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Results for “RNA Stability”

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PubMed2003

RBP38, a novel RNA-binding protein from trypanosomatid mitochondria, modulates RNA stability.

We describe here the isolation and characterization of a novel RNA-binding protein, RBP38, from Leishmania tarentolae mitochondria. This protein does not contain any known RNA-binding motifs and is highly conserved among the trypanosomatids, but no homologues were found in other organisms. Recombinant LtRBP38 binds single and double-stranded (ds) RNA substrates with dissociation constants in the 100 nM range, as determined by fluorescence polarization analysis. Downregulation of expression of the homologous gene, TbRBP38, in procyclic Trypanosoma brucei by using conditional dsRNA interference resulted in 80% reduction of steady-state levels of RNAs transcribed from both maxicircle and minicircle DNA. In organello pulse-chase labeling experiments were used to determine the stability of RNAs in mitochondria that were depleted of TbRBP38. The half-life of metabolically labeled RNA decreased from approximately 160 to approximately 60 min after depletion. In contrast, there was no change in transcriptional activity. These observations suggest a role of RBP38 in stabilizing mitochondrial RNA.

Amino Acid Sequence↗

PubMed2004

The effect of sample degradation and RNA stabilization on classical swine fever virus RT-PCR and ELISA methods.

Classical swine fever (CSF), also known as hog cholera, is a highly contagious viral infection of swine caused by a member of the genus pestivirus of the family, Flaviviridae. The need for accurate laboratory diagnosis of CSF is particularly important as it is more reliable than clinical diagnosis. CSF is endemic in many tropical countries where the climate is characterized by high ambient temperature and humidity. This study details the effect of sample quality on CSF antigen-capture ELISA (AC-ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR) methods. RT-PCR assessment of AC-ELISA-positive spleen samples stored in a conventional glycerol/saline buffer demonstrated that the RT-PCR was detrimentally affected by poor sample quality. To provide a more accurate representation of this effect, a 14 days study was performed to determine the effect of tropical ambient conditions on CSF virus-positive spleen samples stored in two transport media; glycerol/saline and a proprietary RNA preservation solution (RNAlater). A protective effect was demonstrated in both assays with RNAlater as samples were positive in both assays until day 14 post-exposure. Samples stored in glycerol/saline were negative at RT-PCR at day 3 post-exposure although AC-ELISA was still positive at day 14 post-exposure.

Animals↗

PubMed2006

The role of transforming growth factor beta signaling in messenger RNA stability.

Transforming growth factor beta (TGF-beta) is a biologically multipotent regulatory protein implicated in functions that include the regulation of cellular growth, differentiation, extracellular matrix formation, and wound healing. It also plays a role in the pathologies of Alzheimer's disease, cancer and autoimmune disorders. TGF-beta modulates gene expression by affecting transcriptional activation and mRNA turnover rate. Steady-state mRNA levels depend on both the transcriptional activity and mRNA half-life. The stability of mRNA can be modified by the binding of trans-acting factors to cis-elements on the message. These can protect the mRNA from cleavage by RNAses, or they may promote mRNA cleavage. Changes in mRNA stability can lead to changes in the proteome and subsequently in cellular metabolism. The SMAD family of proteins has been implicated in the transduction of the TGF-beta signal, where they regulate transcriptional activity. This review attempts to provide new insights into the role played by TGF-beta in the regulation of mRNA turnover.

3' Untranslated Regions↗

PubMed2006

Differential regulation of the tyrosine hydroxylase and enkephalin neuropeptide transmitter genes in rat PC12 cells by short chain fatty acids: concentration-dependent effects on transcription and RNA stability.

At physiologic concentrations, butyrate regulates the expression of individual genes involving at least three mechanisms: (i) through induction of cis- and trans-acting butyrate-dependent transcription factors for selected genes, (ii) by inhibition of histone deacetylation and attendant chromatin remodeling and (iii) by affecting turnover of mRNAs. Our previous work illustrated gradual accumulation of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis and the neuropeptide transmitter proenkephalin (ppEnk) in butyrate-differentiated PC12 cells (Nankova, B.B., Chua, J., Mishra, R., Kobasiuk, C.D., La Gamma, E.F. 2003. Nicotinic induction of preproenkephalin and tyrosine hydroxylase gene expression in butyrate-differentiated rat PC12 cells: a model for adaptation to gut-derived environmental signals. Pediatr. Res. 53, 113-118.). However, at higher physiological concentrations (6 mM), TH mRNA levels are significantly reduced while ppEnk mRNA transcripts remained elevated. These differential effects suggest suppression of endogenous TH gene transcription, targeted degradation of TH mRNA or both. By using nuclear run-on assays, we found that transcription increased for both endogenous TH and ppEnk genes, even at time points and concentrations when reduced steady-state levels of TH mRNA were observed. The reduction in TH mRNA was blocked by cycloheximide consistent with a protein-dependent mechanism. We also observed a dose-dependent accumulation of luciferase reporter molecules driven by TH promoter in transient transfection experiments, data that provide additional support for separate regulatory pathways. Significantly, butyrate-dependent decreases in TH mRNA were also reflected in a reduction in TH protein. Our results suggest a novel mode of regulation for TH by butyrate operating via both transcriptional and post-transcriptional mechanisms. We speculate that, depending on plasma concentrations of butyrate, this naturally occurring signaling molecule can function as an in vivo molecular switch to alter levels of TH mRNA, its protein and thus the biosynthesis of endogenous catecholamines.

Animals↗

PubMed1999

Calcium ionophore upregulation of AUUUA-specific binding protein activity is contemporaneous with granulocyte macrophage colony-stimulating factor messenger RNA stabilization in AML14.3D10 cells.

Eosinophils produce granulocyte macrophage colony-stimulating factor (GM-CSF), which enhances their survival and function. In T cells and fibroblasts, GM-CSF production is controlled predominantly by variable messenger RNA (mRNA) stability involving 3' untranslated region (3' UTR) adenosine-uridine-rich elements (AREs) and sequence-specific mRNA binding proteins. However, the mode of regulation of this critical cytokine remains unknown in eosinophils. Therefore, we measured GM-CSF mRNA decay in an eosinophil-like cell line (AML14.3D10) and, with a radiolabeled GM-CSF RNA probe, asked whether ARE-specific, mRNA binding proteins were present in cytoplasmic lysates of these cells. Human GM-CSF mRNA transfected into unstimulated AML14.3D10 cells decayed with a half-life of 6 min, which increased to 14 min after 1 h, and to 22 min after 2 h, of ionophore-mediated activation. GM-CSF RNA mobility shift assays using cytoplasmic extracts from resting or ionophore-stimulated AML14.3D10 cells revealed multiple RNA-protein complexes of 55, 60, 85, 100, and 125 kD. A 47-kD complex was also detected with an 80-base RNA probe containing four consecutive AUUUA motifs. On the basis of competition studies, all of the observed binding protein activities interacted with the 3' UTR AREs. In addition, binding activity increased 2.5-fold in cytoplasmic lysates from cells stimulated with calcium ionophore for 2 h, contemporaneous with GM-CSF mRNA stabilization. These data provide direct evidence that ionophore stabilizes GM-CSF mRNA in AML14.3D10 cells and simultaneously increases the activity of a series of AUUUA-specific mRNA binding proteins. We conclude that the interaction of AU-specific binding proteins may stabilize GM-CSF mRNA in activated eosinophil-like cell lines.

3' Untranslated Regions↗

PubMed1990

Cytoskeletal messenger RNA stability in human neocortex: studies in normal aging and in Alzheimer's disease.

Total RNA was extracted from human brain temporal and parietotemporal neocortical grey matter with postmortem intervals (PMI) of up to 13.5 hours. The integrity and rank abundance of heterogeneous nuclear RNA (HnRNA) and messenger RNA (mRNA) were analyzed by Northern gel dot blot hybridization with specific cloned probes of neurobiological interest: the RNA messages for four cytoskeletal components including glial fibrillary acidic protein (GFAP), alpha-tubulin, beta-actin and the human neurofilament light chain (HNF-L) genomic sequence, the Alu repetitive element, the scrapie prion PrP DNA probe and the chromatin condensing agent linker histone H1(0) genomic probe. Our observations indicate that for the cytoskeletal RNA messages studied here: (1) short postmortem intervals (of up to 4.5 hours) had only small effects upon RNA quality in these neocortices, (2) GFAP and HNF-L transcripts were represented at relatively high levels in the cerebral neocortex and (3) each RNA species in normal human brain had both unique and characteristic intracellular levels of abundance and decay kinetics. In the pathological condition, Alzheimer's disease (AD), cells of the temporal and parietotemporal neocortices of afflicted brains showed selective reductions in cytoskeletal RNA pool size which are not attributable to RNA transcript stability.

Adult↗

PubMed2001

Phosphorylation of the Saccharomyces cerevisiae La protein does not appear to be required for its functions in tRNA maturation and nascent RNA stabilization.

An abundant nuclear phosphoprotein, the La autoantigen, is the first protein to bind all newly synthesized RNA polymerase III transcripts. Binding by the La protein to the 3' ends of these RNAs stabilizes the nascent transcripts from exonucleolytic degradation. In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the La protein is required for the normal pathway of tRNA maturation. Experiments in which the human protein was expressed in S. pombe have suggested that phosphorylation of the La protein regulates tRNA maturation. To dissect the role of phosphorylation in La protein function, we used mass spectrometry to identify three sites of serine phosphorylation in the S. cerevisiae La protein Lhp1p. Mutant versions of Lhp1p, in which each of the serines was mutated to alanine, were expressed in yeast cells lacking Lhp1p. Using two-dimensional gel electrophoresis, we determined that we had identified and mutated all major sites of phosphorylation in Lhp1p. Lhp1p lacking all three phosphorylation sites was functional in several yeast strains that require Lhp1p for growth. Northern blotting revealed no effects of Lhp1p phosphorylation status on either pre-tRNA maturation or stabilization of nascent RNAs. Both wild-type and mutant Lhp1 proteins localized to both nucleoplasm and nucleoli, demonstrating that phosphorylation does not affect subcellular location. Thus, although La proteins from yeast to humans are phosphoproteins, phosphorylation does not appear to be required for any of the identified functions of the S. cerevisiae protein.

Amino Acid Sequence↗

PubMed2004

Messenger RNA stability in mitochondria: different means to an end.

Gene expression is regulated at many stages not merely at the level of transcription. Among the important post-transcriptional processes, RNA turnover has a crucial role. The stability of mRNA in the cytosol of eukarya is increased by the addition of a 3' poly(A) extension. By contrast, this process mediates rapid RNA decay in prokarya. How is mRNA turnover regulated in mitochondria? Their monophyletic, alpha-proteobacterial origin predicts that polyadenylation will induce rapid decay by nucleases and associated factors that are similar to their bacterial ancestors. In this article, however, we report that the regulation of mitochondrial mRNA turnover in diverse species is surprisingly different.

Animals↗

PubMed2005

Yeast caspase 1 links messenger RNA stability to apoptosis in yeast.

During the past years, yeasts have been successfully established as models to study the mechanisms of apoptotic regulation. We recently showed that mutations in the LSM4 gene, which is involved in messenger RNA decapping, lead to increased mRNA stability and apoptosis in yeast. Here, we show that mitochondrial function and YCA1, which encodes a budding yeast metacaspase, are necessary for apoptosis triggered by stabilization of mRNAs. Deletion of YCA1 in yeast cells mutated in the LSM4 gene prevents mitochondrial fragmentation and rapid cell death during chronological ageing of the culture, diminishes reactive oxygen species accumulation and DNA breakage, and increases resistance to H2O2 and acetic acid. mRNA levels in lsm4 mutants deleted for YCA1 are still increased, positioning the Yca1 budding yeast caspase as a downstream executor of cell death induced by mRNA perturbations. In addition, we show that mitochondrial function is necessary for fast death during chronological ageing, as well as in LSM4 mutated and wild-type cells.

Apoptosis↗

PubMed1994

Genotoxic stress confers preferential and coordinate messenger RNA stability on the five gadd genes.

The growth arrest and DNA damage-inducible (gadd) genes represent a group of five stress-inducible genes that are coordinately regulated at the transcriptional level. Posttranscriptional regulation of gadd153, gadd45, gadd34, gadd33, and gadd7 was studied after exposure to DNA-damaging agents or other growth arrest treatments in hamster cells. Relative transcript levels were measured following treatment with the transcriptional inhibitor actinomycin D. After exposure to methylmethane sulfonate or UV radiation, all five gadd messages demonstrated a coordinate increase in mRNA stability compared to untreated exponentially growing cells. This enhanced stability was not an universal response to genotoxic stress since other DNA damage-inducible genes, such as c-jun and c-fos, did not show an appreciable increase in mRNA half-life. In contrast, induction of growth arrest by media depletion (starvation) or by treatment with the growth inhibitor prostaglandin A2 did not induce such an increase in mRNA stability in all gadd genes. Comparison of overall RNA turnover by 3H labeling of total cellular RNA also indicated that the preferential stabilization of the gadd transcripts by DNA-damaging agents was not an artifactual response due to variations in overall RNA metabolism within each treatment group. However, DNA-damaging agents were ineffective in inducing stabilization of gadd153 mRNA in growth-arrested cells. This suggest that the signal(s) that give rise to gadd mRNA stability may also be affected by the state of cellular proliferation. Together, these results suggest that the global posttranscriptional response of the gadd genes to DNA-damaging agents is specific and unique to actively growing cells, and further implicates the role of the gadd genes in the DNA damage response of cycling cells.

Animals↗

PubMed2001

The Telomerase/vault-associated protein TEP1 is required for vault RNA stability and its association with the vault particle.

Vaults and telomerase are ribonucleoprotein (RNP) particles that share a common protein subunit, TEP1. Although its role in either complex has not yet been defined, TEP1 has been shown to interact with the mouse telomerase RNA and with several of the human vault RNAs in a yeast three-hybrid assay. An mTep1(-/-) mouse was previously generated which resulted in no apparent change in telomere length or telomerase activity in six generations of mTep1-deficient mice. Here we show that the levels of the telomerase RNA and its association with the telomerase RNP are also unaffected in mTep1(-/-) mice. Although vaults purified from the livers of mTep1(-/-) mice appear structurally intact by both negative stain and cryoelectron microscopy, three-dimensional reconstruction of the mTep1(-/-) vault revealed less density in the cap than previously observed for the intact rat vault. Furthermore, the absence of TEP1 completely disrupted the stable association of the vault RNA with the purified vault particle and also resulted in a decrease in the levels and stability of the vault RNA. Therefore, we have uncovered a novel role for TEP1 in vivo as an integral vault protein important for the stabilization and recruitment of the vault RNA to the vault particle.

Animals↗

PubMed2003

Trimethylamine N-oxide stabilizes RNA tertiary structure and attenuates the denaturating effects of urea.

Trimethylamine N-oxide (TMAO) and urea are osmolytes. Osmolytes allow cells to remain viable in harsh or extreme environments. Both TMAO and urea are found in shark and rays at approximate molar ratios of 1:2, respectively. At this ratio TMAO nearly completely counteracts the destabilizing effects that urea has on proteins. We ask whether RNA, which is denatured by urea, is stabilized by TMAO in a manner similar to that seen for proteins. We found that TMAO stabilizes Escherichia coli tRNAfmet tertiary structure and counteracts the denaturing effects of urea at the same ratios found for proteins. Cation binding usually drives RNA tertiary structure formation. These results suggest that tertiary structure stability is not only sensitive to cations but also to the aqueous composition and properties of the solvent. We propose that tertiary structure folding is driven by unfavorable interactions between TMAO and the phosphodiester backbone.

Escherichia coli↗

PubMed1991

Cytokine messenger RNA stability is enhanced in tumor cells.

Hematopoietic growth factors are produced by a number of human tumors. We extracted RNA from selected human tumor cells known to produce at least one hematopoietic growth factor and found high levels of abnormally stable cytokine messenger (m)RNA. Half-life experiments performed after preventing RNA synthesis by exposing cells to actinomycin D before RNA extraction showed stabilization of cytokine messages in tumor cells in liquid culture as well as in human tumor xenografts grown in mice. Exposure to the phorbol ester phorbol 12-myristate 13-acetate (TPA) caused enhancement of granulocyte-macrophage colony-stimulating factor (GM-CSF) message level in lung cancer cells and in control fibroblasts but elevated levels persisted far longer in the tumor cells. In normal cells, an AU-rich sequence in the 3' untranslated region of cytokine mRNAs confers lability to the message. Although a beta-globin gene expression vector containing this region appears to produce unstable mRNA in lung cancer cells, cytokine mRNAs, which also contain this sequence, are very stable in the tumors we studied. This may indicate that another region of the cytokine mRNA molecule is of greater importance than the AU-rich region in determining mRNA stability in tumor cells.

Adenocarcinoma↗

PubMed1995

Mutational analysis of human U6 RNA: stabilizing the intramolecular helix blocks the spliceosomal assembly pathway.

U6 RNA undergoes several conformational transitions during the spliceosome cycle: after the interaction with U4, the singular form of U6 is converted into the U4-U6 base-paired form, and within the spliceosome, the U4-U6 duplex isomerizes into the active U6-U2 conformation. The secondary structure of the singular form contains an extended 3' stem-loop, the upper part of which (intramolecular helix) most likely reforms in the spliceosome. We have previously shown in the mammalian splicing complementation system that the loop and the three adjacent, highly conserved base pairs of the intramolecular helix function during both the U4-U6 interaction and the first step of splicing. Here we demonstrate that the balanced stability of the lower, less conserved part of the 3' stem-loop is also critical for U4-U6 interaction; however, no specific splicing function could be detected in this region. The analysis of the heterologous interaction between mammalian U4 snRNP and yeast U6 RNA derivatives suggests that there are--in addition to the 3' loop and the stability of the intramolecular helix--specific sequence determinants in the 3' terminal domain of U6 that are important for efficient U4/U6 snRNP assembly.

Base Composition↗

PubMed1989

Enhanced messenger RNA stability and differentiation of HL 60 cells treated with 1,25-dihydroxyvitamin D3 and cordycepin.

The effect of inhibitors of RNA synthesis on 1,25(OH)2 vitamin D3-induced monocytic differentiation was studied in a well-differentiating clone AB 47 of HL 60 cells. The concentrations of these inhibitors were chosen to permit the maintenance of cell viability for at least 48 hours, and resulted in 40-60% inhibition of total cellular RNA synthesis. No impairment of 1,25(OH)2 vitamin D3-induced monocytic differentiation was observed with all inhibitors tested, and the presence of cordycepin actually enhanced differentiation. The phenotypic evidence of monocytic differentiation correlated with the increased levels of mRNA for c-fos and c-fms measured by hybridization to appropriate nick-translated cDNA probes. In contrast, nuclear run-on experiments showed the expected inhibition of transcription of these genes by the compounds used. The data suggest that interference by external agents with transcription of genes essential for a differentiation program brings into play compensatory mechanisms which permit the program to continue. Thus, differentiation appears to have a high priority among various competing intracellular pathways in 1,25(OH)2 vitamin D3-treated HL 60 cells. Stabilization of messenger RNA levels evident in this study may therefore represent a general cellular mechanism for the correction of unwanted effects of xenobiotics on the cell.

Antimetabolites, Antineoplastic↗

PubMed2002

Delivery of unmodified bioactive ribozymes by an RNA-stabilizing polyethylenimine (LMW-PEI) efficiently down-regulates gene expression.

The sequence-specific cleavage of RNA molecules through ribozyme targeting is particularly attractive since it allows the effective abrogation of protein expression. So far, however, use of enzymatically active RNA molecules (ribozymes) has, without chemical modification, been severely hampered by ribozyme instability and poor cellular uptake. In this paper, we present a method for protection and cellular delivery of ribozymes by complexation with a low molecular weight polyethylenimine (LMW-PEI). We show that LMW-PEI almost completely stabilizes ribozymes or any RNA against degradation in vitro. Upon their highly efficient cellular uptake, non-toxic LMW-PEI-complexed ribozymes display intracellular bioactivity already at low concentrations as demonstrated by down-regulation of two different genes in different cell lines. In vivo, LMW-PEI-complexed ribozymes were stabilized after intraperitoneal (i.p.) injections, showed prolonged circulation time and intact ribozymes were detected in the subcutaneous (s.c.) tumor mass 60 min after the injection. In addition, i.p. injections of LMW-PEI-complexed ribozymes targeted against the growth factor pleiotrophin (PTN) resulted in marked reduction of s.c. human melanoma tumor growth and of intratumoral PTN levels in a mouse xenograft model. Thus, this paper describes a novel method for exogenous delivery of any bioactive RNA ribozyme in vitro and in vivo without chemical modification.

Animals↗

PubMed1993

Homology with Saccharomyces cerevisiae RNA14 suggests that phenotypic suppression in Drosophila melanogaster by suppressor of forked occurs at the level of RNA stability.

The suppressor of forked [su(f)] locus of Drosophila melanogaster encodes at least one cell-autonomous vital function. Mutations at su(f) can affect the expression of unlinked genes where retroviral-like transposable elements are inserted. Changes in phenotype are correlated with changes in mRNA profiles, indicating that su(f) affects the production and/or stability of mRNAs. We have cloned the su(f) gene by P-element transposon tagging. Alterations in the DNA map of eight lethal alleles were detected in a 4.3-kb region. P-element-mediated transformation using a fragment including this interval rescued all aspects of the su(f) mutant phenotype. The gene is transcribed to produce a major 2.6-kb RNA and minor RNAs of 1.3 and 2.9 kb, which are present throughout development, being most abundant in embryos, pupae, and adult females. The major predicted gene product is an 84- kD protein that is homologous to RNA14 of Saccharomyces cerevisiae, a vital gene where mutation affects mRNA stability. This suggests that phenotypic modification by su(f) occurs at the level of RNA stability.

Amino Acid Sequence↗

PubMed1985

Regulation of messenger RNA stability in mouse erythroleukemia cells.

The decay rates of several messenger RNA species were determined in mouse erythroleukemia cells. The t1/2 values for the actin and tubulin mRNAs were 16 to 26 hours and about seven hours, respectively. The globin mRNA, and two mRNA species subject to translation repression, the P40 and P21 mRNAs, were about as stable as the ribosomal RNA. A stable tubulin mRNA component also appeared to be present in the cells. Exposure of the cells to dimethylsulfoxide for 48 hours led to considerable increases in the rates of decay of all but the globin mRNA. The induction of erythroid differentiation caused by the drug appears to lead to activation of a mRNA-degradation process that affects individual species to different degrees. The newly synthesized actin and tubulin mRNAs lost their poly(A) rather rapidly. This was accompanied by accumulation of poly(A)-deficient mRNA chains, particularly in the case of actin mRNA. The steady-state distribution of mRNA components, determined by Northern blot analysis, also showed that the actin mRNA and one tubulin mRNA species have a high proportion of poly(A)-deficient molecules. The globin, P40 and P21 mRNAs showed little tendency to lose their poly(A) sequence. The steady-state globin and P40 mRNAs also had a low proportion of chains depleted of poly(A). For all five species, the proportions of poly(A)-deficient chains in newly synthesized mRNA were about the same in uninduced and induced cells, in spite of the large decreases in mRNA stability in the induced cells. The lack of correlation between tendency to lose poly(A) and rate of mRNA decay, and the large accumulation of poly(A)-deficient molecules in the cases of the actin and tubulin mRNAs suggest that the stability of mRNA is not determined solely by the presence of poly(A) on the RNA chains. The behavior of the untranslated species in induced and uninduced cells also fails to support the notion of a relationship between translation and mRNA decay.

Actins↗