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PubMed2007

Relationship between mRNA stability and intron presence.

Introns were found to enhance almost every steps of gene expression except increasing mRNA stability. By analyzing the genome-wide data of mRNA stability published by someone previously, we found that human intron-containing genes have more stable mRNAs than intronless genes, and the Arabidopsis thaliana genes with the most unstable mRNAs have fewer introns than other genes in the genome. After controlling for mRNA length, we found mRNA stability is still positively correlated with intron number in human intron-containing genes. But in yeast Saccharomyces cerevisiae, two different datasets on mRNA half-life gave conflicting results. The components of messenger ribonucleoprotein particles recruited during intron splicing may be retained in cytoplasmic mRNPs and act as signals of mRNA stability or simply insulators to avoid mRNA degradation.

Arabidopsis↗
PubMed2005

The MHC class II transactivator (CIITA) mRNA stability is critical for the HLA class II gene expression in myelomonocytic cells.

The human promyelocytic U937 cells express detectable levels of MHC class II (MHC-II) molecules. Treatment with 12-o--tetradecanoyl phorbol 13-acetate (TPA), inducing macrophage-like differentiation, produces a dramatic decrease of MHC-II expression as result of down-modulation of the activation of immune response gene 1 (AIR-1)-encoded MHC-II transactivator (CIITA). This event is specific, as MHC class I remains unaffected. Similar results are observed with U937 cells expressing an exogenous full-length CIITA. Molecular studies demonstrate that TPA treatment affects the stability of CIITA mRNA rather than CIITA transcription. Importantly, cis-acting elements within the distal 650 bp of the 1035-bp 3' untranslated region (3'UTR, nucleotides 3509-4543) are associated to transcript instability. Transcription inhibitors actinomycin D and 5,6-dichlororibofuranosyl benzimidazole, and the translation inhibitor cycloheximide significantly rescue the accumulation of CIITA mRNA in TPA-treated cells. A similar effect is also observed after treatment with staurosporine and the PKC-specific inhibitor GF109203X. The instability of CIITA mRNA produced by TPA in U937 cells is not seen in B cells. These results demonstrate the presence of an additional level of control of MHC-II expression in the macrophage cell lineage depending upon the control of CIITA mRNA stability, most likely mediated by differentiation-induced, 3'UTR-interacting factors which require kinase activity for their destabilizing function.

3' Untranslated Regions↗
PubMed2004

cAMP controls human renin mRNA stability via specific RNA-binding proteins.

It is now recognized that post-transcriptional mechanisms are pivotal to renin production. These involve factors that modulate renin mRNA stability. In 2003 new data has emerged from work in Australia and Germany that has identified several of the, as many as, 20 or so proteins involved. These include CP1 (hnRNP E1), HuR, HADHB, dynamin, nucleolin, YP-1, hnRNP K and MINT-homologous protein. Cyclic AMP (cAMP) is a crucial regulator of renin secretion as well as transcriptional and post-transcriptional control of expression. Many of the RNA-binding proteins that were identified responded to forskolin, increasing in amount by two to 10-fold. The cAMP mechanisms that regulate renin mRNA target, at least in large part, other genes that presumably encode some of these proteins. The increase in the expression of these then facilitates, sequentially, renin mRNA stabilization and destabilization. Our data, using a battery of different techniques, confirm that CP1 and HuR stabilize renin mRNA, whereas HADHB causes destabilization. These proteins target cis-acting C-rich sequences (in the case of CP1) and AU-rich sequences (HuR) in the distal region of the 3'-untranslated region of renin mRNA. We found HADHB was enriched in juxtaglomerular cells and that that within Calu-6 cells HADHB, HuR and CP1 all localized in nuclear subregions, as well as cytoplasm (HADHB and CP1) and mitochondria (HADHB) commensurate with the role each plays in control of renin mRNA stability. The specific proteins that bind to human renin mRNA have begun to be revealed. Cyclic AMP upregulates the binding of several of these proteins, which in turn affect renin mRNA stability and thus overall expression of renin.

Cyclic AMP↗
PubMed1998

Hypoxic regulation of VEGF mRNA stability by RNA-binding proteins.

Vascular endothelial growth factor (VEGF), is a potent angiogenic factor whose expression is dramatically induced by hypoxia. We have previously demonstrated that the induction of VEGF by hypoxia is in large part the result of an increased stability of VEGF mRNA. The stabilization of VEGF mRNA by hypoxia is mediated by the binding of sequence-specific RNA-binding proteins. This review focuses on one such protein, HuR, an RNA-binding protein which we have recently shown is critical for the hypoxic stabilization of VEGF mRNA.

Journal Article↗
PubMed2001

Poly(rC) binding proteins mediate poliovirus mRNA stability.

The 5'-terminal 88 nt of poliovirus RNA fold into a cloverleaf RNA structure and form ribonucleoprotein complexes with poly(rC) binding proteins (PCBPs; AV Gamarnik, R Andino, RNA, 1997, 3:882-892; TB Parsley, JS Towner, LB Blyn, E Ehrenfeld, BL Semler, RNA, 1997, 3:1124-1134). To determine the functional role of these ribonucleoprotein complexes in poliovirus replication, HeLa S10 translation-replication reactions were used to quantitatively assay poliovirus mRNA stability, poliovirus mRNA translation, and poliovirus negative-strand RNA synthesis. Ribohomopoly(C) RNA competitor rendered wild-type poliovirus mRNA unstable in these reactions. A 5'-terminal 7-methylguanosine cap prevented the degradation of wild-type poliovirus mRNA in the presence of ribohomopoly(C) competitor. Ribohomopoly(A), -(G), and -(U) did not adversely affect poliovirus mRNA stability. Ribohomopoly(C) competitor RNA inhibited the translation of poliovirus mRNA but did not inhibit poliovirus negative-strand RNA synthesis when poliovirus replication proteins were provided in trans using a chimeric helper mRNA possessing the hepatitis C virus IRES. A C24A mutation prevented UV crosslinking of PCBPs to 5' cloverleaf RNA and rendered poliovirus mRNA unstable. A 5'-terminal 7-methylguanosine cap blocked the degradation of C24A mutant poliovirus mRNA. The C24A mutation did not inhibit the translation of poliovirus mRNA nor diminish viral negative-strand RNA synthesis relative to wild-type RNA. These data support the conclusion that poly(rC) binding protein(s) mediate the stability of poliovirus mRNA by binding to the 5'-terminal cloverleaf structure of poliovirus mRNA. Because of the general conservation of 5' cloverleaf RNA sequences among picornaviruses, including C24 in loop b of the cloverleaf, we suggest that viral mRNA stability of polioviruses, coxsackieviruses, echoviruses, and rhinoviruses is mediated by interactions between PCBPs and 5' cloverleaf RNA.

Binding, Competitive↗
PubMed2000

Regulation of Nramp1 mRNA stability by oxidants and protein kinase C in RAW264.7 macrophages expressing Nramp1(Gly169).

The murine Nramp1 (natural-resistance-associated macrophage protein) locus confers innate resistance against intracellular macrophage pathogens. The gene encodes a transporter molecule, which is rapidly recruited to the phagosome. Nramp1 functions as an iron transporter by transporting iron into the phagosome. Within the phagosome iron mediates anti-microbial killing by hydroxyl radical formation through the iron-catalysed Fenton/Haber-Weiss reaction. In addition to its effects on the growth of intracellular pathogens, Nramp1 exerts a wide range of pleiotropic effects in activated macrophages. One of these pleiotropic effects is the increased stability of a variety of mRNA species, including Nramp1 mRNA. In the present study, the stability of Nramp1 mRNA in Mycobacterium avium infected RAW264. 7 macrophages expressing either the Nramp1(Gly169) resistant allele or the Nramp1(Asp169) susceptible allele was examined. Nramp1 mRNA stability was greater in Nramp1(Gly169) macrophages than in Nramp1(Asp169) macrophages. The increase in Nramp1 mRNA stability in resistant macrophages was inhibited by antioxidants and protein kinase C (PKC) inhibitors, suggesting that Nramp1 mRNA stability is regulated by an oxidant-generated signalling pathway that requires PKC activity. This was corroborated by treating Nramp1(Asp169) macrophages with menadione, which generates reactive oxygen species within cells. Menadione increased Nramp1 mRNA stability to the level observed in resistant macrophages; this increase was also inhibited by a PKC inhibitor. Further, PKC activity was found to be greater in M. avium-infected Nramp1(Gly169) macrophages than in infected Nramp1(Asp169) macrophages and inhibited by treatment with an antioxidant.

Animals↗
PubMed2003

Regulation of eotaxin gene expression by TNF-alpha and IL-4 through mRNA stabilization: involvement of the RNA-binding protein HuR.

During inflammatory responses, a major posttranscriptional regulation of early response and inflammatory gene expression occurs through modulation of mRNA turnover. We report that two potent inducers of the CC chemokine eotaxin, TNF-alpha and IL-4, regulate its production in airway epithelial cells by increasing eotaxin mRNA stability. In experiments using the transcriptional inhibitor actinomycin D, eotaxin mRNA half-life was significantly prolonged by cell stimulation with TNF-alpha or IL-4, with the combination of the two cytokines being the most effective in extending the mRNA half-life. Involvement of the eotaxin 3' untranslated region in the mRNA-stabilizing effect was tested by transient transfection of a construct expressing a chimeric transcript carrying a serum-inducible beta-globin reporter linked to the eotaxin 3' untranslated region. The half-life of the chimeric mRNA was markedly increased in cells stimulated with TNF-alpha and IL-4. Evidence that the mRNA-stabilizing protein HuR participated in the cytokine effect was obtained: first, HuR presence in the cytoplasm, believed to be required for HuR-mediated mRNA stabilization, increased in both transformed (BEAS-2B cell line) and primary bronchial epithelial cells following treatment with TNF-alpha and IL-4. Second, endogenous eotaxin mRNA was found to bind to HuR in vivo, as detected by immunoprecipitation of HuR-containing messenger ribonucleoprotein complexes followed by real-time RT-PCR analysis; such association increased after cell treatment with TNF-alpha and IL-4. Third, overexpression of HuR in BEAS-2B cells significantly increased the expression of eotaxin mRNA and protein. Our findings implicate mRNA stabilization in the cytokine-mediated increase in eotaxin expression and strongly suggest a role for HuR in this effect.

3' Untranslated Regions↗
PubMed1999

Evidence for autoregulation of cystathionine gamma-synthase mRNA stability in Arabidopsis.

Control of messenger RNA (mRNA) stability serves as an important mechanism for regulating gene expression. Analysis of Arabidopsis mutants that overaccumulate soluble methionine (Met) revealed that the gene for cystathionine gamma-synthase (CGS), the key enzyme in Met biosynthesis, is regulated at the level of mRNA stability. Transfection experiments with wild-type and mutant forms of the CGS gene suggest that an amino acid sequence encoded by the first exon of CGS acts in cis to destabilize its own mRNA in a process that is activated by Met or one of its metabolites.

Amino Acid Sequence↗
PubMed2002

Nutritional control of mRNA stability is mediated by a conserved AU-rich element that binds the cytoplasmic shuttling protein HuR.

The cationic amino acid transporter, Cat-1, is a high affinity transporter of the essential amino acids, arginine and lysine. Expression of the cat-1 gene increases during nutritional stress as part of the adaptive response to starvation. Amino acid limitation induces coordinate increases in stability and translation of the cat-1 mRNA, at a time when global protein synthesis decreases. It is shown here that increased cat-1 mRNA stability requires an 11 nucleotide AU-rich element within the distal 217 bases of the 3'-untranslated region. When this 217-nucleotide nutrient sensor AU-rich element (NS-ARE) is present in a chimeric mRNA it confers mRNA stabilization during amino acid starvation. HuR is a member of the ELAV family of RNA-binding proteins that has been implicated in regulating the stability of ARE-containing mRNAs. We show here that the cytoplasmic concentration of HuR increases during amino acid starvation, at a time when total cellular HuR levels decrease. In addition, RNA gel shift experiments in vitro demonstrated that HuR binds to the NS-ARE and binding was dependent on the 11 residue AU-rich element. Moreover, HuR binding to the NS-ARE in extracts from amino acid-starved cells increased in parallel with the accumulation of cytoplasmic HuR. It is proposed that an adaptive response of cells to nutritional stress results in increased mRNA stability mediated by HuR binding to the NS-ARE.

3' Untranslated Regions↗
PubMed1995

Estimation of PCNA mRNA stability in cell cycle by a serum-deprivation method.

A simple scheme was developed to study the mRNA stability of the proliferating cell nuclear antigen (PCNA) gene during cellular transition from the G1/S boundary to a quiescent state. By this scheme, CHO.K1 cells were grown to about 80% confluence and then serum-starved for 40 h for synchronization in a quiescent state. The quiescent cells were serum-stimulated for a period of time (between 8 h and 12 h) and then grown in serum-free medium until being harvested for further analyses. The cellular PCNA mRNA level was analyzed by Northern blotting. As compared with that in cells which were continuously incubated in serum-containing medium, the decline of the mRNA level, after reaching the peak, in these serum-deprived cells was virtually devoid of mRNA synthesis. Thus, this mRNA decay was taken for the measurement of mRNA stability. The advantage of the scheme is that, unlike the treatment of transcription inhibitors, it does not prevent the cells from completing the rest of the cell cycle before returning to the resting state, and so the mRNA stability observed is cell cycle dependent. In contrast with the previous report that the stability of PCNA mRNA in quiescent cells is less by severalfold than that in S phase cells, our study shows that the mRNA stability of PCNA remained constant during the cellular transition from G1/S boundary to quiescent state.

Animals↗
PubMed2006

Cisplatin increases TNF-alpha mRNA stability in kidney proximal tubule cells.

Cisplatin induces acute renal injury in part by increasing the production of TNF-alpha. However, the mechanism by which cisplatin increases renal TNF-alpha expression is not known. The transcription, translation, and stability of TNF-alpha mRNA are sites of regulation of TNF-alpha production. This study investigated the effects of cisplatin on TNF-alpha mRNA stability and the role of MAP kinases in this process in cultured renal proximal tubule cells. Cisplatin increased the expression of TNF-alpha mRNA by proximal tubule cells in a time- and dose-dependent manner, as well as activated p42/44 ERK kinase, p38 MAP kinase, and JNK in a dose-dependent manner. The inhibition of these pathways reduced TNF-alpha expression significantly. Cisplatin also increased the stability of TNF-alpha mRNA, but this effect was not mediated by MAP kinases and did not require the synthesis of a new protein. The treatment of cells with cisplatin induced the formation of complexes of cytosolic proteins and the AU-rich region of the TNF-alpha 3'UTR. These results are consistent with the view that cisplatin increases TNF-alpha mRNA stability in a MAP kinase-independent manner. The stabilization of TNF-alpha mRNA by cisplatin may involve the binding of certain proteins to AU-rich regions in the 3'UTR.

Acute Kidney Injury↗
PubMed2004

Reciprocal regulation of beta-adrenergic receptor mRNA stability by mitogen activated protein kinase activation and inhibition.

Genes encoding numerous proto-oncogenes and cytokines, as well as a number of G-protein coupled receptors, are regulated post-transcriptionally at the level of mRNA stability. A common feature of all of these genes is the presence of A + U-rich elements (AREs) within their 3' untranslated regions. We, and others, have demonstrated previously that mRNAs encoding beta-adrenergic receptors (beta-ARs) are destabilized by agonist stimulation of the beta-AR/Galphas/adenylylcyclase pathway. However, in addition to PK-A, beta-ARs can also activate or inhibit mitogen activated kinase (MAPK) cascades, in a cell-type dependent basis. Recent evidence points to an important role for MAPKs in regulating the turnover of cytokine mRNAs, such as TNFalpha. We hypothesized that activation of MAPK's may also regulate beta-AR mRNA stability. The studies conducted herein demonstrate that generalized stimulation of MAPKs (JNK, p38) with anisomycin resulted in marked stabilization of beta-AR mRNA. Reciprocally, selective inhibition of JNK with SP600125 significantly decreased beta-AR mRNA half-life. Similarly, inhibition of the MEK/ERK pathway with either PD98059 or U0126 decreased beta-AR mRNA stability substantially. However, inhibition of p38 MAPK with SB203580 produced destabilization of beta-AR mRNA only at higher, non pharmacologically selective concentrations. In contrast to their effects on several other ARE containing mRNAs, inhibition of tyrosine kinases by genistein or PI3K by wortmannin, had no detectable effect on beta-AR mRNA stability. In summary, these results demonstrate for the first time that modulation of MAPK pathways can bi-directionally influence beta-AR mRNA stability.

3' Untranslated Regions↗
PubMed1998

mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation.

Translation and mRNA stability are enhanced by the presence of a poly(A) tail. In vivo, the tail interacts with a conserved polypeptide, poly(A) binding protein (Pab1p). To examine Pab1p function in vivo, we have tethered Pab1p to the 3' UTR of reporter mRNAs by fusing it to MS2 coat protein and placing MS2 binding sites in the 3' UTR of the reporter. This strategy allows us to uncouple Pab1p function from its RNA binding activity. We show that mRNAs that lack a poly(A) tail in vivo are stabilized by Pab1p, and that the portions of Pab1p required for stabilization are genetically distinct from those required for poly(A) binding. In addition, stabilization by Pab1p requires ongoing translation of the mRNA. We conclude that the primary, or sole, function of poly(A) with respect to mRNA stability is simply to bring Pab1p to the mRNA, and that mRNA stabilization is an intrinsic property of Pab1p. The approach we describe may be useful in identifying and assaying 3' UTR regulatory proteins, as it uncouples analysis of function from RNA binding.

3' Untranslated Regions↗
PubMed2001

Regulation of corticotropin-releasing hormone (CRH) transcription and CRH mRNA stability by glucocorticoids.

1. The increases in corticotropin-releasing hormone (CRH) mRNA following long-term adrenalectomy are associated with low levels of CRH gene transcription, suggesting that glucocorticoids regulate CRH mRNA at the posttranscriptional level. In this study we determined the time course of transcriptional activation after early adrenalectomy by intronic in situ hybridization, and evaluated the effects of glucocorticoids on CRH mRNA stability. 2. Plasma corticosterone was undetectable 3 h after adrenalectomy, but CRH hnRNA increased only by 12 h, and remained elevated for the next 72 h. CRH mRNA increased 18 h after adrenalectomy and reached a plateau lasting from 2 to 6 days, despite very low CRH hnRNA levels. 3. Assessment of CRH mRNA stability, by incubation of slide-mounted hypothalamic sections in an intracellular-like medium at 37 degrees C, prior to measuring CRH mRNA levels by in situ hybridization, revealed a half-life (t1/2) of 11.5 min in sham-operated rats, and a slower decrease adrenalectomized rats (t1/2--26.3 min). Corticosterone administration for 3 days markedly decreased CRH mRNA t1/2 in both sham-operated and adrenalectomized rats (6.5 and 5.0 min, respectively). 4. The data show that adrenalectomy causes transient increases in CRH mRNA transcription, followed by decreases in the rate of CRH mRNA degradation. This suggests that glucocorticoids regulate CRH mRNA at two sites, by inhibiting transcription and by decreasing mRNA stability.

Adrenal Cortex Hormones↗
PubMed1997

mRNA stability and the control of gene expression.

The stability of an mRNA plays a major role in the determination of gene expression. The stability of an mRNA reflects its primary and higher-order structure, as well as its interactions with a variety of trans-acting RNA-binding proteins. Both of these parameters can control the function of the mRNA and its exposure to rate limiting nuclease digestion. The specific determinants of mRNA stability are now being worked out in well defined model systems. The unusual stability of globin mRNAs, necessitated by the prolonged posttranscriptional phase of erythroid differentiation, presents a potentially informative example of stability control.

Cell Differentiation↗
PubMed2025

Assessment of mRNA Decay and Calculation of Codon Occurrence to mRNA Stability Correlation Coefficients after 5-EU Metabolic Labeling.

mRNA translation and decay are tightly connected. This chapter describes a method to assess the influence of each codon identity on mRNA stability in cultured cells. The technique involves metabolic labeling of the nascent mRNAs by addition of the nucleoside analog 5-ethynyluridine (5-EU), purification of the RNA at different time-points after chase of the 5-EU, then biotinylation with Click chemistry, pull-down, and sequencing. The transcripts' half-lives are calculated from the expression level of each mRNA at the different time-points. Finally, the method describes the calculation of the Codon occurrence to mRNA Stability correlation Coefficient, or CSC, as a correlation between the codon occurrence in a transcript and the transcript half-life, for each codon.

RNA Stability↗
PubMed1999

Cytochrome c transcriptional activation and mRNA stability during contractile activity in skeletal muscle.

We evaluated contractile activity-induced alterations in cytochrome c transcriptional activation and mRNA stability with unilateral chronic stimulation (10 Hz, 3 h/day) of the rat tibialis anterior (TA) muscle for 1, 2, 3, 4, 5, and 7 days (n = 3-11/group). Transcriptional activation was assessed by direct plasmid DNA injection into the TA with a chloramphenicol acetyltransferase (CAT) reporter gene linked to 326 bp of the cytochrome c promoter. Cytochrome c mRNA in stimulated muscles increased by 1.3- to 1. 7-fold above control between 1 and 7 days. Cytochrome c protein was increased after 5 days of stimulation to reach levels that were 1. 9-fold higher than control by 7 days. Cytochrome c mRNA stability, determined with an in vitro decay assay, was greater in stimulated TA than in control between 2 and 4 days, likely mediated by the induction of a cytosolic factor. In contrast, cytochrome c transcriptional activation was elevated only after 5 days of stimulation when mRNA stability had returned to control levels. Thus the contractile activity-induced increase in cytochrome c mRNA was due to an early increase in mRNA stability, followed by an elevation in transcriptional activation, leading to an eventual increase in cytochrome c protein levels.

Animals↗
PubMed2005

Cyclic AMP and AKAP-mediated targeting of protein kinase A regulates lactate dehydrogenase subunit A mRNA stability.

Expression of the lactate dehydrogenase A subunit (ldh-A) gene is controlled through transcriptional as well as post-transcriptional mechanisms. Both mechanisms involve activation of protein kinase A (PKA) into its subunits and subsequent phosphorylation and activation of several key regulatory factors. In rat C6 glioma cells, post-transcriptional gene regulation occurs through PKA-mediated stabilization of LDH-A mRNA and subsequent increase of intracellular LDH-A mRNA levels. Previous studies have demonstrated a cAMP-stabilizing region (CSR) located in the LDH-A 3'-untranslated region which, in combination with several phosphorylated CSR-binding proteins (CSR-BP), regulates the PKA-mediated stabilization of LDH-A mRNA. However, the mechanistic details of interaction of CSR with proteins as they pertain to mRNA stabilization by PKA are so far largely unknown. In this study we tested the hypothesis that ribosomal protein extracts (RSW) from glioma cells contain PKA regulatory (RII) and catalytic (C) subunits that, in combination with a protein kinase A anchoring protein (AKAP 95) and CSR-BPs participate in forming CSR-protein complexes that are responsible for mRNA stability regulation. To demonstrate the importance of CSR-protein complex formation, the PKA subunits and AKAP 95 were removed from the RSW by immunoprecipitation, and the antigen-deleted RSW were subjected to CSR binding analysis using gel mobility shift and UV cross-linking. It was shown that AKAP 95 as well as RII formed a direct linkage with CSR during CSR-protein complex formation. In contrast, the catalytic subunit formed part of the CSR-protein complex but did not bind to CSR directly in a covalent linkage. To determine whether formation of CSR complexes that included C, RII, and AKAP 95 constituted a functional event and was necessary for mRNA stabilization, cell-free decay reactions were carried out with RSW extracts, and the kinetics of decay of LDH-A mRNA was determined. Depletion of PKA subunits and AKAP 95 from RSW extracts by immunoprecipitation resulted in a marked loss of mRNA stabilization activity indicating that the presence of the PKA regulatory and catalytic subunits as well as AKAP 95 in the CSR-protein complexes was absolutely necessary to achieve LDH-A mRNA stabilization.

3' Untranslated Regions↗