and are unusually prevalent in mycobacteria.
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- Award ID(s):
- 1652756
- NSF-PAR ID:
- 10203537
- Editor(s):
- Henkin, Tina M.
- Date Published:
- Journal Name:
- Journal of Bacteriology
- Volume:
- 202
- Issue:
- 9
- ISSN:
- 0021-9193
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Background RNA secondary structure (RSS) can influence the regulation of transcription, RNA processing, and protein synthesis, among other processes. 3′ untranslated regions (3′ UTRs) of mRNA also hold the key for many aspects of gene regulation. However, there are often contradictory results regarding the roles of RSS in 3′ UTRs in gene expression in different organisms and/or contexts.
Results Here, we incidentally observe that the primary substrate of miR159a (pri-miR159a), when embedded in a 3′ UTR, could promote mRNA accumulation. The enhanced expression is attributed to the earlier polyadenylation of the transcript within the hybrid pri-miR159a-3′ UTR and, resultantly, a poorly structured 3′ UTR. RNA decay assays indicate that poorly structured 3′ UTRs could promote mRNA stability, whereas highly structured 3′ UTRs destabilize mRNA in vivo. Genome-wide DMS-MaPseq also reveals the prevailing inverse relationship between 3′ UTRs’ RSS and transcript accumulation in the transcriptomes of
Arabidopsis , rice, and even human. Mechanistically, transcripts with highly structured 3′ UTRs are preferentially degraded by 3′–5′ exoribonuclease SOV and 5′–3′ exoribonuclease XRN4, leading to decreased expression inArabidopsis . Finally, we engineer different structured 3′ UTRs to an endogenousFT gene and alter theFT -regulated flowering time inArabidopsis .Conclusions We conclude that highly structured 3′ UTRs typically cause reduced accumulation of the harbored transcripts in
Arabidopsis . This pattern extends to rice and even mammals. Furthermore, our study provides a new strategy of engineering the 3′ UTRs’ RSS to modify plant traits in agricultural production and mRNA stability in biotechnology. -
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Abstract Background The eukaryotic genome is capable of producing multiple isoforms from a gene by alternative polyadenylation (APA) during pre-mRNA processing. APA in the 3′-untranslated region (3′-UTR) of mRNA produces transcripts with shorter or longer 3′-UTR. Often, 3′-UTR serves as a binding platform for microRNAs and RNA-binding proteins, which affect the fate of the mRNA transcript. Thus, 3′-UTR APA is known to modulate translation and provides a mean to regulate gene expression at the post-transcriptional level. Current bioinformatics pipelines have limited capability in profiling 3′-UTR APA events due to incomplete annotations and a low-resolution analyzing power: widely available bioinformatics pipelines do not reference actionable polyadenylation (cleavage) sites but simulate 3′-UTR APA only using RNA-seq read coverage, causing false positive identifications. To overcome these limitations, we developed APA-Scan, a robust program that identifies 3′-UTR APA events and visualizes the RNA-seq short-read coverage with gene annotations.
Methods APA-Scan utilizes either predicted or experimentally validated actionable polyadenylation signals as a reference for polyadenylation sites and calculates the quantity of long and short 3′-UTR transcripts in the RNA-seq data. APA-Scan works in three major steps: (i) calculate the read coverage of the 3′-UTR regions of genes; (ii) identify the potential APA sites and evaluate the significance of the events among two biological conditions; (iii) graphical representation of user specific event with 3′-UTR annotation and read coverage on the 3′-UTR regions. APA-Scan is implemented in Python3. Source code and a comprehensive user’s manual are freely available at
https://github.com/compbiolabucf/APA-Scan .Result APA-Scan was applied to both simulated and real RNA-seq datasets and compared with two widely used baselines DaPars and APAtrap. In simulation APA-Scan significantly improved the accuracy of 3′-UTR APA identification compared to the other baselines. The performance of APA-Scan was also validated by 3′-end-seq data and qPCR on mouse embryonic fibroblast cells. The experiments confirm that APA-Scan can detect unannotated 3′-UTR APA events and improve genome annotation.
Conclusion APA-Scan is a comprehensive computational pipeline to detect transcriptome-wide 3′-UTR APA events. The pipeline integrates both RNA-seq and 3′-end-seq data information and can efficiently identify the significant events with a high-resolution short reads coverage plots.
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Singh, Yogendra (Ed.)ABSTRACT Dynamical properties of gene regulatory networks are tuned to ensure bacterial survival. In mycobacteria, the MprAB-σ E network responds to the presence of stressors, such as surfactants that cause surface stress. Positive feedback loops in this network were previously predicted to cause hysteresis, i.e., different responses to identical stressor levels for prestressed and unstressed cells. Here, we show that hysteresis does not occur in nonpathogenic Mycobacterium smegmatis but does occur in Mycobacterium tuberculosis . However, the observed rapid temporal response in M. tuberculosis is inconsistent with the model predictions. To reconcile these observations, we implement a recently proposed mechanism for stress sensing, namely, the release of MprB from the inhibitory complex with the chaperone DnaK upon the stress exposure. Using modeling and parameter fitting, we demonstrate that this mechanism can accurately describe the experimental observations. Furthermore, we predict perturbations in DnaK expression that can strongly affect dynamical properties. Experiments with these perturbations agree with model predictions, confirming the role of DnaK in fast and sustained response. IMPORTANCE Gene regulatory networks controlling stress response in mycobacterial species have been linked to persistence switches that enable bacterial dormancy within a host. However, the mechanistic basis of switching and stress sensing is not fully understood. In this paper, combining quantitative experiments and mathematical modeling, we uncover how interactions between two master regulators of stress response—the MprAB two-component system (TCS) and the alternative sigma factor σ E —shape the dynamical properties of the surface stress network. The result show hysteresis (history dependence) in the response of the pathogenic bacterium M. tuberculosis to surface stress and lack of hysteresis in nonpathogenic M. smegmatis . Furthermore, to resolve the apparent contradiction between the existence of hysteresis and fast activation of the response, we utilize a recently proposed role of chaperone DnaK in stress sensing. These result leads to a novel system-level understanding of bacterial stress response dynamics.more » « less
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Simon, Anne E. (Ed.)ABSTRACT Regardless of the general model of translation in eukaryotic cells, a number of studies suggested that many mRNAs encode multiple proteins. Leaky scanning, which supplies ribosomes to downstream open reading frames (ORFs) by readthrough of upstream ORFs, has great potential to translate polycistronic mRNAs. However, the mRNA elements controlling leaky scanning and their biological relevance have rarely been elucidated, with exceptions such as the Kozak sequence. Here, we have analyzed the strategy of a plant RNA virus to translate three movement proteins from a single RNA molecule through leaky scanning. The in planta and in vitro results indicate thatthe significantly shorter 5′ untranslated region (UTR) of the most upstream ORF promotes leaky scanning, potentially fine-tuning the translation efficiency of the three proteins in a single RNA molecule to optimize viral propagation. Our results suggest that the remarkably short length of the leader sequence, like the Kozak sequence, is a translational regulatory element with a biologically important role, as previous studies have shown biochemically. IMPORTANCE Potexvirus , a group of plant viruses, infect a variety of crops, including cultivated crops. It has been thought that the three transition proteins that are essential for the cell-to-cell transfer of potexviruses are translated from two subgenomic RNAs, sgRNA1 and sgRNA2. However, sgRNA2 has not been clearly detected. In this study, we have shown that sgRNA1, but not sgRNA2, is the major translation template for the three movement proteins. In addition, we determined the transcription start site of sgRNA1 in flexiviruses and found that the efficiency of leaky scanning caused by the short 5′ UTR of sgRNA1, a widely conserved feature, regulates the translation of the three movement proteins. When we tested the infection of viruses with mutations introduced into the length of the 5′ UTR, we found that the movement efficiency of the virus was affected. Our results provide important additional information on the protein translation strategy of flexiviruses, including Potexvirus , and provide a basis for research on their control as well as the need to reevaluate the short 5′ UTR as a translational regulatory element with an important role in vivo .more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1128/JB.00746-19
