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At least 37 records · Page 2Linked to original sources
PubMed2005

Circadian control of messenger RNA stability. Association with a sequence-specific messenger RNA decay pathway.

Transcriptional and posttranscriptional regulation are well-established mechanisms for circadian gene expression. Among the latter, differential messenger RNA (mRNA) stability has been hypothesized to control gene expression in response to the clock. However, direct proof that the rate of mRNA turnover can be regulated by the clock is lacking. Previous microarray expression data for unstable mRNAs in Arabidopsis (Arabidopsis thaliana) revealed that mRNA instability is associated with a group of genes controlled by the circadian clock. Here, we show that CCR-LIKE (CCL) and SENESCENCE ASSOCIATED GENE 1 transcripts are differentially regulated at the level of mRNA stability at different times of day. In addition, the changes in CCL mRNA stability continue under free-running conditions, indicating that it is controlled by the Arabidopsis circadian clock. Furthermore, we show that these mRNAs are targets of the mRNA degradation pathway mediated by the downstream (DST) instability determinant. Disruption of the DST-mediated decay pathway in the dst1 mutant leads to aberrant circadian mRNA oscillations that correlate with alterations of the half-life of CCL mRNA relative to parental plants in the morning and afternoon. That this is due to an effect on the circadian control is evidenced by mRNA decay experiments carried out in continuous light. Finally, we show that the defects exhibited by dst mutants are reflected by an impact on circadian regulation at the whole plant level. Together, these results demonstrate that regulation of mRNA stability is important for clock-controlled expression of specific genes in Arabidopsis. Moreover, these data uncover a connection between circadian rhythms and a sequence-specific mRNA decay pathway.

Arabidopsis↗
PubMed2002

Improvement of molecular monitoring of residual disease in leukemias by bedside RNA stabilization.

The sensitivity of assays designed to monitor minimal residual disease (MRD) by RT-PCR in leukemia depend on quality and quantity of RNA derived from peripheral blood (PB) and bone marrow (BM) leukocytes. Shipment of material may lead to RNA degradation resulting in a loss of sensitivity and, potentially, false negative results. Furthermore, degradation may lead to inaccurate estimates of MRD in positive specimens. We sought to determine feasibility and efficacy of a novel blood collection and processing system which is based on integrated RNA stabilization at the time of phlebotomy (PAXgene Blood RNA Kit) by comparison with standard methods of RNA extraction (cesium chloride gradient ultracentrifugation and RNeasy Mini Kit) using unstabilized EDTA anticoagulated PB. In 26 patients with chronic myelogenous leukemia (CML) on therapy, PB was processed after a storage time at room temperature of 2 and 72 h according to these protocols. BCR-ABL, total ABL and glucose-6-phosphate dehydrogenase (G6PD) mRNA transcripts of PB samples were quantified as a measure for response to therapy and RNA integrity. RNA yield expressed as the ratio of ABL transcripts after a storage time of 72 h/ABL transcripts after a storage time of 2 h at room temperature was significantly higher with the stabilizing method (median 0.40) compared to the RNeasy method using unstabilized PB (median 0.13, P = 0.01). Furthermore, ratios BCR-ABL/ABL after 72 vs 2 h still correlated well using the PAXgene method (r = 0.99, P < 0.0001) in contrast to the standard method which did not (r = 0.65, P = 0.03). Even investigation of complete cytogenetic responders with very low tumor burden showed a good correlation of ratios BCR-ABL/ABL compared to the reference method. Comparable results were achieved using G6PD transcripts as standard. We conclude that the new PAXgene stabilization method could improve RNA quality and the comparability of molecular monitoring within and between multicenter trials.

Adult↗
PubMed2006

A transient heterochromatic state in Xist preempts X inactivation choice without RNA stabilization.

X chromosome inactivation (XCI) depends on a noncoding sense-antisense transcript pair, Xist and Tsix. At the onset of XCI, Xist RNA accumulates on one of two Xs, coating and silencing the chromosome in cis. The molecular basis for monoallelic Xist upregulation is not known, though evidence predominantly supports a posttranscriptional mechanism through RNA stabilization. Here, we test whether Tsix RNA destabilizes Xist RNA. Unexpectedly, we find that Xist upregulation is not based on transcript stabilization at all but is instead controlled by transcription in a sex-specific manner. Tsix directly regulates its transcription. On the future inactive X, Tsix downregulation induces a transient heterochromatic state in Xist, followed paradoxically by high-level Xist expression. A Tsix-deficient X chromosome adopts the heterochromatic state in pre-XCI cells. This state persists through XCI establishment and "reverts" to a euchromatic state during XCI maintenance. We have therefore identified chromatin marks that preempt and predict asymmetric Xist expression.

Animals↗
PubMed1989

Function of plastid mRNA 3' inverted repeats. RNA stabilization and gene-specific protein binding.

Plastid protein coding regions in plants are generally flanked by 3' inverted repeat (IR) sequences. In a previous work (Stern, D. B., and Gruissem, W. (1987) Cell 51, 1145-1157), we have shown that their role may be in RNA stabilization and as a processing signal that establishes the mature mRNA 3' end. In this report we have investigated the stability and protein interaction of chloroplast mRNA 3' IR-RNA sequences in more detail. Progressive deletions into the 3' IR-RNA sequences for the chloroplast cytochrome b6/f subunit IV (petD) mRNA reduce the stability of the RNA, indicating that the potential to form a stem/loop is a minimum requirement for petD 3' IR-RNA stability in vitro. Specific point mutants also destabilize the processed 3' IR-RNA, suggesting an important role for the primary sequence. Gel mobility shift and UV-cross-linking analysis has shown that 3' IR-RNAs of petD and two other chloroplast mRNAs (rbcL and psbA) interact with proteins in vitro. Comparison of the bound petD 3' IR-RNA proteins with proteins that bind to rbcL and psbA reveals that binding of certain proteins is gene-specific. Also, precursor and processed petD 3' IR-RNAs bind different sets of proteins. A single nucleotide transversion (T----A) near the base of the stem eliminates the binding of a 29-kDa protein to the petD 3' IR-RNA precursor. We discuss the possible role of 3' IR-RNA-protein interactions in plastid mRNA 3' end maturation and differential mRNA stability.

Base Sequence↗
PubMed2005

Progression of secondary hyperparathyroidism involves deregulation of genes related to DNA and RNA stability.

BACKGROUND: Renal secondary hyperparathyroidism in its late stages becomes autonomous, so excessive parathyroid hormone (PTH) secretion no longer responds to physiologic stimuli or to aggressive medical treatment. METHODS: To gain molecular understanding of progression of renal secondary hyperparathyroidism, normal and hyperplastic parathyroid tissue with diffuse and nodular growth were analyzed. The results were also compared to parathyroid adenomas. The analysis was performed by high-density oligonucleotide microarray and bidirectional subtraction library. RESULTS: Analysis of the DNA arrays found 16 overexpressed and 132 repressed genes in the nodules while the subtraction library produced 34 overexpressed and 40 repressed genes. The differentially expressed genes between diffuse and nodular samples included some related to DNA stability and repair (TALDO1, PRDX2, DDB1, XRCC1, and POLB), RNA stability and degradation (OASL and AUF1), protein synthesis and processing (PFDN5, HSPD1, and NACA), cell growth (CDC25C and GRPR), and tumorigenesis and cell cycle (VIL2 and TPD52). CONCLUSION: According to the function described for the deregulated genes, when secondary hyperparathyroidism becomes autonomous and refractory to treatment, RNA degradation may be increased while DNA integrity may be compromised. These two mechanisms, combined with deregulation of genes related to growth and differentiation show the complex pathway of parathyroid glands' evolution in renal hyperparathyroidism and may explain the large amount of molecular cytogenetic aberrations found in refractory hyperparathyroidism. Considering that some of the genes with altered expression in nodular hyperplasia lead to irreversible consequences in the genomic integrity of the cells, an adequate and early management of the secondary hyperparathyroidism of chronic kidney disease becomes mandatory.

Adult↗
PubMed2006

A time course study demonstrating RNA stability in postmortem skin.

Knowledge of the factors regulating the rate of mRNA degradation, including postmortem delay, is important in determining the reliability of gene expression patterns in dermal tissue. Since RNA stability can be tissue dependent, this study evaluates the effect of postmortem interval on the integrity of total RNA or the levels of representative mRNA species in murine cutaneous tissue. Pieces of fresh skin tissue were excised for periods of 0-60 min from SKH-1 female hairless mice that were maintained at room temperature post-sacrifice. Total RNA was subsequently isolated and RNA integrity from each specimen was evaluated. Bioanalyzer profiles showed no apparent change in 28S/18S rRNA ratio or RNA integrity number at time points up to 60 min. Changes in mRNA expression levels of five selected genes were determined by real-time quantitative PCR. There were no statistical differences in the relative gene expressions of Ccnd1, Hif1alpha, cMyc and Cyr61 as a function of postmortem interval. Our data suggest that the molecular quality of cutaneous tissue is well preserved for at least 60 min after death, which can be regarded as important information for consideration of the order for tissue procurement in in vivo studies and acute ex vivo dermal studies.

Animals↗
PubMed2001

Evidence for multiple sequences and factors involved in c-myc RNA stability during amphibian oogenesis.

To investigate the molecular mechanisms regulating c-myc RNA stability during late amphibian oogenesis, a heterologous system was used in which synthetic Xenopus laevis c-myc transcripts, progressively deleted from their 3' end, were injected into the cytoplasm of two different host axolotl (Ambystoma mexicanum) cells: stage VI oocytes and progesterone-matured oocytes (unfertilized eggs; UFE). This in vivo strategy allowed the behavior of the exogenous c-myc transcripts to be followed and different regions involved in the stability of each intermediate deleted molecule to be identified. Interestingly, these specific regions differ in the two cellular contexts. In oocytes, two stabilizing regions are located in the 3' untranslated region (UTR) and two in the coding sequence (exons II and III) of the RNA. In UFE, the stabilizing regions correspond to the first part of the 3' UTR and to the first part of exon II. However, in UFE, the majority of synthetic transcripts are degraded. This degradation is a consequence of nuclear factors delivered after germinal vesicle breakdown and specifically acting on targeted regions of the RNA. To test the direct implication of these nuclear factors in c-myc RNA degradation, an in vitro system was set up using axolotl germinal vesicle extracts that mimic the in vivo results and confirm the existence of specific destabilizing factors. In vitro analysis revealed that two populations of nuclear molecules are implicated: one of 4.4-5S (50-65 kDa) and the second of 5.4-6S (90-110 kDa). These degrading nuclear factors act preferentially on the coding region of the c-myc RNA and appear to be conserved between axolotl and Xenopus. Thus, this experimental approach has allowed the identification of specific stabilizing sequences in c-myc RNA and the temporal identification of the different factors (cytoplasmic and/or nuclear) involved in post-transcriptional regulation of this RNA during oogenesis.

Ambystoma↗
PubMed2001

Introduction of an RNA stability element at the 5'-end of an antisense RNA cassette increases the inhibition of target RNA translation.

This communication describes improvement strategies used on a previously described two-unit antisense RNA cassette system. This cassette system encodes RNA with noncontiguous regions of complementarity to a bacterial target RNA, lacI mRNA. One of the units of complementarity was contained within an RNA stem-loop resembling that of the very efficient, naturally occurring antisense RNA CopA. As relatively low inhibitory activity was obtained previously, we tested variants in which several stem-loops were combined within one RNA, each of them directed against a different stretch of target RNA. One to four stem-loop RNAs were tested and found to be relatively ineffective, likely because of low metabolic stability. To increase the intracellular stability of these and other antisense RNAs, a stabilizer element (stem-loop derived from gene 32 mRNA of phage T4) was inserted at their 5'-ends. The results indicate that addition of this element indeed increased antisense RNA efficiency in vivo. As expected, this effect was primarily due to a longer antisense RNA half-life, as shown by RNA abundance (Northern analysis) and decay rates (rifampicin runout experiments). In summary, the results reported indicate that rational design of antisense RNA is feasible, but that the degree of inhibition (approximately 75% maximum inhibition) accomplished here could still be improved.

Bacterial Proteins↗
PubMed2003

Tumor necrosis factor alpha induces angiogenic factor up-regulation in malignant glioma cells: a role for RNA stabilization and HuR.

Malignant glioma (MG) cells up-regulate angiogenic factor expression in response to different extracellular signals such as hypoxia and cytokines. This up-regulation in turn promotes angiogenesis and tumor progression. Posttranscriptional gene regulation has been implicated as one mechanism for this tumor response, and we have previously shown that HuR, a protein associated with RNA stabilization, is overexpressed in MGs (L. B. Nabors et al., Cancer Res., 61: 2154-2161, 2001). Here, we demonstrate a marked up-regulation (RNA and protein) of tumor necrosis factor alpha (TNF-alpha), interleukin 8, and, to a lesser extent, vascular endothelial growth factor in U251 glioma cells after stimulation with TNF-alpha. RNA kinetic studies indicated that TNF-alpha induced the stabilization of all three transcripts. Using a luciferase reporter assay, we demonstrate that the AU-rich elements (AREs) in the 3'-untranslated region of these genes significantly contribute to this posttranscriptional regulation. UV cross-linking and immunoprecipitation with glioma extracts indicate that HuR binds to all three AREs. When HuR is overexpressed in glioma cells, there is enhanced RNA stabilization of all three angiogenic factor transcripts with a concomitant increase in mRNA and protein expression (up to 7-fold). These findings indicate that TNF-alpha up-regulates angiogenic factor expression in MG cells and that RNA stabilization, via the AREs in the 3'-untranslated region, contributes to this up-regulation.

3' Untranslated Regions↗
PubMed2005

Retroposition of processed pseudogenes: the impact of RNA stability and translational control.

Human processed pseudogenes are copies of cellular RNAs reverse transcribed and inserted into the nuclear genome by the enzymatic machinery of L1 (LINE1) non-LTR retrotransposons. Although it is generally accepted that germline expression is crucial for the heritable retroposition of cellular mRNAs, little is known about the influences of RNA stability, mRNA quality control and compartmentalization of translation on the retroposition of processed pseudogenes. We found that frequently retroposed human mRNAs are derived from stable transcripts with translation-competent functional reading frames that are resistant to nonsense-mediated RNA decay. They are preferentially translated on free cytoplasmic ribosomes and encode soluble proteins. Our results indicate that interactions between mRNAs and L1 proteins seem to occur at free cytoplasmic ribosomes.

Animals↗
PubMedDate not supplied

Controlling messenger RNA stability in bacteria: strategies for engineering gene expression.

Recent advances in the understanding of prokaryotic gene expression have led scientists to look beyond traditional promoter control for new methods of regulating gene expression. A promising, new technique centers on controlling the stability of messenger RNA. To exploit the potential of mRNA stability for gene expression control, it is important to understand the mechanisms of prokaryotic mRNA decay as well as the cellular factors that can be used to enhance bacterial gene expression through mRNA stabilization. Factors involved in controlling prokaryotic mRNA stability such as nucleases, secondary structures, translation influences, and transcription effects are discussed and analyzed within the context of three prevailing mRNA decay theories. Several strategies for manipulating mRNA stability in genetically-engineered cells are developed from these discussions and presented as a future direction in gene expression control. In the near future, it should be possible to use these strategies to control mRNA stability in such applications as pharmaceutical protein production and metabolic pathway design.

Bacteria↗
PubMed2004

Codon optimization reveals critical factors for high level expression of two rare codon genes in Escherichia coli: RNA stability and secondary structure but not tRNA abundance.

Expression patterns in Escherichia coli of two small archaeal proteins with a natural content of about 30% rare codons were analyzed. The proteins, a histone-like protein from Sulfolobus shibatae (Ssh10), and a glutaredoxin-like protein from Methanobacterium thermoautotrophicum (mtGrx), were produced with expression plasmids encoding wild-type genes, codon-optimized synthetic, and GST-fusion genes. These constructs were expressed in BL21 (DE3), its LysS derivative, and modified strains carrying copies for rare codon tRNAs or deletions in the RNAseE gene. Both Ssh10 and mtGrx expression levels were constitutively high in BL21(DE3) and its derivatives, with the exception of the LysS phenotype, which prevented high level expression of the Ssh10 wild-type gene. Surprisingly, a codon-optimized mtGrx gene construct displayed undetectable levels of protein production. The translational block observed with the synthetic mtGrx gene could be circumvented by using a synthetic mtGrx-glutathione S-transferase (GST) fusion construct or by in vitro translation. Taken together, the results underscore the importance of mRNA levels and RNA stability, but not necessarily tRNA abundance for efficient heterologous protein production in E. coli.

Archaeal Proteins↗
PubMed1998

On the role of magnesium ions in RNA stability.

Divalent cations, like magnesium, are crucial for the structural integrity and biological activity of RNA. In this article, we present a picture of how magnesium stabilizes a particular folded form of RNA. The overall stabilization of RNA by Mg2+ is given by the free energy of transferring RNA from a reference univalent salt solution to a mixed salt solution. This term has favorable energetic contributions from two distinct modes of binding: diffuse binding and site binding. In diffuse binding, fully hydrated Mg ions interact with the RNA via nonspecific long-range electrostatic interactions. In site binding, dehydrated Mg2+ interacts with anionic ligands specifically arranged by the RNA fold to act as coordinating ligands for the mental ion. Each of these modes has a strong coulombic contribution to binding; however, site binding is also characterized by substantial changes in ion solvation and other nonelectrostatic contributions. We will show how these energetic differences can be exploited to experimentally distinguish between these two classes of ions using analyses of binding polynomials. We survey a number of specific systems in which Mg(2+)-RNA interactions have been studied. In well-characterized systems such as certain tRNAs and some rRNA fragments these studies show that site-bound ions can play an important role in RNA stability. However, the crucial role of diffusely bound ions is also evident. We emphasize that diffuse binding can only be described rigorously by a model that accounts for long-range electrostatic forces. To fully understand the role of magnesium ions in RNA stability, theoretical models describing electrostatic forces in systems with complicated structures must be developed.

Cations, Divalent↗
PubMed1985

Drosophila ribosomal RNA stability increases during slow growth conditions.

We have developed density labeling pulse-chase methods which, in contrast to a conventional radiolabeling approach, allow us to determine the effectiveness of our chase and to measure RNA stability in vivo without measuring precursor pool specific activities. We have used these methods to determine the stability of the embryonic ribosomal RNA inherited by either normally or slowly growing Drosophila melanogaster larvae. If larvae are raised in a rich growth medium, embryonic rRNA decays with a half-life of 48 h. However, if larvae are raised in a poor growth medium, which slows larval growth and prolongs development, the half-life of rRNA increases to 115 h. This is the only example, of which we are aware, directly showing that rRNA half-life increases during slow growth conditions. We propose that the increased stability of rRNA that we find may enable slowly growing larvae to maintain the ribosome levels necessary to continue growth and development under conditions of nutrient deprivation.

Animals↗
PubMed1996

Angiotensinogen messenger RNA stabilization by angiotensin II.

OBJECTIVE: To further characterize the molecular mechanism whereby angiotensin II stabilizes the angiotensinogen messenger (m)RNA through binding studies of the previously isolated polysomal stabilizing protein to partial and mutagenized sequences of the 3' untranslated region of the gene and to explore its importance to rodent genetic hypertension. DESIGN: Analysis of angiotensinogen mRNA mutants for half-life and binding to a polysomal protein with a molecular weight of 12000. METHODS: Protein/RNA interactions were determined in band shift assays employing radiolabelled 3' untranslated region of angiotensinogen mRNA. Measurement of the mRNA half-life used a cell-free incubation system and 3' untranslated region DNA sequences were polymerase chain reaction (PCR) cloned and sequenced. Sequences of normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rat (SHR) strains were compared. Point mutations were introduced by site directed mutagenesis. RESULTS: The angiotensinogen 3' untranslated region exhibited specific binding to the polysomal 12000 molecular weight protein which, in an in vitro incubation system, increased 10-fold the half-life of full-length angiotensinogen mRNA; no effect was observed with 3' deleted angiotensinogen mRNA indicating a regulatory function of protein at the 3' untranslated region. Sequence analysis of PCR amplified DNA fragments identified a (G-->C) point mutation in the La Jolla colony SHR. Following introduction of this point mutation into wild-type 3' untranslated regions, protein binding significantly increased (wild-type binding constant, 19 mumol/l; mutant binding constant 3.5 mumol/l), indicating that this point mutation affects 3' untranslated region secondary structure, binding of the RNA stabilizing protein and, consequently, the half-life of angiotensinogen mRNA. Deletion of a U-rich region (position 1609-1613, UCCUU) expressed twice in the 3' untranslated region almost completely abolished protein binding suggesting this sequence as one part of the putative binding motif in the 3' untranslated region. CONCLUSIONS: Angiotensin II regulates hepatic angiotensinogen synthesis and secretion by inhibiting degradation of angiotensinogen mRNA by the action of a polysomal protein. Mutations in the 3' untranslated region mRNA coding sequence alter binding and half-life and may significantly affect the half-life of angiotensinogen mRNA thereby altering the secretion rate of angiotensinogen.

Angiotensin II↗
PubMed1990

Induction of RNA-stabilized DNA conformers by transcription of an immunoglobulin switch region.

A deletion DNA rearrangement is associated with immunoglobulin class switching from IgM to IgG, IgA or IgE This recombination occurs in immunoglobulin switch regions, which are complex, highly repetitive regions of DNA. As switch regions become transcriptionally active just before switch recombination, analysis of the behaviour of these sequences during transcription could elucidate the mechanism of switch recombination. Here, we report that transcription of a supercoiled plasmid containing the murine IgA switch region (S alpha) leads to a loss of superhelical turns. The resulting series of less supercoiled plasmids is stabilized by RNA-DNA hybrids formed by the nascent RNA transcripts, which remain base-paired with their DNA templates.

Base Composition↗
PubMed2005

Microarray analysis confirms the specificity of a Chlamydomonas reinhardtii chloroplast RNA stability mutant.

The expression of chloroplast and mitochondrial genes depends on nucleus-encoded proteins, some of which control processing, stability, and/or translation of organellar RNAs. To test the specificity of one such RNA stability factor, we used two known Chlamydomonas reinhardtii nonphotosynthetic mutants carrying mutations in the Mcd1 nuclear gene (mcd1-1 and mcd1-2). We previously reported that these mutants fail to accumulate the chloroplast petD mRNA and its product, subunit IV of the cytochrome b6/f complex, which is essential for photosynthesis. Such mutants are generally presumed to be gene specific but are not tested rigorously. Here, we have used microarray analysis to assess changes in chloroplast, mitochondrial, and nuclear RNAs, and since few other RNAs were significantly altered in these mutants, conclude that Mcd1 is indeed specifically required for petD mRNA accumulation. In addition, a new unlinked nuclear mutation was discovered in mcd1-2, which greatly reduced chloroplast atpA mRNA accumulation. Genetic analyses showed failure to complement mda1-ncc1, where atpA-containing transcripts are similarly affected (D. Drapier, J. Girard-Bascou, D.B. Stern, F.-A. Wollman [2002] Plant J 31: 687-697), and we have named this putative new allele mda1-2. We conclude that DNA microarrays are efficient and useful for characterizing the specificity of organellar RNA accumulation mutants.

Animals↗