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At least 19 recordsLinked to original sources
PubMed1975

Structure of B77 sarcoma virus RNA: stabilization of RNA after packaging.

Extracellular maturation of Bratislava 77(B77) sarcoma virus RNA involves a stabilization of linkage between 35S subunits since the Tm of 60 to 70S RNA in the presence of 0.5 M NaCl increases from 56 to 67.5 C as the age of the virus increases. This stabilization process is strongly temperature dependent; the rate at 45 C is increased fourfold over the rate at 37 C. As a result of the instability of the immature RNA, denaturation to subunits occurs at room temperature during phenol extraction. This dissociation can be prevented by increasing the NaCl concentration during the extraction. The data support a model which proposes that RNA subunits of oncornaviruses exist as assembled 60 to 70S RNA even in immature virions but that the linkages between subunits are stabilized as a function of time. These linkages appear to be maintained by nucleotide base pairing rather than by protein. However, isolated RNA does not undergo stabilization, suggesting that some other component of the virion is necessary for the process to occur.

Avian Sarcoma Viruses↗
PubMed2001

Roles of RNA:DNA hybrid stability, RNA structure, and active site conformation in pausing by human RNA polymerase II.

Human RNA polymerase II recognizes a strong transcriptional pause signal in the initially transcribed region of HIV-1. We report the use of a limited-step transcription assay to dissect the mechanism underlying recognition of and escape from this HIV-1 pause. Our results suggest that the primary determinant of transcriptional pausing is a relatively weak RNA:DNA hybrid that triggers backtracking of RNA polymerase II along the RNA and DNA chains and displaces the RNA 3' OH from the active site. In contrast, two alternative RNA secondary structures, TAR and anti-TAR, are not required for pausing and affect it only indirectly, rather than through direct interaction with RNA polymerase II. TAR accelerates escape from the pause, but anti-TAR inhibits formation of TAR prior to pause escape. The behavior of RNA polymerase II at a mutant pause signal supports a two-step, non-equilibrium mechanism in which the rate-determining step is a conformational change in the enzyme, rather than the changes in nucleic-acid base-pairing that accompany backtracking.

Base Pairing↗
PubMed1983

RNA stabilization and continued RNA processing following nuclear dissolution in maturing Xenopus laevis oocytes.

The Xenopus laevis oocyte provides a convenient system for studying cell cycle-induced changes in RNA metabolism. Initially arrested at first meiotic prophase, it can be induced in vitro to undergo maturation by incubation in the presence of progesterone, progressing to second meiotic metaphase over the course of several hours. The dissolution of the nuclear membrane before the onset of first meiotic metaphase (also known as germinal vesicle breakdown, or GVBD) provides a convenient indication of the overall rate of maturation. It is reported that all ordinarily unstable classes of nuclear RNA show substantial increases in stability following GVBD. However, some radioactivity in rRNA precursors disappears following GVBD and this is quantitatively accounted for by the appearance of label in mature rRNA. These results suggest continued processing of rRNA precursors after GVBD.

Animals↗
PubMed1996

Effect of dexamethasone on insulin receptor mRNA levels, RNA stability and isotype RNA pattern in U-937 human promonocytic cells.

The administration of 5 x 10(-6) mol/l dexamethasone transiently increased insulin receptor (IR) mRNA levels in U-937 human promonocytic cells, which reached the maximum level at 15 h of treatment. Dexamethasone treatment did not affect the IR mRNA half-life (approximately 4 h), suggesting that the increase is regulated at the transcriptional level. The stimulatory action of dexamethasone was not prevented by the simultaneous presence of the protein synthesis inhibitor cycloheximide, indicating that the induction of IR gene transcription occurs as a direct response to the action of the synthetic glucocorticoid. Finally, the A isoform (lacking exon 11) was found to be the only IR isoform present in both untreated and dexamethasone treated-U-937 cells.

Cell Line↗
PubMed2005

Assessment of a new RNA stabilizing reagent (Tempus Blood RNA) for minimal residual disease in onco-hematology using the EAC protocol.

Standardization efforts of real time quantitative PCR (RQ-PCR), such as the Europe Against Cancer (EAC) protocol, are actually essential. However, all the stages of the preanalytical phase (blood collection, conservation and procedures of cellular separation) which influence the ex vivo expression of many genes are not controlled. Various kits for stabilizing the RNA at the time of blood collection were developed: PAXgene Blood kit (Qiagen) and the new Tempus Blood RNA kit (Applied Biosystems). PAXgene Blood was already validated to monitor minimal residual disease (MRD) in onco-hematologic pathologies. In order to evaluate the Tempus Blood RNA kit, it was compared to unstabilized EDTA blood/Ficoll/TRIzol protocol and PAXgene Blood kit. The RNAs extracted by the different methods were assessed for quantity, quality and detection of control genes (GUS and ABL) and fusion gene transcripts BCR-ABL and TEL-AML1 by RQ-PCR. Our study shows that using the EAC protocol, the new Tempus Blood RNA kit allows the detection of fusion gene transcript with the same sensitivity as the two other protocols. Altogether, our data suggest the possible use of such a technology for MRD follow-up in myeloproliferative diseases and acute leukemias. So, further multicentric studies on leukemic patients should be performed to validate this technique in these applications.

Cell Line, Tumor↗
PubMed1987

Transcription and RNA stability are important determinants of higher plant chloroplast RNA levels.

Transcription in lysed barley plastids and Northern slot blot analyses were used to determine the relationship between changes in RNA levels and transcription during plastid development. Transcription in plastids of 4.5-9-day-old dark-grown or illuminated barley seedlings declined up to 10-fold as a function of plant age. Decreased transcription of some plastid genes (rbcL, psaA-psaB) was paralleled by decreased levels of mRNA. In other cases (16SrDNA, psbA) the changes in transcription were not followed by proportional changes in RNA levels indicating that RNA stability is important in establishing the amount of plastid RNA for these genes. Further analysis showed that transcription of the plastid rRNA transcription unit is regulated differently than the transcription of protein coding genes such as psbA or rbcL.

Journal Article↗
PubMed2001

HuR, a RNA stability factor, is expressed in malignant brain tumors and binds to adenine- and uridine-rich elements within the 3' untranslated regions of cytokine and angiogenic factor mRNAs.

Tumors of the central nervous system (CNS) often have sustained expression of labile genes, including angiogenic growth factors and immunosuppressive cytokines, which promote tumor progression. Stabilization of the RNA transcripts for these genes, such as vascular endothelial growth factor (VEGF), is an important molecular pathway for this up-regulation. HuR, a member of the Elav family of RNA-binding proteins, has been implicated in this pathway through its binding to adenine and uridine (AU)-rich stability elements (ARE) located in the 3' untranslated regions (3'-UTRs) of the mRNA. Whereas three of the Elav family members (Hel-N1, HuC, and HuD) are restricted to young and mature neurons, HuR is more broadly expressed, including proliferating cells of the developing CNS. Because RNA stabilization of labile genes may promote tumor growth, we analyzed and compared the expression pattern of HuR in 35 freshly resected and cultured CNS tumors to determine whether there was any correlation with tumor grade or histological type. We found that HuR mRNA was consistently expressed in all of the tumors, regardless of cell origin or degree of malignancy. Using a novel HuR-specific polyclonal antibody, we found that strong HuR protein expression was limited to high-grade malignancies (glioblastoma multiforme and medulloblastoma). Within the glioblastoma multiforme, prominent HuR expression was also detected in perinecrotic areas in which angiogenic growth factors are up-regulated. To further define its role as a potential RNA stabilizer, we analyzed whether HuR could bind to the stability motifs within the 3'-UTRs of cytokines and growth factors linked to brain tumor progression. We used a novel ELISA-based RNA binding assay and focused on the 3'-UTRs of angiogenic factors VEGF, COX-2, and (interleukin) IL-8 as well as the immunomodulating factors IL-6, transforming growth factor (TGF)-beta and tumor necrosis factor (TNF)-alpha as potential RNA ligands. Our results indicated overall a very high binding affinity to these RNA targets. A comparison of these ligands revealed a hierarchy of binding affinities with the angiogenic factors, and TGF-beta showing the highest (Kd of 1.8-3.4 nM), and TNF-alpha the lowest (Kd of 18.3 nM). The expression pattern of HuR, coupled with the RNA binding data, strongly suggests a role for this protein in the posttranscriptional regulation of these genes in CNS tumors.

3' Untranslated Regions↗
PubMed1990

Analysis of RNA stability and (-) strand content in viral infections using biotinylated RNA probes.

Non-radioactive biotinylated RNA probes specific for plus (+) and minus (-) sense RNAs of brome mosaic virus (BMV) were synthesized in vitro from a plasmid bearing a 200 base pair fragment complementary to the 3' terminus of each of the three genomic RNAs of the virus. Using virion RNAs isolated from BMV infected barley plants, the sensitivity of biotinylated RNAs as hybridization probes was compared with that of 32P-labeled probes in Northern hybridization assays. Although the sensitivity of biotinylated and 32P-labeled probes is similar (approximately 5 pg), the time required to detect the RNA bands was much less than for autoradiography; (-) sense RNAs could be detected in 30 min whereas 48 h or more were required by autoradiography. The value of biotinylated probes for following RNA stability was exemplified by the detection of supplied inocula in protoplasts 24 h post-inoculation. Quantitation of relative accumulation of progeny (+) and (-) sense RNAs by densitometry of the Northern blots probed with biotinylated RNAs paralleled that of radiolabeled probes. The application of these probes was extended to the detection of RNAs in barley protoplasts and BMV infected plant sap by dot hybridization. In these tests, viral RNAs were detected in as few as 250 protoplasts and sap dilutions up to 1:2000. The merits of these non-radioactive probes in monitoring the replication events by the detection and quantitation of mutant progeny RNAs of BMV are discussed.

Biotin↗
PubMed1991

Protein overproduction in Escherichia coli: RNA stabilization, cell disruption and recovery with a cross-flow microfiltration membrane.

After optimizing overproduction of a heterologous gene product (chloramphenicol acetyltransferase, CAT) using an RNA stabilization vector * in Escherichia coli (Chan et al., 1988), a single step cell disruption and recovery method * for obtaining a product stream essentially free of cell debris was developed. The behavior of an RNA stabilization plasmid (pKTN-CAT) containing stabilizing intron RNA was investigated in two different media both in batch and chemostat modes. CAT production of pKTN-CAT was consistently higher (3- to 7-fold) than that of the control lacking the stabilization sequences (pK-CAT). Highest CAT production was observed for cells grown in minimal medium in batch mode and induced for CAT expression early in growth. CAT production of cells grown in the chemostat mode exhibited an optimal dilution rate of about 0.1 h-1. Enhancement of protein production by pKTN-CAT as compared to pK-CAT tended to be higher when grown in rich medium rather than in minimal medium. Presence of the RNA stabilization plasmid did not significantly alter the growth rate of the cell. Using a combination of chemical treatment (1 mM EDTA) and shear stress resulting from cross-flow in a stainless steel microfiltration membrane *, CAT was released into the medium through disruption of the E. coli cells. The permeate flux increased from 2000 to 9000 kg m-2 h-1 with increasing axial Reynolds number from 10,000 to 60,000 or increasing mean shear stress from 12 to 47 Pa. The turbidity of the permeate was approximately 4% that of the retentate over this range of axial flow rates, indicating excellent removal of cell debris. Also, the concentration of CAT in the permeate was equal to that in the retentate over this range of axial flow rates, indicating complete passage of protein through the membrane. Thus, using a combination of chemical treatment and fluid-induced shear stress in a cross-flow membrane module, we were able to disrupt and recover the heterologous protein in a stream low in debris.

Biotechnology↗
PubMed1999

Small cis-acting sequences that specify secondary structures in a chloroplast mRNA are essential for RNA stability and translation.

Nucleus-encoded proteins interact with cis-acting elements in chloroplast transcripts to promote RNA stability and translation. We have analyzed the structure and function of three such elements within the Chlamydomonas petD 5' untranslated region; petD encodes subunit IV of the cytochrome b(6)/f complex. These elements were delineated by linker-scanning mutagenesis, and RNA secondary structures were investigated by mapping nuclease-sensitive sites in vitro and by in vivo dimethyl sulfate RNA modification. Element I spans a maximum of 8 nucleotides (nt) at the 5' end of the mRNA; it is essential for RNA stability and plays a role in translation. This element appears to form a small stem-loop that may interact with a previously described nucleus-encoded factor to block 5'-->3' exoribonucleolytic degradation. Elements II and III, located in the center and near the 3' end of the 5' untranslated region, respectively, are essential for translation, but mutations in these elements do not affect mRNA stability. Element II is a maximum of 16 nt in length, does not form an obvious secondary structure, and appears to bind proteins that protect it from dimethyl sulfate modification. Element III spans a maximum of 14 nt and appears to form a stem-loop in vivo, based on dimethyl sulfate modification and the sequences of intragenic suppressors of element III mutations. Furthermore, mutations in element II result in changes in the RNA structure near element III, consistent with a long-range interaction that may promote translation.

Algal Proteins↗
PubMed2001

An essential function of Saccharomyces cerevisiae RNA triphosphatase Cet1 is to stabilize RNA guanylyltransferase Ceg1 against thermal inactivation.

Saccharomyces cerevisiae RNA triphosphatase (Cet1) and RNA guanylyltransferase (Ceg1) interact in vivo and in vitro to form a bifunctional mRNA capping enzyme complex. Here we show that the guanylyltransferase activity of Ceg1 is highly thermolabile in vitro (98% loss of activity after treatment for 10 min at 35 degrees C) and that binding to recombinant Cet1 protein, or a synthetic peptide Cet1(232-265), protects Ceg1 from heat inactivation at physiological temperatures. Candida albicans guanylyltransferase Cgt1 is also thermolabile and is stabilized by binding to Cet1(232-265). In contrast, Schizosaccharomyces pombe and mammalian guanylyltransferases are intrinsically thermostable in vitro and they are unaffected by Cet1(232-265). We show that the requirement for the Ceg1-binding domain of Cet1 for yeast cell growth can be circumvented by overexpression in high gene dosage of a catalytically active mutant lacking the Ceg1-binding site (Cet1(269-549)) provided that Ceg1 is also overexpressed. However, such cells are unable to grow at 37 degrees C. In contrast, cells overexpressing Cet1(269-549) in single copy grow at all temperatures if they express either the S. pombe or mammalian guanylyltransferase in lieu of Ceg1. Thus, the cell growth phenotype correlates with the inherent thermal stability of the guanylyltransferase. We propose that an essential function of the Cet1-Ceg1 interaction is to stabilize Ceg1 guanylyltransferase activity rather than to allosterically regulate its activity. We used protein-affinity chromatography to identify the COOH-terminal segment of Ceg1 (from amino acids 245-459) as an autonomous Cet1-binding domain. Genetic experiments implicate two peptide segments, (287)KPVSLYVW(295) and (337)WQNLKNLEQPLN(348), as likely constituents of the Cet1-binding site on Ceg1.

Acid Anhydride Hydrolases↗
PubMed1994

Rous sarcoma virus RNA stability requires an open reading frame in the gag gene and sequences downstream of the gag-pol junction.

The intracellular accumulation of the unspliced RNA of Rous sarcoma virus was decreased when translation was prematurely terminated by the introduction of nonsense codons within its 5' proximal gene, the gag gene. Subcellular fractionation of transfected cells suggested that nonsense codon-mediated instability occurred in the cytoplasm. Analysis of constructs containing an in-frame deletion in the nucleocapsid domain of gag, which prevents interaction between the Gag protein and viral RNA, showed that an open reading frame extending to approximately 30 nucleotides from the natural gag termination codon was needed for RNA stability. Sequences at the gag-pol junction necessary for ribosomal frameshifting were not required for RNA stability; however, sequences located 100 to 200 nucleotides downstream of the natural gag termination codon were found to be necessary for stable RNA. The stability of RNAs lacking this downstream sequence was not markedly affected by premature termination codons. We propose that this downstream RNA sequence may interact with ribosomes translating gag to stabilize the RNA.

Animals↗
PubMed2006

Relaxed transcription in Arabidopsis mitochondria is counterbalanced by RNA stability control mediated by polyadenylation and polynucleotide phosphorylase.

Plant mitochondrial genomes are extraordinarily large and complex compared to their animal counterparts, due to the presence of large noncoding regions. Multiple promoters are common for plant mitochondrial genes, and transcription exhibits little or no modulation. Mature functional RNAs are produced through various posttranscriptional processes, and control of RNA stability has a major impact on RNA abundance. This control involves polyadenylation which targets RNA for degradation by polynucleotide phosphorylase (PNPase). Here, we have analyzed polyadenylated RNA fragments from Arabidopsis plants down-regulated for PNPase (PNP- plants). Because of their polyadenylated status and the accumulation of the corresponding RNA in PNP- versus wild-type plants, these sequences represent mitochondrial RNA degradation tags. Analysis of these tags revealed that PNPase is involved in degrading rRNA and tRNA maturation by-products but also RNA transcribed from regions that are in some cases highly expressed although lacking known functional genes. Some of these transcripts, such as RNA containing chimeric open reading frames created by recombination or antisense RNA transcribed on the opposite strand of a known gene, may present potential detrimental effects to mitochondrial function. Taken together, our data show that the relaxed transcription in Arabidopsis mitochondria is counterbalanced by RNA stability control mediated by polyadenylation and PNPase.

Arabidopsis↗
PubMed2005

The impact of RNA stabilization on minimal residual disease assessment in chronic myeloid leukemia.

BACKGROUND AND OBJECTIVES: Accurate quantification of BCR-ABL mRNA is of critical importance for managing patients with chronic myeloid leukemia (CML) who are receiving imatinib therapy. RNA degradation thus constitutes a potential problem for laboratories quantifying minimal residual disease (MRD). Patients' samples that take a long time to be transported from the hospital to the analyzing laboratory may be subject to RNA degradation with a corresponding loss in sensitivity and possible generation of false negative results. Recently, RNA preservation systems have been developed in order to improve RNA stability. The aim of the present study was to investigate such a system. DESIGN AND METHODS: We evaluated the performance of the PAXgene Blood RNA Kit in follow-up CML peripheral blood samples and compared the results to those from unstabilized parallel Trizol extracted samples. The different sample processing methods were evaluated by real-time polymerase chain reaction (PCR) analysis. RESULTS: RNA isolated with the PAXgene system gave a superior yield per milliliter of blood than did the routine Trizol extraction method. However, although of comparable quality, the RNA did not PCR-amplify as efficiently as equal amounts of RNA from routinely processed samples. Therefore, RNA processed with the PAXgene system showed decreased sensitivity for MRD detection, resulting in false negative results. The sensitivity was comparable to that of samples processed routinely 20-30 hours after phlebotomy. INTERPRETATION AND CONCLUSIONS: We conclude that routinely processed, i.e. unstabilized, peripheral blood that reaches the laboratory and is processed within 30 hours is preferable for MRD detection. Optimal results were achieved with fresh samples processed within 5 hours with the Trizol method. However, RNA stabilization may be useful if sample transit is expected to exceed 30 hours.

Gene Expression Regulation↗
PubMed2003

Computational methods and evaluation of RNA stabilization reagents for genome-wide expression studies.

Gene expression studies require high quality messenger RNA (mRNA) in addition to other factors such as efficient primers and labeling reagents. To prevent RNA degradation and to improve the quality of gene array expression data, several commercial reagents have become available. We examined a conventional hot-phenol lysis method and RNA stabilization reagents, and generated comparative gene expression profiles from Escherichia coli cells grown on minimal medium. Our data indicate that certain RNA stabilization reagents induce stress responses and proper caution must be exercised during their use. We observed that the laboratory reagent (phenol/EtOH, 5:95, v/v) worked efficiently in isolating high quality mRNA and reproducibility was such that reliable gene expression profiles were generated. To assist in the analysis of gene expression data, we wrote a number of macros that use the most recent gene annotation and process data in accordance with gene function. Scripts were also written to examine the occurrence of artifacts, based on GC content, length of the individual open reading frame (ORF), its distribution on plus and minus DNA strands, and the distance from the replication origin.

Base Composition↗
PubMed2004

Immunostimulating capacities of stabilized RNA molecules.

Since direct injection of naked mRNA induces an immune response, we tested the capacity of RNA to signal danger. We show here that mRNA molecules that are protected from immediate degradation either through interaction with cationic proteins (trans protection) or through chemical modification of the phosphodiester backbone (phosphorothioate RNA; cis protection) act as sequence-independent danger signals on mouse DC. As opposed to CpG DNA, the cis-stabilized RNA is degraded in a few minutes, does not activate B cells and, in contrast to double-stranded RNA, requires MyD88 for activation of the DC. We postulate that phosphorothioate RNA, which mimics trans-stabilized RNA, is a new PAMP.

Adaptor Proteins, Signal Transducing↗
PubMed2005

RNA stability of the E2A-encoded transcription factor E47 is lower in splenic activated B cells from aged mice.

We have demonstrated previously that DNA binding and protein expression of the E2A-encoded transcription factor E47 are lower in nuclear extracts of activated splenic B cells from old mice. In the present study, we address how E47 protein expression is regulated in aging. Results herein show that E2A mRNA levels were decreased in stimulated splenic B cells from old as compared with young mice. RNA stability assays showed that the rate of E2A mRNA decay was accelerated in stimulated splenic B cells from old mice, but E47 protein degradation rates were comparable in young vs aged B cells, indicating that the regulation of E47 expression in activated splenic B cells occurs primarily by mRNA stability. The rates of decay of other mRNAs showed that the increased mRNA degradation in aged splenic activated B cells is not a general phenomenon but restricted to a subset of mRNAs. We next investigated the signal transduction pathways controlling E2A mRNA expression and stability and found that p38 MAPK regulates E2A mRNA expression through increased mRNA stability and is down-regulated in aged activated B cells. Results show that inhibition of p38 MAPK significantly reduces E2A mRNA stability in both young and old B cells, further stressing the role of p38 MAPK in E2A RNA stabilization. These studies demonstrate that the transcription factor E2A, critical for many aspects of B cell function, is regulated by a novel mechanism in aging.

Aging↗
PubMed2004

[Selective effect of inductors of apoptosis on the endoribonuclease activity of 26S proteasomes and alpha-RNP particles in K562 cells: possible involvement of 26S proteasomes and alpha-RNP in the regulation of RNA stability].

It has been shown that endoribonuclease activity of alpha-RNP particles and 26S proteasomes are changed under the action of inductors of programmed cell death. Treatment of K562 cells with inductors of apoptosis--doxorubicin (adriamycin) and diethylmaleate--lead to a significant stimulation of RNAse activity of alpha-RNP and to reduction of proteasome RNase activity. The enzymatic activity under study has been shown to be specifically and selectively dependent on phosphorylation of subunits of alpha-RNP particles and 26S proteasomes. The characteristics of RNAse activity of different subpopulations of proteasomes differ. The specificity of a subpopulation of proteasomes exported from the cell has been demonstrated. Proteasome and alpha-RNP involvement in the coordinated control of stability of various specific messenger RNA molecules is suggested, and one of the mechanisms of this control might be the export of specific subpopulation of proteasomes from the cell.

Apoptosis↗