scholarly journals mRNA Degradation Rates Are Coupled to Metabolic Status in Mycobacteria

2019 ◽  
Author(s):  
Diego A. Vargas-Blanco ◽  
Ying Zhou ◽  
Luis Gutierrez Zamalloa ◽  
Tim Antonelli ◽  
Scarlet S. Shell
Keyword(s):  
Mrna Stability ◽  
Mrna Degradation ◽  
Metabolic Status ◽  
Basic Knowledge ◽  
Metabolic Adaptation ◽  
Nutrient Deprivation ◽  
Mrna Stabilization ◽  
Growth Status ◽  
Metabolic States ◽  
Multiple Drugs

ABSTRACTThe success ofMycobacterium tuberculosis(Mtb) as a human pathogen is due in part to its ability to survive stress conditions, such as hypoxia or nutrient deprivation, by entering non-growing states. In these low-metabolic states, Mtb can tolerate antibiotics and develop genetically encoded antibiotic resistance, making its metabolic adaptation to stress crucial for survival. Numerous bacteria, including Mtb, 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 remains unknown. To better understand the biology of non-growing mycobacteria, we sought to identify the mechanisms by which mRNA stabilization occurs using the non-pathogenic modelMycobacterium smegmatis. We found that mRNA half-life was responsive to energy stress, with carbon starvation and hypoxia causing global mRNA stabilization. This global mRNA stabilization was rapidly reversed when hypoxia-adapted cultures were re-exposed to oxygen, even in the absence of new transcription. The stringent response and RNase protein levels did not explain mRNA stabilization, nor did transcript abundance. This led us to hypothesize that metabolic changes during growth cessation impact the activity 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 seems to be dependent on the status of energy metabolism.IMPORTANCEThe logistics of treating tuberculosis are difficult, requiring multiple drugs for at least six months. Mtb is able to survive within the human host in part by entering non-growing states in which it is metabolically less active, thus rendering it less susceptible to antibiotics. Basic knowledge on how Mtb survives during these low-metabolic states is incomplete, and we postulate that optimized energy resource management –such as transcriptome stabilization—is important for survival. Here we report that mRNA stabilization (increased mRNA half-lives) is a common feature of mycobacteria under stress (e.g. hypoxia and nutrient deprivation) but is not dependent on the mechanisms that have been most often 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 changes per se. Our findings suggest a need to re-orient the study of global mRNA stabilization to identify novel mechanisms that are presumably responsible.

scholarly journals mRNA Degradation Rates Are Coupled to Metabolic Status inMycobacterium smegmatis

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Diego A. Vargas-Blanco ◽  
Ying Zhou ◽  
L. Gregory Zamalloa ◽  
Tim Antonelli ◽  
Scarlet S. Shell
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mrna Stability ◽  
Mrna Degradation ◽  
Metabolic Status ◽  
Metabolic Adaptation ◽  
Content Type ◽  
Mrna Stabilization ◽  
Growth Status ◽  
Energy Stress ◽  
Multiple Drugs

ABSTRACTThe success ofMycobacterium tuberculosisas 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. tuberculosiscan tolerate antibiotics and develop genetically encoded antibiotic resistance, making its metabolic adaptation to stress crucial for survival. Numerous bacteria, includingM. 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 modelMycobacterium 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.IMPORTANCEThe logistics of tuberculosis therapy are difficult, requiring multiple drugs for many months.Mycobacterium tuberculosissurvives in part by entering nongrowing states in which it is metabolically less active and thus less susceptible to antibiotics. Basic knowledge on howM. tuberculosissurvives 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 rateper se. Our findings suggest a need to reorient studies of global mRNA stabilization to identify novel mechanisms that are presumably responsible.

scholarly journals P38 activation induces the dissociation of tristetraprolin from Argonaute 2 to increase ARE-mRNA stabilization

2018 ◽  
Vol 29 (8) ◽  
pp. 988-1002 ◽  
Author(s):  
Mei-Yan Qi ◽  
Jing-Wen Song ◽  
Zhuo Zhang ◽  
Shuang Huang ◽  
Qing Jing
Keyword(s):  
Mrna Stability ◽  
Mrna Decay ◽  
Mrna Degradation ◽  
Processing Bodies ◽  
Mrna Stabilization ◽  
Argonaute 2 ◽  
Core Member ◽  
Mrna Granules

Tristetraprolin (TTP) destabilizes AU-rich element (ARE)-containing mRNA by directly binding with their 3′UTR. P38 stimulation substantially increases ARE-mRNA stability, at least through repressing TTP. However, the mechanism by which P38 keeps TTP inactive has not been fully understood. TTP and ARE-mRNA localize to processing bodies (PBs), the mRNA granules associated with mRNA silencing. Here, we detected the influence of P38 on TTP localization within PBs and found that P38 regulates TTP localization within PBs. Through luciferase-based systems, we demonstrated that PBs depletion significantly increased ARE-mRNA stability inhibited by TTP. Additionally, we provided evidence that the microRNA-induced silencing complex (miRISC) core member Ago2 is required for TTP distribution within PBs. Importantly, the cooperation of TTP and Ago2 is a prerequisite for effective ARE-mRNA degradation. Moreover, Dcp1a and Dcp2 act downstream of Ago2 and TTP engaging in ARE-mRNA decay. Finally, we demonstrated that P38 activation represses the interaction between TTP and Ago2 due to TTP phosphorylation, which impairs TTP localization within PBs and ARE-mRNA degradation. Collectively, our study revealed a novel mechanism through which P38 activation repressed the cooperation of TTP with Ago2, thus ensuring that ARE-mRNA does not associate with PBs and remains stable.

scholarly journals Tetracycline Induces Stabilization of mRNA in Bacillus subtilis

2002 ◽  
Vol 184 (4) ◽  
pp. 889-894 ◽  
Author(s):  
Yi Wei ◽  
David H. Bechhofer
Keyword(s):  
Bacillus Subtilis ◽  
Mrna Stability ◽  
The Other ◽  
Leader Region ◽  
Subinhibitory Concentration ◽  
Coding Sequence ◽  
Mrna Stabilization ◽  
Threefold Increase ◽  
The Stability

ABSTRACT The tet(L) gene of Bacillus subtilis confers low-level tetracycline (Tc) resistance. Previous work examining the >20-fold-inducible expression of tet(L) by Tc demonstrated a 12-fold translational induction. Here we show that the other component of tet(L) induction is at the level of mRNA stabilization. Addition of a subinhibitory concentration of Tc results in a two- to threefold increase in tet(L) mRNA stability. Using a plasmid-borne derivative of tet(L) with a large in-frame deletion of the coding sequence, the mechanism of Tc-induced stability was explored by measuring the decay of tet(L) mRNAs carrying specific mutations in the leader region. The results of these experiments, as well as experiments with a B. subtilis strain that is resistant to Tc due to a mutation in the ribosomal S10 protein, suggest different mechanisms for the effects of Tc on translation and on mRNA stability. The key role of the 5" end in determining mRNA stability was confirmed in these experiments. Surprisingly, the stability of several other B. subtilis mRNAs was also induced by Tc, which indicates that addition of Tc may result in a general stabilization of mRNA.

scholarly journals Distinct PKC-mediated posttranscriptional events set cytokine production kinetics in CD8+ T cells

2017 ◽  
Vol 114 (36) ◽  
pp. 9677-9682 ◽  
Author(s):  
Fiamma Salerno ◽  
Nahuel A. Paolini ◽  
Regina Stark ◽  
Marieke von Lindern ◽  
Monika C. Wolkers
Keyword(s):  
T Cells ◽  
Mrna Stability ◽  
Cytokine Production ◽  
De Novo ◽  
Translation Efficiency ◽  
Mrna Stabilization ◽  
Relative Contribution ◽  
Production Kinetics ◽  
Tnf Α ◽  
Ifn Γ

Effective T cell responses against invading pathogens require the concerted production of three key cytokines: TNF-α, IFN-γ, and IL-2. The cytokines functionally synergize, but their production kinetics widely differ. How the differential timing of expression is regulated remains, however, poorly understood. We compared the relative contribution of transcription, mRNA stability, and translation efficiency on cytokine production in murine effector and memory CD8+ T cells. We show that the immediate and ample production of TNF-α is primarily mediated by translation of preformed mRNA through protein kinase C (PKC)-induced recruitment of mRNA to polyribosomes. Also, the initial production of IFN-γ uses translation of preformed mRNA. However, the magnitude and subsequent expression of IFN-γ, and of IL-2, depends on calcium-induced de novo transcription and PKC-dependent mRNA stabilization. In conclusion, PKC signaling modulates translation efficiency and mRNA stability in a transcript-specific manner. These cytokine-specific regulatory mechanisms guarantee that T cells produce ample amounts of cytokines shortly upon activation and for a limited time.

scholarly journals Cx32 mRNA in rat liver: effects of inflammation on poly(A) tail distribution and mRNA degradation

1999 ◽  
Vol 276 (5) ◽  
pp. R1249-R1257 ◽  
Author(s):  
Nicholas G. Theodorakis ◽  
Antonio de Maio
Keyword(s):  
Inflammatory Response ◽  
Mrna Stability ◽  
Actinomycin D ◽  
Mrna Degradation ◽  
Connexin 32 ◽  
Detectable Change ◽  
Final Size ◽  
Tail Distribution ◽  
The Stability ◽  
Lps Treatment

Previous studies showed that the expression of connexin 32 (Cx32), the polypeptide subunit component of the major hepatic gap junction, is reduced in liver by changes in mRNA stability during bacterial lipopolysaccharide (LPS)-induced inflammation. In this study, we examined the distribution of Cx32 mRNA poly(A) tail lengths during LPS-induced inflammation, because this is considered the first step in the degradation of many mRNAs. During LPS treatment the first detectable change in Cx32 mRNA was a gradual shortening of its poly(A) tail, which reached a final size of ∼20 nucleotides. However, the poly(A) tail did not disappear entirely before the bulk of Cx32 mRNA was degraded. Treatment with actinomycin D, which blocks the degradation of Cx32 mRNA after LPS administration, resulted in the appearance of a completely deadenylated mRNA, which otherwise could not be detected. On the contrary, treatment with cycloheximide resulted in a decrease in the stability of Cx32 mRNA without an apparent change of the poly(A) tail size. The effect of cycloheximide on Cx32 mRNA stability seems to be due indirectly to the induction of an inflammatory response by this drug. These results suggest that, similar for many mRNAs, shortening of the poly(A) tail is one of the first steps in the degradation of Cx32 mRNA during inflammation.

scholarly journals mRNA Degradation Plays a Significant Role in the Program of Gene Expression Regulated by Phosphatidylinositol 3-Kinase Signaling

2010 ◽  
Vol 30 (22) ◽  
pp. 5295-5305 ◽  
Author(s):  
Julie R. Graham ◽  
Melissa C. Hendershott ◽  
Jolyon Terragni ◽  
Geoffrey M. Cooper
Keyword(s):  
Gene Expression ◽  
Mrna Stability ◽  
Actinomycin D ◽  
Mrna Degradation ◽  
Akt Pathway ◽  
Kinase Signaling ◽  
Phosphatidylinositol 3 Kinase ◽  
Control Of Gene Expression ◽  
Steady State Levels ◽  
Interfering Rna

ABSTRACT Control of gene expression by the phosphatidylinositol (PI) 3-kinase/Akt pathway plays an important role in mammalian cell proliferation and survival, and numerous transcription factors and genes regulated by PI 3-kinase signaling have been identified. Because steady-state levels of mRNA are regulated by degradation as well as transcription, we have investigated the importance of mRNA degradation in controlling gene expression downstream of PI 3-kinase. We previously performed global expression analyses that identified a set of approximately 50 genes that were downregulated following inhibition of PI 3-kinase in proliferating T98G cells. By blocking transcription with actinomycin D, we found that almost 40% of these genes were regulated via effects of PI 3-kinase on mRNA stability. Analyses of β-globin-3′ untranslated region (UTR) fusion transcripts indicated that sequences within 3′ UTRs were the primary determinants of rapid mRNA decay. Small interfering RNA (siRNA) experiments further showed that knockdown of BRF1 or KSRP, both ARE binding proteins (ARE-BPs) regulated by Akt, stabilized the mRNAs of a majority of the downregulated genes but that knockdown of ARE-BPs that are not regulated by PI 3-kinase did not affect degradation of these mRNAs. These results show that PI 3-kinase regulation of mRNA stability, predominantly mediated by BRF1, plays a major role in regulating gene expression.

scholarly journals Convergent Actions of IκB Kinase β and Protein Kinase Cδ Modulate mRNA Stability through Phosphorylation of 14-3-3β Complexed with Tristetraprolin

2005 ◽  
Vol 25 (15) ◽  
pp. 6454-6463 ◽  
Author(s):  
Sonja I. Gringhuis ◽  
Juan Jesús García-Vallejo ◽  
Bert van het Hof ◽  
Willem van Dijk
Keyword(s):  
Protein Kinase ◽  
Mrna Stability ◽  
Umbilical Vein ◽  
Regulation Of Gene Expression ◽  
Necrosis Factor Alpha ◽  
Mrna Stabilization ◽  
Iκb Kinase ◽  
Umbilical Vein Endothelial Cells ◽  
And Function ◽  
Protein Kinase Cδ

ABSTRACT Regulation of gene expression at the level of mRNA stability is a major topic of research; however, knowledge about the regulatory mechanisms affecting the binding and function of AU-rich element (ARE)-binding proteins (AUBPs) in response to extracellular signals is minimal. The β1,4-galactosyltransferase 1 (β4GalT1) gene enabled us to study the mechanisms involved in binding of tristetraprolin (TTP) as the stability of its mRNA is regulated solely through one ARE bound by TTP in resting human umbilical vein endothelial cells. Here, we provide evidence that the complex formation of TTP with 14-3-3β is required to bind β4GalT1 mRNA and promote its decay. Furthermore, upon tumor necrosis factor alpha stimulation, the activation of both Iκβ kinase and protein kinase Cδ is involved in the phosphorylation of 14-3-3β on two serine residues, paralleled by release of binding of TTP and 14-3-3β from β4GalT1 mRNA, nuclear sequestration of TTP, and β4GalT1 mRNA stabilization. Thus, a key mechanism regulating mRNA binding and function of the destabilizing AUBP TTP involves the phosphorylation status of 14-3-3β.

scholarly journals Hormonal regulation of stability of glutamine synthetase mRNA in cultured 3T3-L1 adipocytes

1990 ◽  
Vol 267 (1) ◽  
pp. 241-244 ◽  
Author(s):  
K Saini ◽  
P Thomas ◽  
B Bhandari
Keyword(s):  
Gene Expression ◽  
Glutamine Synthetase ◽  
Gene Transcription ◽  
Mrna Stability ◽  
Half Life ◽  
Hormonal Regulation ◽  
Molecular Mechanisms ◽  
Mrna Degradation ◽  
Dibutyryl Cyclic Amp ◽  
Mrna Content

In 3T3-L1 adipocytes, glutamine synthetase (GS; EC 6.3.1.2) is subject to regulation by dexamethasone, insulin and dibutyryl cyclic AMP (Bt2cAMP). Dexamethasone increases GS-mRNA content and GS-gene transcription, whereas insulin and Bt2cAMP prevent these increases. The effects of these modulators on the control of GS-mRNA stability were investigated. We report here that GS mRNA has a half-life of about 110 min. Bt2cAMP increases GS-mRNA degradation by greater than 2-fold (half-life 50 min), whereas insulin or dexamethasone have little effect on GS-mRNA stability. Down-regulation of GS-gene expression by Bt2cAMP will involve a co-ordinate response at the level of gene transcription and mRNA stability. However, the molecular mechanisms by which insulin and dexamethasone regulate GS-gene expression in cultured adipocytes remains to be elucidated.

scholarly journals Roles for interleukin-1β, phorbol ester and a post-transcriptional regulator in the control of bradykinin B1 receptor gene expression

1998 ◽  
Vol 330 (1) ◽  
pp. 361-366 ◽  
Author(s):  
Xiaofeng ZHOU ◽  
Peter POLGAR ◽  
Linda TAYLOR
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Mrna Level ◽  
Mrna Degradation ◽  
Mrna Stabilization ◽  
B1 Receptor ◽  
Kinase C ◽  
Bradykinin B1 Receptor

Bradykinin B1 receptor (BKB1R) is involved in a variety of pathophysiological processes, particularly those related to inflammation. The gene for this receptor is known to be upregulated by interleukin (IL)-1β, a proinflammatory cytokine. However, the molecular mechanisms involved in the regulation of the BKB1R gene expression have not been defined. We demonstrated that IL-1β induces a rapid increase in BKB1R mRNA level and the binding of desArg10-kallidin in human embryo lung fibroblasts (IMR90). This increase in BKB1R mRNA level is protein synthesis-independent as indicated by treatment of cells with cycloheximide (CHX) or puromycin (PUR). By testing the IL-1β effect on BKB1R mRNA degradation, we showed that the IL-1β upregulation of BKB1R expression is achieved through both transcriptional activation and post-transcriptional mRNA stabilization. In addition to the IL-1β effects, translation inhibitors, CHX and PUR increase the steady state BKB1R mRNA level by inhibiting BKB1R mRNA degradation. Removal of the CHX block with subsequent resumption of protein synthesis results in a sizable increase of desArg10-kallidin binding. Using signalling pathway inhibitors, we show that IL-1β functions through a protein tyrosine kinase, not protein kinase C or protein kinase A. However, activation of protein kinase C by phorbol 12-myristate 13-acetate increases the level of BKB1R mRNA and the binding of desArg10-kallidin. This increase is blocked by NF-κB activation inhibitors.

scholarly journals Poly(A) binding KPAF4/5 complex stabilizes kinetoplast mRNAs in Trypanosoma brucei

2020 ◽  
Vol 48 (15) ◽  
pp. 8645-8662
Author(s):  
Inna Aphasizheva ◽  
Tian Yu ◽  
Takuma Suematsu ◽  
Qiushi Liu ◽  
Mikhail V Mesitov ◽  
...  
Keyword(s):  
Quality Control ◽  
Trypanosoma Brucei ◽  
Mrna Stability ◽  
Critical Element ◽  
Pentatricopeptide Repeat ◽  
Present Evidence ◽  
Mrna Stabilization ◽  
Translational Activation ◽  
Pyrophosphate Hydrolysis ◽  
Editing Process

Abstract In Trypanosoma brucei, mitochondrial pre-mRNAs undergo 3′-5′ exonucleolytic processing, 3′ adenylation and uridylation, 5′ pyrophosphate removal, and, often, U-insertion/deletion editing. The 3′ modifications are modulated by pentatricopeptide repeat (PPR) Kinetoplast Polyadenylation Factors (KPAFs). We have shown that KPAF3 binding to the 3′ region stabilizes properly trimmed transcripts and stimulates their A-tailing by KPAP1 poly(A) polymerase. Conversely, poly(A) binding KPAF4 shields the nascent A-tail from uridylation and decay thereby protecting pre-mRNA upon KPAF3 displacement by editing. While editing concludes in the 5′ region, KPAF1/2 dimer induces A/U-tailing to activate translation. Remarkably, 5′ end recognition and pyrophosphate hydrolysis by the PPsome complex also contribute to mRNA stabilization. Here, we demonstrate that KPAF4 functions as a heterodimer with KPAF5, a protein lacking discernable motifs. We show that KPAF5 stabilizes KPAF4 to enable poly(A) tail recognition, which likely leads to mRNA stabilization during the editing process and impedes spontaneous translational activation of partially-edited transcripts. Thus, KPAF4/5 represents a poly(A) binding element of the mitochondrial polyadenylation complex. We present evidence that RNA editing substrate binding complex bridges the 5′ end-bound PPsome and 3′ end-bound polyadenylation complexes. This interaction may enable mRNA circularization, an apparently critical element of mitochondrial mRNA stability and quality control.

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