Accelerated mRNA Degradation Research Articles

Comprehensive chromatographic assessment of forced degraded in vitro transcribed mRNA

Heightened interest in messenger RNA (mRNA) therapeutics has accelerated the need for analytical methodologies that facilitate the production of supplies for clinical trials. Forced degradation studies are routinely conducted to provide an understanding of potential weak spots in the molecule that are exploited by stresses encountered during bulk purification, production, shipment, and storage. Consequently, temperature fluctuations and excursions are often experienced during these unit operations and may accelerate mRNA degradation. Here, we present a concise panel of chromatography-based stability-indicating assays for evaluating thermally stressed in vitro transcribed (IVT) mRNA as part of a forced degradation study. We found that addition of EDTA to the mRNAs prior to heat exposure reduced the extent of degradation, suggesting that transcripts may be fragmenting via a divalent metal-ion mediated pathway. Trace divalent metal contamination that can accelerate RNA instability is likely carried over from upstream steps. We demonstrate the application of these methods to evaluate the critical quality attributes (CQAs) of mRNAs as well as to detect intrinsic process- and product-related impurities.

11,12-EET suppressed LPS induced TF expression and thrombus formation by accelerating mRNA degradation rate via strengthening PI3K-Akt signaling pathway and inhibiting p38-TTP pathway

Epoxyeicosatrienoic acids (EETs), which are synthesized from arachidonic acid by cytochrome P450 epoxygenases, function primarily as autocrine and paracrine effectors in the cardiovascular system. So far, most research has focused on the vasodilatory, anti-inflammatory, anti-apoptotic and mitogenic properties of EETs in the systemic circulation. However, whether EETs could suppress tissue factor (TF) expression and prevent thrombus formation remains unknown. Here we utilized in vivo and in vitro models to investigate the effects and underlying mechanisms of exogenously EETs on LPS induced TF expression and inferior vein cava ligation induced thrombosis. We observed that the thrombus formation rate and the size of the thrombus were greatly reduced in 11,12-EET treated mice，accompanied by decreased TF and inflammatory cytokines expression. Further in vitro studies showed that by enhancing p38 MAPK activation and subsequent tristetraprolin (TTP) phosphorylation, LPS strengthened the stability of TF mRNA and induced increased TF expression. However, by strengthening PI3K-dependent Akt phosphorylation, which acted as a negative regulator of p38-TTP signaling pathway，11,12-EET reduced LPS-induced TF expression in monocytes. In addition, 11,12-EET inhibited LPS-induced NF-κB nuclear translocation by activating the PI3K/Akt pathway. Further study indicated that the inhibitory effect of 11,12-EET on TF expression was mediated by antagonizing LPS-induced activation of thromboxane prostanoid receptor. In conclusion, our study demonstrated that 11,12-EET prevented thrombosis by reducing TF expression and targeting the CYP2J2 epoxygenase pathway may represent a novel approach to mitigate thrombosis related diseases.

11,12-Eet Suppressed Lps Induced Tf Expression and Thrombus Formation by Accelerating Mrna Degradation Rate Via Strengthening Pi3k-Akt Signaling Pathway and Inhibiting P38-Ttp Pathway

11,12-Eet Suppressed Lps Induced Tf Expression and Thrombus Formation by Accelerating Mrna Degradation Rate Via Strengthening Pi3k-Akt Signaling Pathway and Inhibiting P38-Ttp Pathway

RNF219 attenuates global mRNA decay through inhibition of CCR4-NOT complex-mediated deadenylation

The CCR4-NOT complex acts as a central player in the control of mRNA turnover and mediates accelerated mRNA degradation upon HDAC inhibition. Here, we explored acetylation-induced changes in the composition of the CCR4-NOT complex by purification of the endogenously tagged scaffold subunit NOT1 and identified RNF219 as an acetylation-regulated cofactor. We demonstrate that RNF219 is an active RING-type E3 ligase which stably associates with CCR4-NOT via NOT9 through a short linear motif (SLiM) embedded within the C-terminal low-complexity region of RNF219. By using a reconstituted six-subunit human CCR4-NOT complex, we demonstrate that RNF219 inhibits deadenylation through the direct interaction of the α-helical SLiM with the NOT9 module. Transcriptome-wide mRNA half-life measurements reveal that RNF219 attenuates global mRNA turnover in cells, with differential requirement of its RING domain. Our results establish RNF219 as an inhibitor of CCR4-NOT-mediated deadenylation, whose loss upon HDAC inhibition contributes to accelerated mRNA turnover.

Systematic identification of factors mediating accelerated mRNA degradation in response to changes in environmental nitrogen.

Cellular responses to changing environments frequently involve rapid reprogramming of the transcriptome. Regulated changes in mRNA degradation rates can accelerate reprogramming by clearing or stabilizing extant transcripts. Here, we measured mRNA stability using 4-thiouracil labeling in the budding yeast Saccharomyces cerevisiae during a nitrogen upshift and found that 78 mRNAs are subject to destabilization. These transcripts include Nitrogen Catabolite Repression (NCR) and carbon metabolism mRNAs, suggesting that mRNA destabilization is a mechanism for targeted reprogramming of the transcriptome. To explore the molecular basis of destabilization we implemented a SortSeq approach to screen the pooled deletion collection library for trans factors that mediate rapid GAP1 mRNA repression. We combined low-input multiplexed Barcode sequencing with branched-DNA single-molecule mRNA FISH and Fluorescence-activated cell sorting (BFF) to identify the Lsm1-7p/Pat1p complex and general mRNA decay machinery as important for GAP1 mRNA clearance. We also find that the decapping modulators EDC3 and SCD6, translation factor eIF4G2, and the 5’ UTR of GAP1 are factors that mediate rapid repression of GAP1 mRNA, suggesting that translational control may impact the post-transcriptional fate of mRNAs in response to environmental changes.

A translational silencing function of MCPIP1/Regnase-1 specified by the target site context.

The expression of proteins during inflammatory and immune reactions is coordinated by post-transcriptional mechanisms. A particularly strong suppression of protein expression is exerted by a conserved translational silencing element (TSE) identified in the 3′ UTR of NFKBIZ mRNA, which is among the targets of the RNA-binding proteins Roquin-1/2 and MCPIP1/Regnase-1. We present evidence that in the context of the TSE MCPIP1, so far known for its endonuclease activity toward mRNAs specified by distinct stem–loop (SL) structures, also suppresses translation. Overexpression of MCPIP1 silenced translation in a TSE-dependent manner and reduced ribosome occupancy of the mRNA. Correspondingly, MCPIP1 depletion alleviated silencing and increased polysomal association of the mRNA. Translationally silenced NFKBIZ or reporter mRNAs were mostly capped, polyadenylated and ribosome associated. Furthermore, MCPIP1 silenced also cap-independent, CrPV-IRES-dependent translation. This suggests that MCPIP1 suppresses a post-initiation step. The TSE is predicted to form five SL structures. SL4 and 5 resemble target structures reported for MCPIP1 and together were sufficient for MCPIP1 binding and mRNA destabilization. Translational silencing, however, required SL1–3 in addition. Thus the NFKBIZ TSE functions as an RNA element in which sequences adjacent to the site of interaction with MCPIP1 and dispensable for accelerated mRNA degradation extend the functional repertoire of MCPIP1 to translational silencing.

The Role of mRNA Degradation in Dynamic Nitrogen Environments in Saccharomyces cerevisiae

Studying the reprogramming of gene expression is fundamental to understanding cellular growth, development and stem cell differentiation. In Saccharomyces cerevisiae cells undergo reprogramming of gene expression when they are grown in nitrogen‐poor conditions and then switched to nitrogen‐rich conditions. This is controlled by the nitrogen catabolite repression (NCR) response. In a previous study, NCR transcripts were shown to undergo accelerated degradation when the cells where switched from growth on proline in a nitrogen‐poor environment to growth in a nitrogen‐rich environment upon the addition of glutamine. This study aims to determine the benefit of accelerated mRNA degradation in dynamic nitrogen environments by studying the effects of mutants of NCR repressors during this transition. We suspect that knocking out the repressors will stop the accelerated degradation once glutamine is added. This will in turn affect the growth rates and cause the mutants to have longer lag phase, a period where there is no detectable change in the growth. The general amino acid permease (GAP1) was used in representation of NCR transcripts to study the effects. This gene encodes a permease that senses the presence of amino acids substrates to regulate localization to the plasma membrane when needed. We identified mutants where GAP1 is stabilized in a nitrogen‐rich environment by using knock‐outs (KO) of the NCR repressor genes Ure2, Dal80 and Gzf3. The Ure2KO mutant showed depression of GAP1 in nitrogen‐rich conditions, but it caused a growth defect when the cells were grown in nitrogen‐poor conditions. In addition, we tested the effect of the GAP1 5′ untranslated region (UTR) mutant on growth dynamics because it has been shown to stabilize the GAP1 transcript during the transition from nitrogen‐poor environment to a nitrogen‐rich environment. A T‐test was performed to see if there was any difference between the growth rates of the wildtype and the 5′UTR deletion. Turns out that there was no significant difference between them before (p value=0.13) and after the glutamine addition (p value= 0.15), despite there being a notable distinction between the slopes. When the graphs for the nitrogen up shift were generated a lag phase was detected and it appeared to be an hour longer in the mutant. Which indicates that accelerated degradation is an important process in dynamic nitrogen environments.Support or Funding InformationNIH‐MARC Program undergraduate fellowship (5T34GM00781‐37)

Hypolipidemic effect of oleanolic acid is mediated by the miR-98-5p/PGC-1β axis in high-fat diet-induced hyperlipidemic mice.

Oleanolic acid (OA) is an active component of the traditional Chinese herb Olea europaea L. and has been found to exhibit a significant lipid-lowering effect; however, its direct molecular target is still unknown, which limits its clinical application and the possible structure modification to improve its beneficial functions. In this regard, we carried out the present study to identify potential hepatic targets of OA to mediate its lipid-lowering effect. We found that both acute and chronic OA treatments reduced serum levels of triglycerides, total cholesterol, and LDL cholesterol, and decreased hepatic expression levels of peroxisome proliferator-activated receptor-γ coactivator-1β (PGC-1β), which is an important regulator in maintaining hepatic lipid homeostasis, and its downstream target genes. Of note, liver-specific knockdown of PGC-1β recapitulated the hypolipidemic effects of OA. At the molecular level, OA accelerated mRNA degradation of PGC-1β. Microarray analysis revealed a host of microRNAs that potentially mediate OA-induced PGC-1β mRNA degradation, among which, miR-98-5p significantly inhibited activity of Pgc-1β 3' UTR as well as PGC-1β expression and promoted its mRNA degradation. Conversely, miR-98-5p inhibitors blunted the inhibitory effects of OA on PGC-1β expression. Collectively, our data demonstrated that OA ameliorated hyperlipidemia, likely via regulation of the miR-98-5p/PGC-1β axis.-Chen, S., Wen, X., Zhang, W., Wang, C., Liu, J., Liu, C. Hypolipidemic effect of oleanolic acid is mediated by the miR-98-5p/PGC-1β axis in high-fat diet-induced hyperlipidemic mice.

TGF-β and NF-κB signaling pathway crosstalk potentiates corneal epithelial senescence through an RNA stress response.

The corneal epithelium plays important roles in the maintenance of corneal transparency for good vision, and acts as a protective barrier against foreign insults. Structural and functional changes with aging in the corneal epithelium have been documented. Here we found that transforming growth factor-β (TGF-β) is highly expressed in the elderly donor corneal epithelium, as are senescence-associated genes, such as p16 and p21. In human corneal epithelial cell (HCEC) models, TGF-β induces cellular senescence, characterized by increased SA-β-gal positive cells and elevated expression of p16 and p21. Pharmacological inhibition of TGF-β signaling alleviates TGF-β-induced cellular senescence. In addition, we determined that senescence-associated inflammation was significantly aggravated in TGF-β-induced cellular senescence by detecting the expression of interleukin-6 (IL-6), IL-8, and tumor necrosis factor alpha (TNFα). Both genetic and pharmacological approaches revealed that blocking nuclear factor-κB (NF-κB) signaling not only inhibited the production of inflammatory factors, but also rescued the senescent phenotype induced by TGF-β in HCECs. Mechanistically, TGF-β induced an atypical RNA stress responses, leading to accelerated mRNA degradation of IκBα, an inhibitor of NF-κB. Together, our data indicate that TGF-β-driven NF-κB activation contributes to corneal epithelial senescence via RNA metabolism and the inflammation blockade can attenuate TGF-β-induced senescence.

Steady-state and dynamic gene expression programs in Saccharomyces cerevisiae in response to variation in environmental nitrogen.

Cell growth rate is regulated in response to the abundance and molecular form of essential nutrients. InSaccharomyces cerevisiae(budding yeast), the molecular form of environmental nitrogen is a major determinant of cell growth rate, supporting growth rates that vary at least threefold. Transcriptional control of nitrogen use is mediated in large part by nitrogen catabolite repression (NCR), which results in the repression of specific transcripts in the presence of a preferred nitrogen source that supports a fast growth rate, such as glutamine, that are otherwise expressed in the presence of a nonpreferred nitrogen source, such as proline, which supports a slower growth rate. Differential expression of the NCR regulon and additional nitrogen-responsive genes results in >500 transcripts that are differentially expressed in cells growing in the presence of different nitrogen sources in batch cultures. Here we find that in growth rate-controlled cultures using nitrogen-limited chemostats, gene expression programs are strikingly similar regardless of nitrogen source. NCR expression is derepressed in all nitrogen-limiting chemostat conditions regardless of nitrogen source, and in these conditions, only 34 transcripts exhibit nitrogen source-specific differential gene expression. Addition of either the preferred nitrogen source, glutamine, or the nonpreferred nitrogen source, proline, to cells growing in nitrogen-limited chemostats results in rapid, dose-dependent repression of the NCR regulon. Using a novel means of computational normalization to compare global gene expression programs in steady-state and dynamic conditions, we find evidence that the addition of nitrogen to nitrogen-limited cells results in the transient overproduction of transcripts required for protein translation. Simultaneously, we find that that accelerated mRNA degradation underlies the rapid clearing of a subset of transcripts, which is most pronounced for the highly expressed NCR-regulated permease genesGAP1,MEP2,DAL5,PUT4, andDIP5 Our results reveal novel aspects of nitrogen-regulated gene expression and highlight the need for a quantitative approach to study how the cell coordinates protein translation and nitrogen assimilation to optimize cell growth in different environments.

Evaluation of the role of miR-31-dependent reduction in dystrophin and nNOS on atrial-fibrillation-induced electrical remodelling in man

BackgroundThe management of atrial fibrillation remains a challenge. This condition remodels atrial electrical properties, which promote resistance to treatment. Although remodelling has long been a therapeutic target in atrial fibrillation, its causes remain incompletely understood. We aimed to evaluate the role of miR-31-dependent reduction in dystrophin and neuronal nitric oxide synthase (nNOS, also known as NOS1) on atrial electrical properties and atrial fibrillation inducibility. MethodsWe recruited 258 patients (209 patients in sinus rhythm and 49 with permanent atrial fibrillation) from the John Radcliffe Hospital, Oxford, UK; written informed consent was obtained from each participant. We also used a goat model of pacing-induced atrial fibrillation (24 with atrial fibrillation vs 20 controls in normal sinus rythm) and nNos-knock-out mice (n=28 compared with 27 wild-type littermates). Gene expression of miR-31, dystrophin, and nNOS was assessed by quantitative RT-PCR; protein content was measured by immunoblotting; NOS activity was evaluated with high-performance liquid chromatography; action potential duration (APD) and rate dependent adaptation were assessed by single-cell patch-clamping, and atrial fibrillation inducibility was evaluated by transoesophageal atrial burst stimulation. FindingsWe found that atrial-specific upregulation of miR-31 in human atrial fibrillation caused dystrophin (DYS) translational repression and accelerated mRNA degradation of nNOS leading to a profound reduction in atrial DYS and nNOS protein content and in nitric oxide availability. In human atrial myocytes obtained from patients in sinus rhythm, nNOS inhibition was sufficient to recapitulate hallmark features of remodelling induced by atrial fibrillation, such as shortening of APD and loss of APD rate-dependency, but had no effect in patients with atrial fibrillation. In mice, nNos gene deletion or inhibition shortened atrial APD and increased atrial fibrillation inducibility in vivo. Inhibition of miR-31 in human atrial fibrillation recovered DYS and nNOS, and normalised APD and APD rate-dependency. Prevention of miR-31 binding to nNOS 3′UTR recovered both nNOS protein and gene expression but had no effect on the DYS protein or mRNA level (consistent with the mRNA degradation of nNOS by miR-31). Prevention of miR-31 binding to DYS 3′UTR increased DYS protein but not mRNA is consistent with translation repression of DYS by miR-31; recovery of DYS protein increased nNOS protein but not mRNA in keeping with a stabilising effect of DYS on nNOS protein. In goats, a reduction in dystrophin and nNOS protein content was associated with upregulation of miR-31 in the atria but not in the ventricles. InterpretationThe findings suggest that atrial-specific upregulation of miR-31 in human atrial fibrillation is a key mechanism causing atrial loss of dystrophin and nNOS; this loss leads to the electrical phenotype induced by atrial fibrillation. FundingBritish Heart Foundation (BHF) Programme grant (for BC and XL), BHF Centre of Excellence in Oxford (SR), Leducq Foundation (in part for BC and SR), the European Union's seventh Framework Programme Grant Agree.

Gammaherpesviral Gene Expression and Virion Composition Are Broadly Controlled by Accelerated mRNA Degradation

Lytic gammaherpesvirus infection restricts host gene expression by promoting widespread degradation of cytoplasmic mRNA through the activity of the viral endonuclease SOX. Though generally assumed to be selective for cellular transcripts, the extent to which SOX impacts viral mRNA stability has remained unknown. We addressed this issue using the model murine gammaherpesvirus MHV68 and, unexpectedly, found that all stages of viral gene expression are controlled through mRNA degradation. Using both comprehensive RNA expression profiling and half-life studies we reveal that the levels of the majority of viral mRNAs but not noncoding RNAs are tempered by MHV68 SOX (muSOX) activity. The targeting of viral mRNA by muSOX is functionally significant, as it impacts intracellular viral protein abundance and progeny virion composition. In the absence of muSOX-imposed gene expression control the viral particles display increased cell surface binding and entry as well as enhanced immediate early gene expression. These phenotypes culminate in a viral replication defect in multiple cell types as well as in vivo, highlighting the importance of maintaining the appropriate balance of viral RNA during gammaherpesviral infection. This is the first example of a virus that fails to broadly discriminate between cellular and viral transcripts during host shutoff and instead uses the targeting of viral messages to fine-tune overall gene expression.

MicroRNAs Reduce Gene Expression Noise

MicroRNAs regulate a large number of genes in metazoan organisms by accelerating mRNA degradation and inhibiting translation. Although the physiological function of some microRNAs is known in detail, it is not clear why microRNA regulation is so ubiquitous and conserved, since individual microRNAs only weakly repress the vast majority of their target genes and knockouts rarely result in mutant phenotypes. Compelling reasons for this widespread regulation have been suggested, including the ability of microRNAs to provide robustness to gene expression - e.g. by buffering stochastic variations in gene expression. Here we use mathematical modeling and a single cell reporter assay to quantify the effect of microRNA regulation on protein expression variability, or noise, in individual murine embryonic stem cells. We find that microRNA regulation decreases noise for lowly expressed - inherently variable - targets, whereas it increases noise for highly expressed targets. We show that decreased noise results from a reduction in intrinsic noise, which is the dominant noise for lowly expressed genes. However, additional extrinsic noise propagates from the microRNA-pool to the regulated gene and can tip the overall balance towards increased noise at high expression levels. We find that this propagated microRNA-pool noise can be effectively dampened if the target is regulated by multiple independently transcribed microRNAs, and we can verify this for two endogenous microRNA targets. Our findings show how microRNAs confer robustness to gene expression and offer a plausible explanation for the commonly observed combinatorial targeting of endogenous genes by multiple microRNAs.

Uncoupling of mRNA synthesis and degradation impairs adaptation to host temperature in Cryptococcus neoformans

The pathogenic fungus Cryptococcus neoformans must overcome multiple stressors to cause disease in its human host. In this study, we report that C. neoformans rapidly and transiently repressed ribosomal protein (RP) transcripts during a transition from 30°C to host temperature. This repression was accompanied by accelerated mRNA degradation mediated by the major deadenylase, Ccr4, and influenced by the dissociable RNA polymerase II subunit, Rpb4. Destabilization and deadenylation of RP transcripts were impaired in an rpb4Δ mutant, suggesting that Rpb4 may be involved in host temperature-induced Ccr4-mediated decay. Accelerated decay of ER stress transcripts 1 h following a shift to host temperature was also impaired in the rpb4Δ mutant. In response to host temperature, Rpb4 moved from the nucleus to the cytoplasm, supporting a role for Rpb4 in coupling transcription and degradation. The PKH signalling pathway was implicated as a regulator of accelerated degradation of the RP transcripts, but not of the ER stress transcripts, revealing a further level of specificity. When transcription and degradation were uncoupled by deletion of Rpb4, growth at host temperature was impaired and virulence was attenuated. These data suggest that mRNA synthesis and decay are coupled in C. neoformans via Rpb4, and this tight coordination promotes host-temperature adaptation and pathogenicity.

Identification of regulatory mechanisms and RNA targets of human Pumilio proteins

Proper gene expression requires a balance between synthesis, modification, and degradation of mRNAs. PUF proteins are a conserved family of RNA binding proteins that regulate specific mRNAs to control many biological processes including development and proliferation. PUFs bind specific sequence elements in the 3′ UTRs of specific mRNAs via an RNA binding domain. PUFs in model organisms repress targets via deadenylation and degradation and/or direct inhibition of translation. The mechanisms by which human PUFs repress their mRNA targets as well as their physiological targets remain unclear. Humans have two related PUFs, PUM1 and PUM2. We found that human PUFs employ a conserved mechanism of repression via direct recruitment of the CCR4‐NOT deadenylase complex to target mRNAs, leading to translational inhibition and accelerated mRNA degradation. Further, PUMs could repress a non‐adenylated mRNA; therefore, PUMs exhibit a second deadenylation independent repression mechanism.Few specific mRNAs regulated by PUMs have been identified. To better ascertain the roles of human PUMs, we sought to identify mRNAs regulated by them. We depleted PUMs from human cells and measured changes in mRNA expression levels by RNA‐Seq. We identified more than of 200 PUM target RNAs meeting the following criteria: >;1.5‐fold change, >;1 PUM site, and FDR = 0.05. Our results begin to reveal PUM regulated pathways in human networks.University of Michigan internal grant

Neural Regulation of the Stress Response: The Many Faces of Feedback.

The mammalian stress response is an integrated physiological and psychological reaction to real or perceived adversity. Glucocorticoids (GCs) are an important component of this response, acting to redistribute energy resources to both optimize survival in the face of challenge and restore homeostasis after the immediate threat has subsided. Release of GCs is mediated by the hypothalamo-pituitary-adrenocortical (HPA) axis, driven by a neural signal originating in the paraventricular nucleus (PVN). Stress levels of GCs bind to glucocorticoid receptors (GRs) in multiple body compartments, including brain, and consequently have wide-reaching actions. For this reason, GCs serve a vital function in feedback inhibition of their own secretion. Fast, non-genomic feedback inhibition of the HPA axis is mediated at least in part by GC signaling in the PVN, acting by a cannabinoid-dependent mechanism to rapidly reduce both neural activity and GC release. Delayed feedback termination of the HPA axis response is mediated by forebrain GRs, presumably by genomic mechanisms. GCs also act in the brainstem to attenuate neuropeptidergic excitatory input to the PVN via acceleration of mRNA degradation, providing a mechanism to attenuate future responses to stressors. Thus, rather than having a single defined feedback switch, GCs work through multiple neurocircuits and signaling mechanisms to coordinate HPA axis activity to suit the overall needs of multiple body systems.

MiR-206-mediated dynamic mechanism of the mammalian circadian clock.

BackgroundAs a group of highly conserved small non-coding RNAs with a length of 21~23 nucleotides, microRNAs (miRNAs) regulate the gene expression post-transcriptionally by base pairing with the partial or full complementary sequences in target mRNAs, thus resulting in the repression of mRNA translation and the acceleration of mRNA degradation. Recent work has revealed that miRNAs are essential for the development and functioning of the skeletal muscles where they are. In particular, miR-206 has not only been identified as the only miRNA expressed in skeletal muscles, but also exhibited crucial roles in regulation of the muscle development. Although miRNAs are known to regulate various biological processes ranging from development to cancer, much less is known about their role in the dynamic regulation of the mammalian circadian clock.ResultsA detailed dynamic model of miR-206-mediated mammalian circadian clock system was developed presently by using Hill-type terms, Michaelis-Menten type and mass action kinetics. Based on a system-theoretic approach, the model accurately predicts both the periodicity and the entrainment of the circadian clock. It also explores the dynamics properties of the oscillations mediated by miR-206 by means of sensitivity analysis and alterations of parameters. Our results show that miR-206 is an important regulator of the circadian clock in skeletal muscle, and thus by study of miR-206 the main features of its mediation on the clock may be captured. Simulations of these processes display that the amplitude and frequency of the oscillation can be significantly altered through the miR-206-mediated control.ConclusionsMiR-206 has a profound effect on the dynamic mechanism of the mammalian circadian clock, both by control of the amplitude and control or alteration of the frequency to affect the level of the gene expression and to interfere with the temporal sequence of the gene production or delivery. This undoubtedly uncovers a new mechanism for regulation of the circadian clock at a post-transcriptional level and provides important insights into the normal development as well as the pathological conditions of skeletal muscles, such as the aging, chronic disease and cancer.

Rare codons play a positive role in the expression of the stationary phase sigma factor RpoS (σS) in Escherichia coli

Rare codons can influence the stability of messenger RNAs, promote regular spacing of ribosomes on a transcript, or modulate stability and proper folding of nascent proteins. The mRNA specifying the stationary phase master regulator RpoS, which belongs to the RpoD family of sigma factors, contains a high number of rare codons, including many codons at positions corresponding to more frequent codons encoding the same amino acids in the homologous RpoD sequence. Substituting these rare codons in rpoS by the more frequent synonymous rpoD codons resulted in decreased transcript and protein levels compared to the natural rare-codon wildtype version of rpoS. The frequent-codon mutant rpoS transcript exhibited faster turnover than the rare-codon wildtype mRNA. Studies with endoribonuclease-deficient strains revealed RNase E to be crucial for this accelerated mRNA degradation. Thus, in the case of RpoS expression, ‘less is obviously more‘, as our data suggest a model, in which slowing down translational speed by ribosomal pausing at many rare codons along a transcript could reduce ribosome spacing and thereby protect the transcript against ribonucleolytic attack by RNase E. Such a mechanism may be especially important for translationally controlled genes like rpoS where the formation of secondary structure in the translational initiation region competes with (therefore relatively inefficient) ribosome loading. Moreover, strong codon differences in genes encoding isoenzymes expressed in exponential and stationary phase suggest that transcript protection by repetitive ribosome pausing at multiple rare codons in stationary phase-expressed transcripts may be a general principle to save resources under nutrient-limited conditions.

Lon Peptidase 1 (LONP1)-dependent Breakdown of Mitochondrial 5-Aminolevulinic Acid Synthase Protein by Heme in Human Liver Cells

5-Aminolevulinic acid synthase (ALAS-1) is the first rate controlling enzyme that controls cellular heme biosynthesis. Negative feedback regulation of ALAS-1 by the end product heme is well documented and provides the foundation for heme treatment of acute porphyrias, a group of diseases caused by genetic defects in the heme biosynthesis pathway and exacerbated by controlled up-regulation of ALAS-1. Heme is known to affect ALAS-1 activity by repressing gene transcription, accelerating mRNA degradation, and impeding pre-ALAS-1 mitochondrial translocation. In the current study, we examined the effect of heme on the rate of mature ALAS-1 protein turnover in human cells and tissues and explored the mediator involved in this new regulatory mechanism. We found that heme and other metalloporphyrins such as CoPP and CrPP decreased mitochondrial ALAS-1 protein through proteolysis. This degradative effect cannot be emulated by iron or free protoporphyrin, two major chemical components of the heme ring, and is independent of oxidative stress. Down-regulating the activity of mitochondrial LONP1, an ATP-dependent protease that controls the selective turnover of mitochondrial matrix proteins, with potent inhibitors and specific siRNA diminished the negative effect of heme on mitochondrial ALAS-1. Therefore, our data support the existence of a conserved heme feedback regulatory mechanism that functions on the mature form of ALAS-1 protein through the activity of a mitochondrial proteolytic system.

Expression hierarchy in the Yersinia type III secretion system established through YopD recognition of RNA.

The Yersinia type III secretion system (T3SS) is environmentally responsive to enable its rapid induction upon contact with host cells and is necessary for Yersiniae to establish a replicative niche and cause disease. YopD, a translocator protein, represses the expression of T3SS genes until signalled by environmental cues, a mechanism known as the low calcium response. In this work, we investigated recognition of target genes by Yersinia pestis YopD. Expression of all genes of the T3SS was induced in a yopD mutant, though not to the same degree, with effector Yops most affected. Two, short AU-rich sequence elements up- and downstream of start codons of target genes were necessary but not sufficient for YopD mediated repression. Purified YopD-LcrH bound specifically to target RNAs in vitro with different relative affinities, with effector Yops having greater affinity. Together, the data suggest YopD binds to T3SS transcripts where it may prevent ribosome binding causing accelerated mRNA degradation. This regulatory mechanism may ensure an expression hierarchy during the low calcium response as low affinity YopD targets such as chaperones would be translated prior to high affinity targets such as effector Yops allowing the bacteria another layer of control over Yop translocation during infection.