mRNA Decay Rates Research Articles

Conceptual Advances in Control of Inflammation by the RNA-Binding Protein Tristetraprolin.

Regulated changes in mRNA stability are critical drivers of gene expression adaptations to immunological cues. mRNA stability is controlled mainly by RNA-binding proteins (RBPs) which can directly cleave mRNA but more often act as adaptors for the recruitment of the RNA-degradation machinery. One of the most prominent RBPs with regulatory roles in the immune system is tristetraprolin (TTP). TTP targets mainly inflammation-associated mRNAs for degradation and is indispensable for the resolution of inflammation as well as the maintenance of immune homeostasis. Recent advances in the transcriptome-wide knowledge of mRNA expression and decay rates together with TTP binding sites in the target mRNAs revealed important limitations in our understanding of molecular mechanisms of TTP action. Such orthogonal analyses lead to the discovery that TTP binding destabilizes some bound mRNAs but not others in the same cell. Moreover, comparisons of various immune cells indicated that an mRNA can be destabilized by TTP in one cell type while it remains stable in a different cell linage despite the presence of TTP. The action of TTP extends from mRNA destabilization to inhibition of translation in a subset of targets. This article will discuss these unexpected context-dependent functions and their implications for the regulation of immune responses. Attention will be also payed to new insights into the role of TTP in physiology and tissue homeostasis.

HnRNP F and hnRNP H1 regulate mRNA stability of amyloid precursor protein.

Amyloid precursor protein (APP) is a transmembrane protein that plays a crucial role in the production of amyloid-β peptides. Any disruption in APP protein production, its mRNA decay rate or processing may result in abnormal production of amyloid-β peptides and subsequent development of protein aggregation diseases. Therefore, the equilibrium is crucial for neuronal function. An association study of heterogeneous nuclear ribonucleoprotein (hnRNP)-F and hnRNP H1 with APP was carried out in Neuro-2a (N2a) cells. In the present study, we found that hnRNP F and hnRNP H1 were significantly upregulated in the hippocampus of APP/PS1 mice. The changes in APP expression were positively associated with hnRNP F and hnRNP H1 when hnRNP F and hnRNP H1 were depleted or increased in N2a cells. Importantly, cross-linked RNA immunoprecipitation demonstrated binding affinities of hnRNP F and hnRNP H1 for App mRNA. Mechanistically, mRNA stability assay revealed that overexpression of hnRNP F or hnRNP H1 increases the APP level by stabilizing App mRNA half-life, implying that levels of hnRNP F and hnRNP H1 can change the production of APP. Further understanding of the regulatory mechanism of APP expression in association with hnRNP F and hnRNP H1 would provide insights into the mechanism underlying the maintenance of brain health and cognition. This study provides a theoretical basis for the development of hnRNP-stabilizing compounds to regulate APP.

Inhibition of GATA3 and Th2 cytokines expression in type 2 high asthmatics by a small molecule targeting RNA-binding protein HuR

Abstract RNA-binding protein human antigen R (HuR) is a master regulator of gene expression in human pathophysiology. Our animal studies in the past showed multiple critical roles for HuR in regulating proinflammatory markers in CD4+ T cells. We hypothesized HuR plays an important role in expression of GATA3 and Th2 cytokines in type 2 high asthma and its inhibition impairs these Th2 inflammatory responses. Peripheral CD4+ T cells were isolated from 26 healthy controls and 32 type 2 high asthmatics (blood eosinophil ≥300 cells/μl or FeNO levels ≥25ppb) and stimulated with anti-CD3/CD28 for 4 days, followed by determination of GATA3 and cytokines expression by flow cytometry and LEGENDplex. HuR levels were quantitated by Western blot, flow cytometry, and qPCR. Inhibition of inflammatory responses was evaluated using a cumarin-derived small molecule named CMLD-2 (a HuR-specific inhibitor). Actinomycin D treatment was used to measure mRNA decay rates of targeted HuR mRNAs. HuR protein levels in CD4+ T cells was significantly higher in type 2 high asthmatics compared to healthy controls. The expression and secretion of Th2 cytokines were significantly higher in asthmatics compared to controls. Our further mRNA and protein studies in type 2 high asthmatics showed reduced expression of GATA3 and Th2 cytokines after CMLD-2 treatment. There was also a significant reduction in gata3 mRNA stability with CMLD-2 treatment, compared with vehicle in the asthmatics. Our study is the first to demonstrate the role of RNA-binding protein HuR in type 2 high asthmatics and that interfering with HuR action using CMLD-2 ameliorates GATA3 and Th2 cytokines production. This data suggests HuR may serve as a novel and powerful therapeutic target to treat type 2 high asthmatics.

METTL3-Mediated N6-Methyladenosine Modification Is Involved in the Dysregulation of NRIP1 Expression in Down Syndrome.

Down syndrome (DS) is a common genetic condition in which a person is born with an extra copy of chromosome 21. Intellectual disability is the most common characteristic of DS. N6-methyladenosine (m6A) is a common RNA modification that is implicated in many biological processes. It is highly enriched within the brain and plays an essential role in human brain development. However, the mRNA m6A modification in the fetal brain of DS has not been explored. Here, we report m6A mRNA profiles and mRNA expression profiles of fetal brain cortex tissue from DSs and controls. We observed that the m6A modification in DS brain tissues was reduced genome-wide, which may be due to decreased the m6A methyltransferase like 3 (METTL3) protein expression. The nuclear receptor-interacting protein 1 (NRIP1/RIP140) is coded by a highly conserved chromosome 21 (Hsa21) gene. Overexpression of NRIP1 is associated with mitochondrial dysfunction in DS. The NRIP1 mRNA increased in fetal brain tissues of DS, whereas the m6A modification of the NRIP1 mRNA significantly decreased. METTL3 knockdown reduced the m6A modification of NRIP1 mRNA and increased its expression, and an increase in NRIP1 m6A modification and a decrease in its expression were observed in METTL3-overexpressed cells. The Luciferase reporter assay confirmed that METTL3 regulates NRIP1 expression in an m6A-dependent manner. The decay rate of NRIP1 mRNA was significantly reduced in METTL3-knockdown cells but increased in METTL3-overexpressed cells. We proposed that the m6A modification of NRIP1 mRNA in DS fetal brain tissue is reduced, reducing its transcript degradation rate, resulting in abnormally increased expression of NRIP1, at least partially, in the DS brain. It provides a new mechanism for the molecular pathology of DS and leads to a new insight that may become therapeutically relevant.

Systematic Quantification of Sequence and Structural Determinants Controlling mRNA stability in Bacterial Operons.

mRNA degradation is a central process that affects all gene expression levels, and yet, the determinants that control mRNA decay rates remain poorly characterized. Here, we applied a synthetic biology, learn-by-design approach to elucidate the sequence and structural determinants that control mRNA stability in bacterial operons. We designed, constructed, and characterized 82 operons in Escherichia coli, systematically varying RNase binding site characteristics, translation initiation rates, and transcriptional terminator efficiencies in the 5' untranslated region (UTR), intergenic, and 3' UTR regions, followed by measuring their mRNA levels using reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays during exponential growth. We show that introducing long single-stranded RNA into 5' UTRs reduced mRNA levels by up to 9.4-fold and that lowering translation rates reduced mRNA levels by up to 11.8-fold. We also found that RNase binding sites in intergenic regions had much lower effects on mRNA levels. Surprisingly, changing the transcriptional termination efficiency or introducing long single-stranded RNA into 3' UTRs had no effect on upstream mRNA levels. From these measurements, we developed and validated biophysical models of ribosome protection and RNase activity with excellent quantitative agreement. We also formulated design rules to rationally control a mRNA's stability, facilitating the automated design of engineered genetic systems with desired functionalities.

Suppressors of mRNA Decapping Defects Restore Growth Without Major Effects on mRNA Decay Rates or Abundance.

Faithful degradation of mRNAs is a critical step in gene expression, and eukaryotes share a major conserved mRNA decay pathway. In this major pathway, the two rate-determining steps in mRNA degradation are the initial gradual removal of the poly(A) tail, followed by removal of the cap structure. Removal of the cap structure is carried out by the decapping enzyme, containing the Dcp2 catalytic subunit. Although the mechanism and regulation of mRNA decay is well understood, the consequences of defects in mRNA degradation are less clear. Dcp2 has been reported as either essential or nonessential. Here, we clarify that Dcp2 is not absolutely required for spore germination and extremely slow growth, but in practical terms it is impossible to continuously culture dcp2∆ under laboratory conditions without suppressors arising. We show that null mutations in at least three different genes are each sufficient to restore growth to a dcp2∆, of which kap123∆ and tl(gag)g∆ appear the most specific. We show that kap123∆ and tl(gag)g∆ suppress dcp2 by mechanisms that are different from each other and from previously isolated dcp2 suppressors. The suppression mechanism for tL(GAG)G is determined by the unique GAG anticodon of this tRNA, and thus likely by translation of some CUC or CUU codons. Unlike previously reported suppressors of decapping defects, these suppressors do not detectably restore decapping or mRNA decay to normal rates, but instead allow survival while only modestly affecting RNA homeostasis. These results provide important new insight into the importance of decapping, resolve previously conflicting publications about the essentiality of DCP2, provide the first phenotype for a tl(gag)g mutant, and show that multiple distinct mechanisms can bypass Dcp2 requirement.

Theory of transcription bursting: stochasticity in the transcription rates

Transcription bursting creates variation among the individuals of a given population. Bursting emerges as the consequence of turning on and off the transcription process randomly. There are at least three sub-processes involved in the bursting phenomenon with different timescale regimes viz. flipping across the on–off state channels, microscopic transcription elongation events and the mesoscopic transcription dynamics along with the mRNA recycling. We demonstrate that when the flipping dynamics is coupled with the microscopic elongation events, then the distribution of the resultant transcription rates will be over-dispersed. This in turn reflects as the transcription bursting with over-dispersed non-Poisson type distribution of mRNA numbers. We further show that there exist optimum flipping rates (αC, βC) at which the stationary state Fano factor and variance associated with the mRNA numbers attain maxima. These optimum points are connected via $$ \alpha_{C} = \sqrt {\beta_{C} \left( {\beta_{C} + \gamma_{r} } \right)} $$ . Here α is the rate of flipping from the on-state to the off-state, β is the rate of flipping from the off-state to the on-state and γr is the decay rate of mRNA. When α = β = χ with zero rate in the off-state channel, then there exist optimum flipping rates at which the non-stationary Fano factor and variance attain maxima. Here $$ \chi_{C,v} \simeq {{3k_{r}^{ + } } \mathord{\left/ {\vphantom {{3k_{r}^{ + } } {2\left( {1 + k_{r}^{ + } t} \right)}}} \right. \kern-0pt} {2\left( {1 + k_{r}^{ + } t} \right)}} $$ (here $$ k_{r}^{ + } $$ is the rate of transcription purely through the on-state elongation channel) is the optimum flipping rate at which the variance of mRNA attains a maximum and $$ \chi_{C,\kappa } \simeq {{1.72} \mathord{\left/ {\vphantom {{1.72} t}} \right. \kern-0pt} t} $$ is the optimum flipping rate at which the Fano factor attains a maximum. Close observation of the transcription mechanism reveals that the RNA polymerase performs several rounds of stall-continue type dynamics before generating a complete mRNA. Based on this observation, we model the transcription event as a stochastic trajectory of the transcription machinery across these on–off state elongation channels. Each mRNA transcript follows different trajectory. The total time taken by a given trajectory is the first passage time (FPT). Inverse of this FPT is the resultant transcription rate associated with the particular mRNA. Therefore, the time required to generate a given mRNA transcript will be a random variable. For a stall-continue type dynamics of RNA polymerase, we show that the overall average transcription rate can be expressed as $$ k_{r} \simeq h_{\infty }^{ + } k_{r}^{ + } $$ where $$ k_{r}^{ + } \simeq {{\lambda_{r}^{ + } } \mathord{\left/ {\vphantom {{\lambda_{r}^{ + } } L}} \right. \kern-0pt} L} $$ , λ+ is the microscopic transcription elongation rate in the on-state channel and L is the length of a complete mRNA transcript and h∞+ = [β/(α + β)] is the stationary state probability of finding the transcription machinery in the on-state channel.

BR-Bodies Provide Selectively Permeable Condensates that Stimulate mRNA Decay and Prevent Release of Decay Intermediates

Biomolecular condensates play a key role in organizing RNAs and proteins into membraneless organelles. Bacterial RNP-bodies (BR-bodies) are a biomolecular condensate containing the RNA degradosome mRNA decay machinery, but the biochemical function of such organization remains poorly defined. Here, we define the RNA substrates of BR-bodies through enrichment of the bodies followed by RNA sequencing (RNA-seq). We find that long, poorly translated mRNAs, small RNAs, and antisense RNAs are the main substrates, while rRNA, tRNA, and other conserved non-coding RNAs (ncRNAs) are excluded from these bodies. BR-bodies stimulate the mRNA decay rate of enriched mRNAs, helping to reshape the cellular mRNA pool. We also observe that BR-body formation promotes complete mRNA decay, avoiding the buildup of toxic endo-cleaved mRNA decay intermediates. The combined selective permeability of BR-bodies for both enzymes and substrates together with the stimulation of the sub-steps of mRNA decay provide an effective organization strategy for bacterial mRNA decay.

SRSF3 Depletion Leads to an Increase in SF3B4 Expression in SNU-368 HCC Cells.

SF3B4, a critical component of U2 pre-mRNA spliceosomal complex, has been recently indicated as a potential oncogene in hepatocellular carcinoma (HCC). However, limited information exists on how SF3B4 expression is regulated in HCC. To determine the regulatory factor for SF3B4 expression, small interfering RNA (siRNA), real-time polymerase chain reaction (qRT-PCR) and western blotting assay were performed. The in vivo expression profiles of SRSF3 and SF3B4 were analyzed using public datasets and clinical samples. Among 10 liver-specific splicing factors, only SRSF3 knockdown resulted in a significant increase in SF3B4 mRNA and consequently protein levels in SNU-368 HCC cells, probably via the retardation of SF3B4 mRNA decay rates. Using green fluorescent protein-SF3B4 fusion construct, the coding region of SF3B4 was found to be involved in SRSF3-mediated regulation of SF3B4 expression. Publicly available data from paired normal and tumor tissues in HCC and results from patients with HCC suggest that SRSF3 and SF3B4 possess an inverse relationship. SRSF3 is a key molecule for determining SF3B4 levels in HCC cells.

A cell-based probabilistic approach unveils the concerted action of miRNAs.

Mature microRNAs (miRNAs) regulate most human genes through direct base-pairing with mRNAs. We investigate the underlying principles of miRNA regulation in living cells. To this end, we overexpressed miRNAs in different cell types and measured the mRNA decay rate under a paradigm of a transcriptional arrest. Based on an exhaustive matrix of mRNA-miRNA binding probabilities, and parameters extracted from our experiments, we developed a computational framework that captures the cooperative action of miRNAs in living cells. The framework, called COMICS, simulates the stochastic binding events between miRNAs and mRNAs in cells. The input of COMICS is cell-specific profiles of mRNAs and miRNAs, and the outcome is the retention level of each mRNA at the end of 100,000 iterations. The results of COMICS from thousands of miRNA manipulations reveal gene sets that exhibit coordinated behavior with respect to all miRNAs (total of 248 families). We identified a small set of genes that are highly responsive to changes in the expression of almost any of the miRNAs. In contrast, about 20% of the tested genes remain insensitive to a broad range of miRNA manipulations. The set of insensitive genes is strongly enriched with genes that belong to the translation machinery. These trends are shared by different cell types. We conclude that the stochastic nature of miRNAs reveals unexpected robustness of gene expression in living cells. By applying a systematic probabilistic approach some key design principles of cell states are revealed, emphasizing in particular, the immunity of the translational machinery vis-a-vis miRNA manipulations across cell types. We propose COMICS as a valuable platform for assessing the outcome of miRNA regulation of cells in health and disease.

Thiouridine-to-Cytidine Conversion Sequencing (TUC-Seq) to Measure mRNA Transcription and Degradation Rates.

The study of RNA dynamics, specifically RNA transcription and decay rates, has gained increasing attention in recent years because various mechanisms have been discovered that affect mRNA half-life, thereby ultimately controlling protein output. Therefore, there is a need for methods enabling minimally invasive, simple and high-throughput determination of RNA stability that can be applied to determine RNA transcription and decay rates in cells and organisms. We have recently developed a protocol which we named TUC-seq to directly distinguish newly synthesized transcripts from the preexisting pool of transcripts by metabolic labeling of nascent RNAs with 4-thiouridine (4sU) followed by osmium tetroxide-mediated conversion of 4sU to cytidine (C) and direct sequencing. In contrast to other related methods (SLAM-seq, TimeLapse-seq), TUC-seq converts 4sU to a native C instead of an alkylated or otherwise modified nucleoside derivative. TUC-seq can be applied to any cell type that is amenable to 4sU labeling. By employing different labeling strategies (pulse or pulse-chase labeling), it is suitable for a broad field of applications and provides a fast and highly efficient means to determine mRNA transcription and decay rates.

NFKBIZ3′ UTR Mutations Confer Selective Growth Advantage and Activate Genes with Therapeutic Implications in Diffuse Large B-Cell Lymphoma

Introduction: The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) is characterized by activation of NF-κB signaling and increased mortality. Recurrent mutations affecting genes such as MYD88, CD79A/B and TNFAIP3 have been shown to be involved in activation of the NF-κB pathway in ABC DLBCL; however there still remain cases with no known genetic basis for this pathway activation suggesting that our understanding of the drivers of ABC DLBCL remains incomplete. Previously, NFKBIZ was shown to be amplified in ~10% of ABC DLBCLs and to contribute to activation of NF-κB signaling. We have recently described a novel pattern of mutations affecting the 3′ UTR of NFKBIZ resulting in an overall mutation rate of 34% for this gene (UTR mutations or amplifications) in ABC DLBCL. These NFKBIZ UTR mutations are mutually exclusive with MYD88 mutations, thus suggesting they may also lead to activation of NF-κB signaling. NFKBIZ encodes the IκB-ζ protein, which interacts with NF-κB transcription factors and is thought to regulate canonical NF-κB signaling. We hypothesized that NFKBIZ UTR mutations affect the normally rapid degradation of mRNA by disrupting secondary structures recognized by RNA-binding proteins such as ribonucleases. The resulting elevated mRNA levels would in turn lead to accumulation of IκB-ζ protein as a novel mechanism to promote cell growth and survival in ABC DLBCL. Methods: NFKBIZ 3′ UTR mutations were introduced into a DLBCL cell line (WSU-DLCL2) using the CRISPR-Cas9 system. NFKBIZ mRNA and protein levels were evaluated using custom designed droplet digital PCR assays and western blot analyses. Cells were stimulated with LPS to induce NFKBIZ expression and mRNA and protein levels were measured in wild-type (WT) and CRISPR-mutant lines to compare rates of mRNA decay and protein expression. A competitive growth assay with WT and CRISPR-mutant lines was performed to assess whether UTR mutations provide a growth advantage in culture (in vitro) and in mouse xenografts (in vivo). The composition of the pool of WT and mutant lines was determined by comparing WT and mutant DNA sequence proportions. RNA-sequencing was then performed on WT and a subset of CRISPR-mutant cell lines to identify genes up-regulated by IκB-ζ in mutant lines. Findings from these models were compared to the effects of NFKBIZ over-expression in patient tissues. Results: Introduction of NFKBIZ mutations into a DLBCL cell line confirmed that UTR deletions lead to increased mRNA and protein levels. LPS stimulation showed prolonged mRNA elevation in mutant lines, consistent with our model wherein these mutations disrupt post-transcriptional regulatory mechanisms. NFKBIZ UTR deletions gave DLBCL cells a selective growth advantage over WT both in vitro (cell culture) and in vivo (xenograft mouse model). RNA-sequencing of mutant and WT lines revealed possible transcriptional targets of IκB-ζ including some NF-κB targets and genes commonly over-expressed in ABC DLBCL (BATF, MAML2, and TNFRSF13B). HCK, a gene known to be activated by MYD88 was also upregulated in NFKBIZ mutant lines. This is consistent with our hypothesis that mutations in MYD88 and the NFKBIZ UTR are mutually exclusive because they activate similar pathways. HCK is also a target of ibrutinib, suggesting the potential utility of ibrutinib in these patients. Novel targets of IκB-ζ were also discovered through this analysis including CD274, the gene encoding PD-L1. This could be a novel mechanism for DLBCL tumours to express PD-L1 and therefore suggest that these tumours may be susceptible to anti-PD1/PD-L1 immunotherapies. Conclusions: This work highlights the role of NFKBIZ and 3′ UTR mutations in driving ABC DLBCL. We demonstrate that these mutations can lead to over-expression of NFKBIZ and provide a selective growth advantage to cells both in vitro and in vivo. In addition, we described multiple targets of IκB-ζ that may have implications in treatment susceptibility and/or resistance in ABC DLBCL. These findings contribute to a better understanding of the genetic basis of DLBCL, which is necessary to guide personalized therapeutic strategies. Disclosures Scott: Roche/Genentech: Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding; Janssen: Consultancy, Research Funding; Celgene: Consultancy. Steidl:Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Juno Therapeutics: Consultancy; Tioma: Research Funding; Bayer: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy.

Alterations of signaling pathways in response to chemical perturbations used to measure mRNA decay rates in yeast.

Assessing variations in mRNA stability typically involves inhibiting transcription either globally or in a gene-specific manner. Alternatively, mRNA pulse-labeling strategies offer a means to calculate mRNA stability without inhibiting transcription. However, key stress-responsive cell signaling pathways, which affect mRNA stability, may themselves be perturbed by the approaches used to measure mRNA stability, leading to artifactual results. Here, we have focused on common strategies to measure mRNA half-lives in yeast and determined that commonly used transcription inhibitors thiolutin and 1,10 phenanthroline inhibit TORC1 signaling, PKC signaling, and partially activate HOG signaling. Additionally, 4-thiouracil (4tU), a uracil analog used in mRNA pulse-labeling approaches, modestly induces P-bodies, mRNA–protein granules implicated in storage and decay of nontranslating mRNA. Thiolutin also induces P-bodies, whereas phenanthroline has no effect. Doxycycline, which controls “Tet On/Tet Off” regulatable promoters, shows no impact on the above signaling pathways or P-bodies. In summary, our data argues that broad-acting transcriptional inhibitors are problematic for determining mRNA half-life, particularly if studying the impacts of the TORC1, HOG, or PKC pathway on mRNA stability. Regulatable promoter systems are a preferred approach for individual mRNA half-life studies, with 4tU labeling representing a good approach to global mRNA half-life analysis, despite modestly inducing P-bodies.

Evaluation of RNA degradation in pure culture and field Microcystis samples preserved with various treatments

RNA-based molecular technique (RT-qPCR) is a promising method for microcystin monitoring in lakes and reservoirs, but great lability of RNA in cyanobacterial samples limits its application. To date, no studies have investigated how to effectively preserve RNA in cyanobacterial samples. In this study, four different treatments (−80 °C freezer, −196 °C liquid nitrogen, 4 °C or 25 °C preservation after adding RNA protective fluid) were employed to preserve RNA in pure culture and field Microcystis samples, and RNA degradation in these treatments were systematically evaluated. Results showed liquid nitrogen was the most effective treatment to preserve RNA in pure culture and field Microcystis samples. RNA preservation using RNA protective fluid was temperature dependent. Low temperature (4 °C) could effectively slow down RNA degradation within a short time (1–7 d), since decay rate of mcyH mRNA (k = 0.00094 d−1) was much lower at 4 °C than that at 25 °C (0.0549 d−1) (P < 0.05). However, for field samples, RNA degradation was much faster than pure culture samples with the same treatment. Therefore, to better preserve RNA in field samples, a practical strategy for RNA preservation combining RNA protective fluid and liquid nitrogen, was proposed. Tests of field experiments showed it was more effective than individual treatment for RNA preservation in Microcystis samples during field sampling. Thus, this strategy could be employed to preserve RNA in cyanobacterial samples during field sampling, which will contribute to the application of RT-qPCR technique for microcystin monitoring in lakes and reservoirs.

Unfolded Protein Response Differentially Regulates TLR4-Induced Cytokine Expression in Distinct Macrophage Populations.

Cellular stress responses are often engaged at sites of inflammation and can alter macrophage cytokine production. We now report that macrophages in distinct states of differentiation or in different temporal stages of inflammatory response exhibit differential sensitivity to cell stress mediated alterations in M1-like polarized inflammatory cytokine production. Tunicamycin (Tm) treatment of bone marrow derived macrophages (BMDM) cultured with M-CSF cultured bone marrow derived macrophages (M-BMDM) had markedly amplified M1-like responses to LPS, exhibiting higher levels of IL12p40 and IL12p35 mRNAs while BMDM cultured with GM-CSF, which normally express high IL12 subunit production in response to LPS, were relatively unaltered. Anti-inflammatory IL10 mRNA production in LPS-stimulated M-BMDM was greatly reduced by cell stress. These changes in cytokine mRNA levels resulted from altered rates of transcription and mRNA decay. Stress also altered cytokine protein production. Resident liver macrophages isolated from mice treated with Tm showed elevated levels of IL12 subunit mRNA production following LPS stimulation. Furthermore, macrophages infiltrating the liver during the early phase of acetaminophen injury (24 h) had little stress-mediated change in cytokine mRNA production while cells isolated in the later phase (48–72 h) exhibited higher sensitivity for stress elevated cytokine production. Hence cultured macrophages developed using different growth/differentiation factors and macrophages from different temporal stages of injury in vivo show markedly different sensitivity to cell stress for altered inflammatory cytokine production. These findings suggest that cellular stress can be an important modulator of the magnitude and character of myeloid inflammatory activity.

Tuning of mRNA stability through altering 3\u2032-UTR sequences generates distinct output expression in a synthetic circuit driven by p53 oscillations

Synthetic biological circuits that can generate outputs with distinct expression dynamics are useful for a variety of biomedical and industrial applications. We present a method to control output dynamics by altering output mRNA decay rates. Using oscillatory expression of the transcription factor p53 as the circuit regulator, we use two approaches for controlling target gene transcript degradation rates based on the output gene’s 3′-untranslated region (3′-UTR): introduction of copies of destabilizing AU-rich elements into the 3′-UTR or swapping in naturally occurring 3′-UTRs conferring different transcript stabilities. As a proof of principle, we apply both methods to control the expression dynamics of a fluorescent protein and visualize the circuit output dynamics in single living cells. We then use the naturally occurring 3′-UTR approach to restore apoptosis in a tunable manner in a cancer cell line deficient for caspase-3 expression. Our method can be readily adapted to regulate multiple outputs each with different expression dynamics under the control of a single naturally occurring or synthetically constructed biological oscillator.

Inhibiting transcription in cultured metazoan cells with actinomycin D to monitor mRNA turnover.

Decay of transcribed mRNA is a key determinant of steady state mRNA levels in cells. Global analysis of mRNA decay in cultured cells has revealed amazing heterogeneity in rates of decay under normal growth conditions, with calculated half-lives ranging from several minutes to many days. The factors that are responsible for this wide range of decay rates are largely unknown, although our knowledge of trans-acting RNA binding proteins and non-coding RNAs that can control decay rates is increasing. Many methods have been used to try to determine mRNA decay rates under various experimental conditions in cultured cells, and transcription inhibitors like actinomycin D have probably the longest history of any technique for this purpose. Despite this long history of use, the actinomycin D method has been criticized as prone to artifacts, and as ineffective for some promoters. With appropriate guidelines and controls, however, it can be a versatile, effective technique for measuring endogenous mRNA decay in cultured mammalian and insect cells, as well as the decay of exogenously-expressed transcripts. It can be used readily on a genome-wide level, and is remarkably cost-effective. In this short review, we will discuss our utilization of this approach in these cells; we hope that these methods will allow more investigators to apply this useful technique to study mRNA decay under the appropriate conditions.

Dynamic modelling of the iron deficiency modulated transcriptome response in Arabidopsis thaliana roots

The iron deficiency response in plants is a complex biological process with a host of influencing factors. The ability to precisely modulate this process at the transcriptome level would enable genetic manipulations allowing plants to survive in nutritionally poor soils and accumulate increased iron content in edible tissues. Despite the collected experimental data describing different aspects of the iron deficiency response in plants, no attempts have been made towards aggregating this information into a descriptive and predictive model of gene expression changes over time. We formulated and trained a dynamic model of the iron deficiency induced transcriptional response in Arabidopsis thaliana. Gene activity dynamics were modelled with a set of ordinary differential equations that contain biologically tractable parameters. The trained model was able to capture and account for a significant difference in mRNA decay rates under iron sufficient and iron deficient conditions, approximate the expression behaviour of currently unknown gene regulators, unveil potential synergistic effects between the modulating transcription factors and predict the effect of double regulator mutants. The presented modelling approach illustrates a framework for experimental design, data analysis and information aggregation in an effort to gain a deeper understanding of various aspects of a biological process of interest.

Beyond transcription factors: roles of mRNA decay in regulating gene expression in plants.

Gene expression is typically quantified as RNA abundance, which is influenced by both synthesis (transcription) and decay. Cytoplasmic decay typically initiates by deadenylation, after which decay can occur through any of three cytoplasmic decay pathways. Recent advances reveal several mechanisms by which RNA decay is regulated to control RNA abundance. mRNA can be post-transcriptionally modified, either indirectly through secondary structure or through direct modifications to the transcript itself, sometimes resulting in subsequent changes in mRNA decay rates. mRNA abundances can also be modified by tapping into pathways normally used for RNA quality control. Regulated mRNA decay can also come about through post-translational modification of decapping complex subunits. Likewise, mRNAs can undergo changes in subcellular localization (for example, the deposition of specific mRNAs into processing bodies, or P-bodies, where stabilization and destabilization occur in a transcript- and context-dependent manner). Additionally, specialized functions of mRNA decay pathways were implicated in a genome-wide mRNA decay analysis in Arabidopsis. Advances made using plants are emphasized in this review, but relevant studies from other model systems that highlight RNA decay mechanisms that may also be conserved in plants are discussed.

270 - Down regulation of glutathione and glutamate cysteine ligase in the inflammatory response of macrophages

Glutathione (GSH), the most abundant non-protein antioxidant thiol in cells, also plays a key role in mediating the inflammatory response. Its content is reduced in the early phase of the inflammatory response of macrophages due to decreased expression of the catalytic subunit of glutamate cysteine ligase (GCLC). How GCLC is down regulated during the inflammation process remains largely unknown. In this study we explored the underlying mechanism of GCLC down regulation in the inflammatory response of human THP1-differentiated macrophages to lipopolysaccharide (LPS). GCLC mRNA decay rate was significantly increased in the inflammatory response to LPS (GCLC mRNA half life was 5 h in vehicle control vs 2 h in LPS). The half-life of GCLC protein in THP-1 macrophages was about 1.5 h and not changed upon LPS exposure. When cells were treated with transcription inhibitor actinomycin D, the decrease in GCLC mRNA was well correlated with that of GCLC protein, further suggesting that the decrease in GCLC protein in the inflammatory response was a result of the rapid degradation of GCLC mRNA. Supplementing with gamma-glutamylcysteine, the product of GCL, restored GSH level depleted by LPS and suppressed the pro-inflammatory response in THP-1 macrophages. In conclusion, these data demonstrated that GCLC was down regulated through increased mRNA decay rate instead of increased protein degradation in the inflammatory response of macrophages, and that the regulation on GSH/GCLC was an important component of the regulatory mechanism of the pro-inflammatory response.