Changes In mRNA Stability Research Articles

Involvement of an RNA binding protein containing Alba domain in the stage-specific regulation of beta-amastin expression in Trypanosoma cruzi

Amastins are surface glycoproteins, first identified in amastigotes of T. cruzi but later found to be expressed in several Leishmania species, as well as in T. cruzi epimastigotes. Amastins are encoded by a diverse gene family that can be grouped into four subfamilies named α, β, γ, and δ amastins. Differential expression of amastin genes results from regulatory mechanisms involving changes in mRNA stability and/or translational control. Although distinct regulatory elements were identified in the 3′ UTR of T. cruzi and Leishmania amastin mRNAs, RNA binding proteins involved with amastin gene regulation have only being characterized in L. infantum where an Alba-domain protein (LiAlba20) able to bind to the 3′ UTR of a δ-amastin mRNA was identified. Here we investigated the role of TcAlba30, the LiAlba20 homologue in T. cruzi, in the post transcriptional regulation of amastin genes. TcAlba30 transcripts are present in all stages of the T. cruzi life cycle. RNA immunoprecipitation assays using a transfected cell line expressing a cMyc tagged TcAlba30 revealed that TcAlba30 can interact with β-amastin mRNA. In addition, over-expression of TcAlba30 in epimastigotes resulted in 50% decreased levels of β-amastin mRNAs compared to wild type parasites. Since luciferase assays indicated the presence of regulatory elements in the 3′ UTR of β-amastin mRNA and reduced levels of luciferase mRNA were found in parasites over expressing TcAlba30, we conclude that TcAlba30 acts as a T. cruzi RNA binding protein involved in the negative control of β-amastin expression through interactions with its 3′UTR.

Regulation of viral gene expression by the herpes simplex virus 1 UL24 protein (HSV-1 UL24 inhibits accumulation of viral transcripts)

UL24 is conserved among all Herpesviridae. In herpes simplex virus 1 (HSV-1), UL24 mutations lead to reduced viral titers both in cell culture and in vivo, and reduced pathogenicity. The human cytomegalovirus ortholog of UL24 has a gene regulatory function; however, it is not known whether other UL24 orthologs also affect gene expression. We discovered that in co-transfection experiments, expression of UL24 correlated with a reduction in the expression of several viral proteins and transcripts. Substitution mutations targeting conserved residues in UL24 impaired this function. Reduced transcript levels did not appear attributable to changes in mRNA stability. The UL24 ortholog of Herpes B virus exhibited a similar activity. An HSV-1 mutant that does not express UL24 produced more viral R1 and R2 transcripts than the wild type or rescue virus relative to the amount of viral DNA. These results reveal a new role for HSV-1UL24 in regulating viral mRNA accumulation.

Abstract A09: ACSL4 is a target of the 17beta-estradiol/estrogen-receptor alpha pathway in mammary carcinoma cell lines

Abstract Polyunsaturated fatty acids (PUFA) are important building blocks for cellular membranes and are incorporated into membrane glycerophospholipids to sustain many cellular functions. Newly absorbed PUFA like arachidonic acid (AA) and eicosapentaenoic acid (EPA) are rapidly transformed to acyl-CoAs by members of the acyl-CoA synthetase family (ACSL1, 3, 4, 5 and 6) prior to their incorporation into membrane phospholipids. However, there is little information on the impact of estrogen on PUFA uptake and ACSL expression in estrogen receptor-positive (ER+ve) breast cancer cells. In the current study, the effect of 17 beta-estradiol on PUFA uptake and ACSL expression was measured in (ER+ve) mammary carcinomas cell lines. Stimulation of estrogen-starved ER+ve MCF-7 and T47D mammary carcinomas cells with 17 beta-estradiol resulted in a significant increase in the cellular uptake of exogenous AA and EPA. This was accompanied by a three-fold increase in the expression the ACSL4 isoform measured by western blot in both MCF-7 and T47D cells, but not in immortalized MCF-10A mammary epithelial cells. There was no measured change in the expression of the other ACSL isoforms. ER alpha silencing with siRNA reversed the 17 beta-estradiol-induced ACSL4 expression in both cells lines showing for the first time that hormonal stimulation of ER+ve breast cancer cells is a driver for ACSL4 expression. However, ACSL4 mRNA levels measured by qPCR were unchanged by 17 beta-estradiol treatment, and no change in mRNA stability was measured with 17beta-estradiol treatment as assessed in actinomycin D-treated cells. Conversely, the half-life of ACSL4 protein after treatment with 17 beta-estradiol was extended from 8±0.5 hours in untreated cells to 26±2 hours in 17 beta-estradiol treated cells as assessed following incubation in the absence or presence of cycloheximide. ER alpha silencing with specific siRNA reversed this half-life extension. SHP2 is a tyrosine phosphatase that regulates ACSL4 expression, however the investigation of possible SHP2-mediated changes in ACSL4 in 17 beta-estradiol-treated cells incubated or not with the SHP2 inhibitor NSC87877 showed no difference in ACSL4 expression. Importantly, ACSL4 silencing with siRNAs reversed the 17 beta-estradiol-induced uptake of AA and EPA in both MCF-7 and T47D cells, indicating that ACSL4 was responsible for the enhanced uptake of these PUFA in ER+ve breast cancer cells. Moreover, ACSL4 silencing completely reversed 17 beta-estradiol-induced cell migration as assessed by an in vitro wound healing assay. Overall, these results demonstrate that 17 beta-estradiol enhances the ability of ER+ve mammary carcinoma cells to incorporate PUFA that are important membrane building blocks, via the induction of ACSL4 protein stability. The requirement of enhanced ASCL4 expression for 17 beta-estradiol-induced cell migration suggests that it is a potential therapeutic target in ER+ve mammary carcinoma cells. This work was supported by the Canadian Breast Cancer Foundation, the Canadian Institutes of Health Research and the Canada Research Chairs Program. Citation Format: Anissa Belkaid, Rodney J. Ouellette, Marc E. Surette. ACSL4 is a target of the 17beta-estradiol/estrogen-receptor alpha pathway in mammary carcinoma cell lines. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A09.

Nitric oxide stress and activation of AMP-activated protein kinase impair β-cell sarcoendoplasmic reticulum calcium ATPase 2b activity and protein stability.

The sarcoendoplasmic reticulum Ca2+ ATPase 2b (SERCA2b) pump maintains a steep Ca2+ concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl l-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-d,l-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca2+ imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca2+ and decreased ER Ca2+ levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b protein stability is decreased under inflammatory conditions through NO- and AMPK-dependent pathways and provide novel insight into pathways leading to altered β-cell calcium homeostasis and reduced β-cell survival in diabetes.

Impact of high pH stress on yeast gene expression: A comprehensive analysis of mRNA turnover during stress responses

Environmental alkalinisation represents a stress condition for yeast Saccharomyces cerevisiae, to which this organism responds with extensive gene expression remodelling. We show here that alkaline pH causes an overall decrease in the transcription rate (TR) and a fast destabilisation of mRNAs, followed by a more prolonged stabilisation phase. In many cases, augmented mRNA levels occur without the TR increasing, which can be attributed to mRNA stabilisation. In contrast, the reduced amount of mRNAs is contributed by both a drop in the TR and mRNA stability. A comparative analysis with other forms of stress shows that, unlike high pH stress, heat-shock, osmotic and oxidative stresses present a common transient increase in the TR. An analysis of environmentally-responsive (ESR) genes for the four above stresses suggests that up-regulated genes are governed mostly by TR changes and complex transient bidirectional changes in mRNA stability, whereas the down-regulated ESR gene set is driven by mRNA destabilisation and a lowered TR. In all the studied forms of stress, mRNA stability plays an important role in ESR. Overall, changes in mRNA levels do not closely reflect the rapid changes in the TR and stability upon exposure to stress, which highlights the existence of compensatory mechanisms.

Global analysis of posttranscriptional gene expression in response to sodium arsenite.

Background:Inorganic arsenic species are potent environmental toxins and causes of numerous health problems. Most studies have assumed that arsenic-induced changes in mRNA levels result from effects on gene transcription.Objectives:We evaluated the prevalence of changes in mRNA stability in response to sodium arsenite in human fibroblasts.Methods:We used microarray analyses to determine changes in steady-state mRNA levels and mRNA decay rates following 24-hr exposure to noncytotoxic concentrations of sodium arsenite, and we confirmed some of these changes using real-time reverse-transcription polymerase chain reaction (RT-PCR).Results:In arsenite-exposed cells, 186 probe set–identified transcripts were significantly increased and 167 were significantly decreased. When decay rates were analyzed after actinomycin D treatment, only 4,992 (9.1%) of probe set–identified transcripts decayed by > 25% after 4 hr. Of these, 70 were among the 353 whose steady-state levels were altered by arsenite, and of these, only 4 exhibited significantly different decay rates between arsenite and control treatment. Real-time RT-PCR confirmed a major, significant arsenite-induced stabilization of the mRNA encoding δ aminolevulinate synthase 1 (ALAS1), the rate-limiting enzyme in heme biosynthesis. This change presumably accounted for at least part of the 2.7-fold increase in steady-state ALAS1 mRNA levels seen after arsenite treatment. This could reflect decreases in cellular heme caused by the massive induction by arsenite of heme oxygenase mRNA (HMOX1; 68-fold increase), the rate-limiting enzyme in heme catabolism.Conclusions:We conclude that arsenite modification of mRNA stability is relatively uncommon, but in some instances can result in significant changes in gene expression.Citation:Qiu LQ, Abey S, Harris S, Shah R, Gerrish KE, Blackshear PJ. 2015. Global analysis of posttranscriptional gene expression in response to sodium arsenite. Environ Health Perspect 123:324–330; http://dx.doi.org/10.1289/ehp.1408626

Abstract 582: Inflammatory Cytokines Reduce Thrombin Activatable Fibrinolysis Inhibitor Protein Levels in HepG2 Cells via TTP-Mediated Destabilization of TAFI mRNA

Thrombin activatable fibrinolysis inhibitor (TAFI) is the zymogen form of a basic carboxypeptidase (TAFIa) with both anti-fibrinolytic and anti-inflammatory properties. The role of TAFI in inflammatory disease is multifaceted and involves both recognition of specific pro-inflammatory substrates by TAFIa as well as regulation of TAFI gene expression by inflammatory mediators. In this study we addressed the hypothesis that decreased TAFI levels observed in inflammation are due to destabilization of its mRNA via the A/U-rich responsive element binding protein tristetraprolin (TTP). TAFI protein levels were measured in conditioned medium of HepG2 cells with recently developed assay specific for TAFIa. Treatment of cells with pro-inflammatory cytokines TNFα, IL-6 in combination with IL-1β, or with bacterial lipopolysaccharide (LPS) decreased TAFI protein levels by approximately 2-fold over 24 to 48 hours of treatment. Conversely, treatment of HepG2 cells with the anti-inflammatory cytokine IL-10 increased TAFI protein levels by 2-fold at both 24 and 48 hour time points. We employed luciferase reporter gene studies using human TAFI promoter constructs and found no change in promoter activity with these treatments. We then hypothesized that changes in mRNA stability may be involved through binding of TTP to the TAFI 3’-UTR, as we recently characterized the role of TTP in mediating TAFI mRNA stability. Using constructs expressing β-globin fusion mRNAs containing the TAFI 3’-UTR, we found that TNFα, IL-6, IL-1β, and LPS reduce TAFI mRNA half-life by 30%. This effect appears to be dependent on TTP binding, since these cytokines did not alter the stability of a fusion transcript lacking the TTP binding site. IL-10 caused an increase in fusion transcript stability by 38%, an effect that was also observed when the TTP binding site was mutated. Using reporter constructs expressing fusion mRNA’s containing the luciferase coding region and the TAFI 3’-UTR, we found that translation rate remained unaffected with both pro- and anti-inflammatory treatments. In conclusion, destabilization of TAFI mRNA appears to be the main mechanism behind decreased TAFI protein levels observed in the presence of pro-inflammatory mediators.

Transcriptional Defect of an Inherited NKX2-5 Haplotype Comprising a SNP, a Nonsynonymous and a Synonymous Mutation, Associated with Human Congenital Heart Disease

Germline mutations in cardiac-specific transcription factor genes have been associated with congenital heart disease (CHD) and the homeodomain transcription factor NKX2-5 is an important member of this group. Indeed, more than 40 heterozygous NKX2-5 germline mutations have been observed in individuals with CHD, and these are spread along the coding region, with many shown to impact protein function. In pursuit of understanding causes of CHD, we analyzed n = 49 cardiac biopsies from 28 patients and identified by direct sequencing two nonsynonymous NKX2-5 alterations affecting alanine 119, namely c.356C>A (p.A119E) and c.355G>T, (p.A119S), in patients with AVSD and HLHS, respectively. In functional assays, a significant reduction in transcriptional activities could be determined for the NKX2-5 variants. Importantly, in one family the mother, besides p.A119E, carried a synonymous mutant allele in the homeodomain (c.543G>A, p.Q181), and a synonymous dbSNP (c.63A>G, p.E21) in the transactivation domain of the protein, that were transmitted to the CHD daughter. The presence of these variants in-cis with the p.A119E mutation led to a further reduction in transcriptional activities. Such difference in activity may be in part related to reduced protein expression for the double variant c.356C>A and c.543G>A. We propose changes in mRNA stability and folding, due to a silent mutation and a dbSNP in the NKX2-5 coding region to contribute to the functional defect. Although the clinical significance of the NKX2-5 haplotype identified in the CHD patients remains to be ascertained, we provide evidence of an interaction of a dbSNP, with synonymous and nonsynonymous mutations to negatively impact NKX2-5 transcriptional activity.

Molecular Effects of Autoimmune-Risk Promoter Polymorphisms on Expression, Exon Choice, and Translational Efficiency of Interferon Regulatory Factor 5

The rs2004640 single nucleotide polymorphism and the CGGGG copy-number variant (rs77571059) are promoter polymorphisms within interferon regulatory factor 5 (IRF5). They have been implicated as susceptibility factors for several autoimmune diseases. IRF5 uses alternative promoter splicing, where any of 4 first exons begin the mRNA. The CGGGG indel is in exon 1A's promoter; the rs2004640 allele creates a splicing recognition site, enabling usage of exon 1B. This study aimed at characterizing alterations in IRF5 mRNA due to these polymorphisms. Cells with risk polymorphisms exhibited ~2-fold higher levels of IRF5 mRNA and protein, but demonstrated no change in mRNA stability. Quantitative PCR demonstrated decreased usage of exons 1C and 1D in cell lines with the risk polymorphisms. RNA folding analysis revealed a hairpin in exon 1B; mutational analysis showed that the hairpin shape decreased translation 5-fold. Although translation of mRNA that uses exon 1B is low due to a hairpin, increased IRF5 mRNA levels in individuals with the rs2004640 risk allele lead to higher overall protein expression. In addition, several new splice variants of IRF5 were sequenced. IRF5's promoter polymorphisms alter first exon usage and increase transcription levels. High levels of IRF5 may bias the immune system toward autoimmunity.

Changes in mRNA Stability Associated with Cold Stress in Arabidopsis Cells

Control of mRNA half-life is a powerful strategy to adjust individual mRNA levels to various stress conditions, because the mRNA degradation rate controls not only the steady-state mRNA level but also the transition speed of mRNA levels. Here, we analyzed mRNA half-life changes in response to cold stress in Arabidopsis cells using genome-wide analysis, in which mRNA half-life measurements and transcriptome analysis were combined. Half-lives of average transcripts were determined to be elongated under cold conditions. Taking this general shift into account, we identified more than a thousand transcripts that were classified as relatively stabilized or relatively destabilized. The relatively stabilized class was predominantly observed in functional categories that included various regulators involved in transcriptional, post-transcriptional and post-translational processes. On the other hand, the relatively destabilized class was enriched in categories related to stress and hormonal response proteins, supporting the idea that rapid decay of mRNA is advantageous for swift responses to stress. In addition, pentatricopeptide repeat, cyclin-like F-box and Myb transcription factor protein families were significantly over-represented in the relatively destabilized class. The global analysis presented here demonstrates not only the importance of mRNA turnover control in the cold stress response but also several structural characteristics that might be important in the control of mRNA stability.

The PARN Deadenylase Targets a Discrete Set of mRNAs for Decay and Regulates Cell Motility in Mouse Myoblasts

PARN is one of several deadenylase enzymes present in mammalian cells, and as such the contribution it makes to the regulation of gene expression is unclear. To address this, we performed global mRNA expression and half-life analysis on mouse myoblasts depleted of PARN. PARN knockdown resulted in the stabilization of 40 mRNAs, including that encoding the mRNA decay factor ZFP36L2. Additional experiments demonstrated that PARN knockdown induced an increase in Zfp36l2 poly(A) tail length as well as increased translation. The elements responsible for PARN-dependent regulation lie within the 3′ UTR of the mRNA. Surprisingly, changes in mRNA stability showed an inverse correlation with mRNA abundance; stabilized transcripts showed either no change or a decrease in mRNA abundance. Moreover, we found that stabilized mRNAs had reduced accumulation of pre–mRNA, consistent with lower transcription rates. This presents compelling evidence for the coupling of mRNA decay and transcription to buffer mRNA abundances. Although PARN knockdown altered decay of relatively few mRNAs, there was a much larger effect on global gene expression. Many of the mRNAs whose abundance was reduced by PARN knockdown encode factors required for cell migration and adhesion. The biological relevance of this observation was demonstrated by the fact that PARN KD cells migrate faster in wound-healing assays. Collectively, these data indicate that PARN modulates decay of a defined set of mRNAs in mammalian cells and implicate this deadenylase in coordinating control of genes required for cell movement.

O025 Triggering mRNA stability switch by a miRNA inhibitor: Normalization of aberrant chemokine response in invasive breast cancer

Introduction Aberrant mRNA stability for a number of genes coding for AU-rich (ARE) mRNAs, including chemokines and growth factors, occurs in many cancer types. The CXCR4-CXCL12 (SDF-1 α ) axis is among the most deregulated chemokine responses that promote the progression of invasive breast cancer. Normalization of the aberrant CXCR4-CXCL12 response by triggering CXCR4 ARE-mRNA destabilization mediated by the zinc finger mRNA binding protein, tristetraprolin (TTP, ZFP36 ), is a potential and intriguing approach. Methods Several breast cancer cell lines were used for comparison with the MDA-MB-231 cell line as an invasive model of breast cancer. We treated the cells with a cell-permeable miR-29a inhibitor which triggers TTP expression, and monitored mRNA stability changes for key RNA binding proteins using RT-QPCR and reporter assays. RNA-immunoprecipitation was also employed to study the mRNA–protein interactions. Subsequent alterations in protein levels and associated functional attributes, including invasion and migration assays were investigated. Further, clinical data from a public database were used to understand these relationships. Results We found that CXCR-4 over-expression in invasive breast cancer is causally linked to deficient TTP expression. TTP deficiency was partly due to the high miR-29a levels that target TTP 3’UTR- this is observed in the highly-invasive MDA-MB-231 cells when compared to other non-invasive or non-tumorigenic cell lines. We also show that CXCR-4 is an mRNA target for TTP but not its zinc finger mutant, C124R, as the wild type was able to bind to CXCR-4 mRNA. TTP deficiency in MDA-MB-231 cells led to increased stability of mRNA coding for the RNA stabilizing protein, HuR, and subsequently HuR protein levels were increased. We demonstrated that HuR is able to bind to and increase CXCR-4 mRNA stability. Thus, the aberrant TTP-HuR balance results in CXCR-4 mRNA stabilization in invasive breast cancer. To overcome this aberration, we employed a cell-permeable peptide-linked miR-29a inhibitor to promote TTP mRNA upregulation and HuR mRNA down-regulation. Indeed this was the case, which subsequently led to CXCR-4 mRNA destabilization and reduction of CXCR-4 levels. This paralleled a reduction in the invasiveness of the cells and migration towards CXCR4 ligand, CXCL12. Clinical data showed that TTP-HuR mRNA ratios which reflect ARE-mRNA stability changes are perturbed in invasive breast cancer samples when compared to breast tissues ( p p Conclusion We establish that the chemokine receptor CXCR-4 is a novel mRNA target for TTP and HuR, and that its over-expression and breast cancer invasiveness is normalized by the miR29a inhibitor. The study shows that aberrant ARE-mediated mRNA stability in invasive breast cancer can be corrected by switching TTP-HuR balance towards mRNA destabilization in the CXCR4/CXCRL12 axis, indicating a potential therapeutic approach.

MRNA stability as a function of striated muscle oxidative capacity

A change in mRNA stability alters the abundance of mRNA available for translation and is emerging as a critical pathway influencing gene expression. Variations in the stability of functional and regulatory mitochondrial proteins may contribute to the divergent mitochondrial densities observed in striated muscle. Thus we hypothesized that the stability of mRNAs encoding for regulatory nuclear and mitochondrial transcription factors would be inversely proportional to muscle oxidative capacity and would be facilitated by the activity of RNA binding proteins (RBPs). The stability of mitochondrial transcription factor A (Tfam), peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), and nuclear respiratory factor 2α (NRF-2α) mRNA was assessed in striated muscles with distinct oxidative capacities using in vitro decay assays. All three mitochondrial regulators were rapidly degraded in cardiac and slow-twitch red (STR) muscle, resulting in a ∼60-65% lower (P < 0.05) mRNA half-life (t(1/2)) compared with fast-twitch white (FTW) fibers. This accelerated rate of Tfam mRNA decay was matched by a 2.5-fold increase in Tfam transcription in slow- compared with fast-twitch muscle (P = 0.05). Protein expression of four unique RBPs [AU-rich binding factor 1 (AUF1), human antigen R (HuR), KH-homology splicing regulatory protein (KSRP), and CUG binding protein 1 (CUGBP1)] believed to modulate mRNA stability was elevated in cardiac and STR muscles (P < 0.05) and was moderately associated with the decay of Tfam, PGC-1α, and NRF-2α mRNA. Variable rates of transcript degradation were apparent when comparing all transcripts within the same muscle type. Thus the distribution of RBPs appears to follow a fiber-type specific pattern and subsequently functions to alter the stability of specific mitochondrial regulators in a transcript- and tissue-specific fashion.

Transcriptome Kinetics Is Governed by a Genome-Wide Coupling of mRNA Production and Degradation: A Role for RNA Pol II

Transcriptome dynamics is governed by two opposing processes, mRNA production and degradation. Recent studies found that changes in these processes are frequently coordinated and that the relationship between them shapes transcriptome kinetics. Specifically, when transcription changes are counter-acted with changes in mRNA stability, transient fast-relaxing transcriptome kinetics is observed. A possible molecular mechanism underlying such coordinated regulation might lay in two RNA polymerase (Pol II) subunits, Rpb4 and Rpb7, which are recruited to mRNAs during transcription and later affect their degradation in the cytoplasm. Here we used a yeast strain carrying a mutant Pol II which poorly recruits these subunits. We show that this mutant strain is impaired in its ability to modulate mRNA stability in response to stress. The normal negative coordinated regulation is lost in the mutant, resulting in abnormal transcriptome profiles both with respect to magnitude and kinetics of responses. These results reveal an important role for Pol II, in regulation of both mRNA synthesis and degradation, and also in coordinating between them. We propose a simple model for production-degradation coupling that accounts for our observations. The model shows how a simple manipulation of the rates of co-transcriptional mRNA imprinting by Pol II may govern genome-wide transcriptome kinetics in response to environmental changes.

RNA-binding Protein Insulin-like Growth Factor mRNA-binding Protein 3 (IMP-3) Promotes Cell Survival via Insulin-like Growth Factor II Signaling after Ionizing Radiation

Ionizing radiation (IR) induces proapoptotic gene expression programs that inhibit cell survival. These programs often involve RNA-binding proteins that associate with their mRNA targets to elicit changes in mRNA stability and/or translation. The RNA-binding protein IMP-3 is an oncofetal protein overexpressed in many human malignancies. IMP-3 abundance correlates with tumor aggressiveness and poor prognosis. As such, IMP-3 is proving to be a highly significant biomarker in surgical pathology. Among its many mRNA targets, IMP-3 binds to and promotes translation of insulin-like growth factor II (IGFII) mRNA. Our earlier studies showed that reducing IMP-3 abundance with siRNAs reduced proliferation of human K562 chronic myeloid leukemia cells because of reduced IGF-II biosynthesis. However, the role of IMP-3 in apoptosis is unknown. Here, we have used IR-induced apoptosis of K562 cells as a model to explore a role for IMP-3 in cell survival. Knockdown of IMP-3 with siRNA increased susceptibility of cells to IR-induced apoptosis and led to reduced IGF-II production. Gene reporter assays revealed that IMP-3 acts through the 5' UTR of IGFII mRNA during apoptosis to promote translation. Finally, culture of IR-treated cells with recombinant IGF-II partially reversed the effects of IMP-3 knockdown on IR-induced apoptosis. Together, these results indicate that IMP-3 acts in part through the IGF-II pathway to promote cell survival in response to IR. Thus, IMP-3 might serve as a new drug target to increase sensitivity of CML cells or other cancers to IR therapy.

Regulation of Retinoid Receptors by Retinoic Acid and Axonal Contact in Schwann Cells

BackgroundSchwann cells (SCs) are the cell type responsible for the formation of the myelin sheath in the peripheral nervous system (PNS). As retinoic acid (RA) and other retinoids have a profound effect as regulators of the myelination program, we sought to investigate how their nuclear receptors levels were regulated in this cell type.Methodology/Principal FindingsIn the present study, by using Schwann cells primary cultures from neonatal Wistar rat pups, as well as myelinating cocultures of Schwann cells with embryonic rat dorsal root ganglion sensory neurons, we have found that sustained expression of RXR-γ depends on the continuous presence of a labile activator, while axonal contact mimickers produced an increase in RXR-γ mRNA and protein levels, increment that could be prevented by RA. The upregulation by axonal contact mimickers and the transcriptional downregulation by RA were dependent on de novo protein synthesis and did not involve changes in mRNA stability. On the other hand, RAR-β mRNA levels were only slightly modulated by axonal contact mimickers, while RA produced a strong transcriptional upregulation that was independent of de novo protein synthesis without changes in mRNA stability.Conclusions/SignificanceAll together, our results show that retinoid receptors are regulated in a complex manner in Schwann cells, suggesting that they could have a prominent role as regulators of Schwann cell physiology.

There is a steady‐state transcriptome in exponentially growing yeast cells

The growth of yeast cells in batches in glucose-based media is a standard condition in most yeast laboratories. Most gene expression experiments are done by taking this condition as a reference. Presumably, cells are in a stable physiological condition that can be easily reproduced in other laboratories. With this assumption, however, it is necessary to consider that the average amount of the mRNAs per cell for most genes does not change during exponential growth. That is to say, there is a steady-state condition for the transcriptome. However, this has not been rigorously demonstrated to date. In this work we take several cell samples during the exponential phase growth to perform a kinetic study using the genomic run-on (GRO) technique, which allows simultaneous measurement of the amount of mRNA and transcription rate variation at the genomic level. We show here that the steady-state condition is fulfilled for almost all the genes during most exponential growth in yeast extract-peptone-dextrose medium (YPD) and, therefore, that simultaneous measures of the transcription rates and the amounts of mRNA can be used for indirect mRNA stability calculations. With this kinetic approach, we were also able to determine the relative influence of the transcription rate and the mRNA stability changes for the mRNA variation for those genes that deviate from the steady state.

Regulação da expressão gênica na depressão metabólica sazonal em mamíferos

Many organisms exhibit a marked decrease of resting rates of metabolism during the annual cycle, associated with lack of oxygen, water, food, or heat. Inhibition of the processes of transcription and translation may contribute significantly to the metabolic depression, although some proteins have increased expression during this condition. In hibernating mammals, regulation of transcription apparently occurs by adjustments at distinct levels, such as chromatin structure, RNA polymerase II activity, transcription factors, alternative splicing, microRNAs, besides changes in mRNA stability.

Regulation of Tfam mRNA stability in skeletal muscle fiber types

Changes in mRNA stability may serve as a regulatory mechanism for mitochondrial biogenesis in skeletal muscle. We hypothesized that oxidative capacity would be closely associated with a greater mRNA stability of proteins vital to organelle biogenesis, prompting an enhanced mitochondrial content. Using an in vitro decay assay, the mRNA stability of mitochondrial transcription factor A (Tfam) was assessed, since it regulates the expression of mitochondrial DNA (mtDNA). The decay of Tfam mRNA was slowest in low oxidative fast‐twitch white (FTW) muscle. Decay rates were 1.5‐ and 2.3‐fold greater in fast‐twitch red (FTR) and slow‐twitch red (STR) fibers. Despite this, steady Tfam mRNA levels were not different between fiber types, suggesting increases in transcription in oxidative muscle which parallel mRNA decay. Degradation of mRNA in fiber types matched similar differences in the mRNA destabilizing protein AUF1, particularly the p42 isoform, with a 4.5‐fold difference in protein expression between STR and FTW muscle. A more dramatic difference in the expression of the mRNA stabilizing protein HuR existed between fiber types, which may serve to restrain uncontrolled mRNA decay in highly oxidative muscle. Thus, oxidative muscle types exhibit faster rates of Tfam mRNA turnover than low oxidative muscle, suggesting more precise control of Tfam expression, and thus mtDNA levels, in response to metabolic stress.

Regulation of p53 mRNA by AMP Kinase (AMPK) activation in C2C12 myoblasts

p53 protein is important for basal and exercise‐induced mitochondrial biogenesis in skeletal muscle. As shown previously, pifithrin‐α (15 μM) a specific inhibitor of p53, reduced the AICAR‐induced increase in mitochondrial content in myoblasts. This demonstrates that p53 partly mediates AMPK‐induced mitochondrial adaptations. Here, we investigated whether p53 mRNA content and stability were modulated by AMPK. Two transcript variants (TV) of murine p53 have been discovered. p53 TV2 mRNA was expressed at 60% lower levels than p53 TV1 in C2C12 myoblasts. AICAR treatment (3 days) reduced p53 TV1 and TV2 mRNA levels by 28% and 13%, respectively, compared to control. To investigate whether this reduced mRNA was due to changes in mRNA stability, we generated reporter constructs containing the full length (R2) 3′untranslated region (UTR), or a shorter construct without the AU‐rich region and the poly‐adenylation signal (R1) of p53 TV2 mRNA. Following transfection, luciferase activity of both R1 and R2 was only ~15% of control, indicating that even the shorter 3′UTR could mediate p53 mRNA decay. AICAR treatment further decreased luciferase activity of both constructs by ~35% compared to untreated cells. This suggests that AMPK regulates p53 by reducing mRNA stability and content. Our findings shed light on the complexity of p53 regulation and have significance for p53‐based therapeutic interventions. (Funded by NSERC)