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PubMed2004

Nonsense mediated decay induced by tethered human UPF3B is restricted to the cytoplasm.

The subcellular localization of the nonsense mediated decay (NMD) of mRNA transcripts bearing premature termination codons has been controversial. Recently, it has been demonstrated that RNA tethering of key mediators of NMD, including human UPF3B, accurately recreates NMD. Here, we have used tethered UPF3B, combined with regulation of nuclear mRNA export using the retroviral Rev/RRE system, to examine where UPF3B-mediated mRNA degradation occurs. Our data clearly demonstrate that nuclear mRNA export is required for UPF3B-mediated degradation and moreover show that this degradation is exclusively cytoplasmic, despite the fact that the UPF3B fusion protein used is a nucleocytoplasmic shuttle protein localized predominantly to the nucleus. Moreover, exclusively cytoplasmic NMD occurred whether the target mRNA was exported via the retroviral Rev/RRE pathway or via the Tap: Nxt pathway used by most cellular mRNAs. These data may suggest that truly nuclear NMD, if it occurs, is at least in part mechanistically distinct from cytoplasmic NMD.

Active Transport, Cell Nucleus↗
PubMed1990

[Confirmation of the presence of a transcription termination site in a DNA segment downstream from gene 31 of bacteriophage T4].

Two open reading frames followed by a rho-independent transcription terminator and early promoter were established in a sequenced region downstream from gene 31 of T4 phage. The mRNA hairpin suggested by the terminator sequence has the structure (x)8CUUCGG(y)8, the loop sequence UUCG having been shown to significantly stabilize RNA hairpins.

Amino Acid Sequence↗
PubMed1985

Regulation of cytochrome P-45011 beta gene expression by adrenocorticotropin.

Recombinant DNA clones corresponding to 11 beta-hydroxylase cytochrome P-450 (P-450(11)beta) have been identified in a bovine adrenocortical cDNA library. These clones, pB11 beta-1 and pB11 beta-2, hybridize to at least three RNA species, 7.2, 6.2, and 4.3 kilobases in length, present in the adrenal cortex. All three RNA species directed cytochrome P-450(11)beta synthesis in an in vitro translation system. Expression of the cytochrome P-450(11)beta gene is tissue specific in that these transcripts were not detected in liver, heart, kidney, or corpus luteum. In cultured bovine adrenocortical cells, adrenocorticotropin (ACTH) or dibutyryl cAMP increased the concentration of cytochrome P-450(11)beta transcripts. This increase appears to be at least partially due to de novo transcription as shown by the action of actinomycin D which effectively blocked the ACTH-induced increase in cytochrome P-450(11)beta RNA level. Furthermore, cycloheximide administered prior to or along with ACTH resulted in the blockage of any new transcription of the cytochrome P-450(11)beta gene as evidenced from the level of RNA. Thus, in addition to potential effects on RNA stabilization, a primary action of ACTH in the regulation of the level of cytochrome P-450(11)beta may be to mediate the induction of an unidentified adrenocortical protein which is required for the activation of cytochrome P-450(11)beta gene expression. These results, together with previous studies (Kramer, R.E., Rainey, W.E., Funkenstein, B., Dee, A., Simpson, E. R., and Waterman, M. R. (1984) J. Biol. Chem. 259, 707-713) that showed increased intracellular levels of cAMP upon treatment of bovine adrenocortical cell cultures with ACTH, suggest a role for cAMP-mediated events in the regulation of cytochrome P-450(11)beta gene expression.

Adrenocorticotropic Hormone↗
PubMed1978

Stabilization of adenovirus nuclear RNA by intercalating drugs.

The effect of the intercalating drugs proflavine, ethidium bromide and daunomycin on the rate of degradation of newly synthesized adenovirus RNA was examined. As shown previously for heterogeneous nuclear RNA (HnRNA) and 45 S precursor ribosomal RNA (pre-rRNA), proflavine immediately stabilizes newly synthesized adenovirus RNA, while ethidium bromide stabilizes the RNA after a 30--60 min lag period. In contrast to its effect on HnRNA and pre-rRNA, daunomycin also stabilizes newly synthesized adenovirus RNA. These results are consistent with the hypothesis that the processing of late adenovirus nuclear RNA involves cleavage of base-paired regions by specific cellular nucleases.

Acridines↗
PubMed1996

Role of highly conserved pyrimidine-rich sequences in the 3' untranslated region of the GAP-43 mRNA in mRNA stability and RNA-protein interactions.

We have shown previously that the mRNA for the growth-associated protein GAP-43 is selectively stabilized during neuronal differentiation. In this study, we explored the role of its highly conserved 3' untranslated region (3'UTR) in mRNA stability and RNA-protein interactions. The 3'UTRs of the rat and chicken GAP-43 mRNAs show 78% sequence identity, which is equivalent to the conservation of their coding regions. In rat PC12 cells stably transfected with the full-length rat or chicken GAP-43 cDNAs, the transgene mRNAs decayed with same half-life of about 3 h. The GAP-43 3'UTR also caused the rabbit beta-globin mRNA to decay with a half-life of 4 h, indicating that the major determinants for GAP-43 mRNA stability are localized in its highly conserved 3'UTR. Three brain cytosolic RNA-binding proteins (molecular mass 40, 65 and 95 kDa) were found to interact with both the rat and chicken GAP-43 mRNAs. These RNA-protein interactions were specific and involved pyrimidine-rich sequences in the 3'UTR. Like the GAP-43 mRNA, the activity of these proteins was enriched in brain and increased during development. We propose that highly conserved pyrimidine-rich sequences in the 3'UTR of this mRNA regulate GAP-43 gene expression via interactions with specific RNA-binding proteins.

Animals↗
PubMed2002

Postmortem RNA and protein stability in perinatal human lungs.

The availability of fetal and neonatal lung tissue is an invaluable resource to elucidate the molecular regulation of human lung development. In this study, we have investigated the mRNA and protein stability of perinatal lung tissues treated with RNA (Ambion Inc., Austin, TX) or snap frozen in liquid nitrogen (LN ). Lung samples were obtained from 25 consecutive perinatal autopsies of live-born and stillborn infants (median gestational age, 23 weeks) with various clinical presentations. Treatment of lung tissue with RNA yielded more total RNA and protein than LN freezing. The integrity of RNA, assessed by spectrophotometry and gel electrophoresis, was equivalent between both tissue preservation methods, and both methods produced RNA suitable for reverse transcriptase-polymerase chain reaction analysis of representative genes (beta-actin and surfactant protein-B [SP-B]). Similarly, the protein integrity of RNA -treated tissues was equivalent to that of LN -frozen tissues, as judged by Western blot analysis of SP-B/actin protein expression. Although the total yield was similar in live-born, nonmacerated stillborn and macerated stillborn infants, only RNA and protein from live-born or nonmacerated stillborn infants was suitable for subsequent molecular analyses. Within the 41-hour range studied, the duration of the postmortem interval did not affect the yield or integrity of RNA and protein with either tissue preservation method. In summary, high-quality RNA and protein, suitable for routine molecular analyses, can be obtained from postmortem lung tissue from live-born and nonmacerated stillborn infants, even with prolonged postmortem intervals. RNA is equivalent, if not superior, to LN for preservation of postmortem RNA and protein in developing human lungs.

Autolysis↗
PubMed2001

A thermodynamic framework for Mg2+ binding to RNA.

We present a model describing how Mg(2+) binds and stabilizes specific RNA structures. In this model, RNA stabilization arises from two energetically distinct modes of Mg(2+) binding: diffuse- and site-binding. Diffusely bound Mg(2+) are electrostatically attracted to the strong anionic field around the RNA and are accurately described by the Poisson-Boltzmann equation as an ensemble distributed according to the electrostatic potentials around the nucleic acid. Site-bound Mg(2+) are strongly attracted to specifically arranged electronegative ligands that desolvate the ion and the RNA binding site. Thus, site-binding is a competition between the strong coulombic attraction and the large cost of desolvating the ion and its binding pocket. By using this framework, we analyze three systems where a single site-bound Mg(2+) may be important for stability: the P5 helix and the P5b stem loop from the P4-P6 domain of the Tetrahymena thermophila group I intron and a 58-nt fragment of the Escherichia coli 23S ribosomal RNA. Diffusely bound Mg(2+) play a dominant role in stabilizing these RNA structures. These ions stabilize the folded structures, in part, by accumulating in regions of high negative electrostatic potential. These regions of Mg(2+) localization correspond to ions that are observed in the x-ray crystallographic and NMR structures of the RNA. In contrast, the contribution of site-binding to RNA stability is often quite small because of the large desolvation penalty. However, in special cases, site-binding of partially dehydrated Mg(2+) to locations with extraordinarily high electrostatic potential can also help stabilize folded RNA structures.

Magnesium↗
PubMed2002

Structure and stability of wild-type and mutant RNA internal loops from the SL-1 domain of the HIV-1 packaging signal.

The packaging signal (Psi) of the human immunodeficiency virus type 1 (HIV-1) enables encapsidation of the full-length genomic RNA against a background of a vast excess of cellular mRNAs. The core HIV-1 Psi is approximately 109 nucleotides and contains sequences critical for viral genomic dimerisation and splicing, in addition to the packaging signal. It consists of a series of stem-loops (termed SL-1 to SL-4), which can be arranged in a cloverleaf secondary structure. Using a combination of NMR spectroscopy, UV melting experiments, molecular modeling and phylogenetic analyses, we have explored the structure of two conserved internal loops proximal to the palindromic sequence of SL-1. Internal loop A, composed of six purines, forms a flexible structure that is strikingly similar to the Rev responsive element motif when bound to Rev protein. This result suggests that it may function as a protein-binding site. The absolutely conserved four-purine internal loop B is instead conformationally and thermodynamically unstable, and exhibits multiple conformations in solution. By introducing a double AGG to GGA mutation within this loop, its conformation is stabilised to form a new intra-molecular G:A:G base-triplet. The structure of the GGA mutant explains the relative instability of the wild-type loop. In a manner analogous to SL-3, we propose that conformational flexibility at this site may facilitate melting of the structure during Gag protein capture or genomic RNA dimerisation.

Base Pairing↗
PubMed2002

Stability of human immunodeficiency virus type 1 RNA in cerebrospinal fluid determined with the AMPLICOR HIV-1 MONITOR test, version 1.5 (ultrasensitive).

We evaluated the effects of time, temperature, freezing, and thawing on the cerebrospinal fluid viral load by using the Roche AMPLICOR HIV-1 MONITOR test, version 1.5 (ultrasensitive). While a statistically significant decrease from the baseline was observed at 24 h, but not at 6 or 12 h, and with one freeze-thaw cycle, all changes were within the range of intra-assay variability.

HIV-1↗
PubMed2006

Characterization of the secondary structure and stability of an RNA aptamer that binds vascular endothelial growth factor.

Thermal denaturation studies and spectroscopic studies were employed to investigate the secondary structure and stability of an RNA-PEG conjugate commercially called Macugen. The RNA aptamer is conjugated to a pegylated moiety, and the majority of its 2'-hydroxyl groups are methylated or otherwise modified. UV optical melting studies and differential scanning calorimetry (DSC) were carried out under different conditions to evaluate the effects of Na+ and oligomer concentrations on the stability of the secondary structure of the RNA oligomer. The results of these studies indicated that the T(m) of the RNA is independent of oligomer concentration but dependent on the salt concentration, in a predictable fashion. Further, the DSC melting profiles obtained under all conditions were highly reversible. Circular dichroism (CD) studies were determined under different salt concentrations, various RNA concentrations, and temperatures as well. Together, the thermal denaturation and CD studies provide evidence that the secondary structure of the RNA oligonucleotide is a stable hairpin at 25 degrees C and that the thermally induced hairpin to single strand transition is highly reversible.

Aptamers, Nucleotide↗
PubMed1994

The role of posttranscriptional modification in stabilization of transfer RNA from hyperthermophiles.

The influence of posttranscriptional modification on structural stabilization of tRNA from hyperthermophilic archaea was studied, using Pyrococcus furiosus (growth optimum 100 degrees C) as a primary model. Optical melting temperatures (Tm) of unfractionated tRNA in 20 mM Mg2+ are 97 degrees C for P. furiosus and 101.5 degrees C for Pyrodictium occultum (growth optimum, 105 degrees C). These values are approximately 20 degrees C higher than predicted solely from G-C content and are attributed primarily to posttranscriptional modification. Twenty-three modified nucleosides were determined in total digests of P. furiosus tRNA by combined HPLC-mass spectrometry. From cells cultured at 70, 85, and 100 degrees C, progressively increased levels of modification were observed within three families of nucleosides, the most highly modified forms of which were N4-acetyl-2'-O-methylcytidine (ac4Cm), N2,N2,2'-O-trimethylguanosine (m2(2)Gm), and 5-methyl-2-thiouridine (m5s2U). Nucleosides ac4Cm and m2(2)Gm, which are unique to the archaeal hyperthermophiles, were shown in earlier NMR studies to exhibit unusually high conformational stabilities that favor the C3'-endo form [Kawai, G., et al. (1991) Nucleic Acids Symp. Ser. 21, 49-50; (1992) Nucleosides Nucleotides 11, 759-771]. The sequence location of m5s2U was determined by mass spectrometry to be primarily at tRNA position 54, a site of known thermal stabilization in the bacterial thermophile Thermus thermophilus [Horie, N., et al. (1985) Biochemistry 24, 5711-5715]. It is concluded that selected posttranscriptional modifications in archaeal thermophiles play major stabilizing roles beyond the effects of Mg2+ binding and G-C content, and are proportionally more important and have evolved with greater structural diversity at the nucleoside level in the bacterial thermophiles.

Archaea↗
PubMed1976

Stabilization of an RNA molecule by 3'-terminal poly (A)-induced inhibition of RNase activity.

The inhibitory properties of poly(A) on human spleen ribonuclease have been investigated. Hydrolytic activity has been shown to be strongly inhibited by poly(A) contained within RNAs isolated from a variety of natural sources. Furthermore, poly(A) segments of varying length have been covalently linked at the 3' terminus of Escherichia coli 5 S rRNA by polynucleotide phosphorylase in an attempt to construct an in vitro demonstration of the stabilization of RNA which contains poly(A). The extent to which these poly(A) tracts, varying from 4 to 132 nucleotides in length, could inhibit endonucleolytic attack on the 5 S rRNA to which they are linked was found to be dependent upon their length and upon small changes in spermidine concentration. The consequences of these findings are discussed in terms of a possible role for poly(A).

Enzyme Activation↗
PubMed2002

Evolution of Tetrahymena ribozyme mutants with increased structural stability.

Determining how large RNA molecules stabilize their tertiary structures is critical for understanding how they perform their biological functions. Here we use in vitro selection to identify active variants of the Tetrahymena ribozyme with increased stability. The mutant pool converged to a single family that shared nine mutations; an RNA representing the consensus sequence was structurally more stable by 10.5 degrees C and catalytically active at elevated temperatures. Remarkably, of the nine altered sites, most are already known to be involved in tertiary interactions, and the stabilizing mutations primarily improve the packing interactions in the molecular interior. The wild type ribozyme and the selected mutants provide pairs of mesophilic and thermophilic homologs for studying the origin of their thermal stability.

Animals↗
PubMed2005

Failure of the PAXgene Blood RNA System to maintain mRNA stability in whole blood.

In multicentre studies of malignant and inflammatory diseases, whole blood, cell or tissue samples are often collected for analyses of gene expression to predict or monitor treatment effects. For correct analysis, sample stability during handling and transport is crucial. In developing the logistics for multicentre studies in malignant melanoma and inflammatory bowel disease, we found poor stability of a number of transcripts using the PAXgene Blood RNA System, which was advertised to maintain RNA stability for several days at room temperature. The results indicate that general statements on sample stability are not reliable and have to be verified for the specific transcripts of interest.

Artifacts↗
PubMed2004

A synonymous SNP of the corneodesmosin gene leads to increased mRNA stability and demonstrates association with psoriasis across diverse ethnic groups.

Psoriasis is a chronic skin disorder with multifactorial aetiology. Genome-wide scans have provided unambiguous evidence for a major disease susceptibility locus on chromosome 6p21 (PSORS1). A minimal PSORS1 interval has been defined which encompasses three genes (HLA-C, HCR and CDSN) carrying psoriasis-associated SNPs. On the basis of this genetic evidence, we have undertaken an assessment of CDSN allele functional impact. A comparison of CDSN intragenic haplotypes showed that SNPs exclusive to disease-associated chromosomes are located in regions implicated in the stabilization of RNA transcripts. As CDSN is over-expressed in psoriatic lesions, we hypothesised that disease-associated intragenic SNPs may alter the rate of its mRNA decay. Here, we demonstrate that mRNAs transcribed from a CDSN risk haplotype present a 2-fold increase in stability, compared with those transcribed from a neutral haplotype (t-test P=0.004). Site-directed mutagenesis revealed that a single synonymous SNP (CDSN*971T) accounts for the observed increase in RNA stability. CDSN*971T maps to a RNA stability motif and UV cross-linking analysis demonstrated that the SNP affects the transcript affinity for a 39 kDa RNA binding protein. Association analyses show that haplotypes bearing CDSN*971T confer psoriasis susceptibility in a wide range of ethnic groups. These results demonstrate the effect of synonymous variation upon allele specific gene expression, a finding of relevance to future studies of the pathogenesis of common and complex traits.

Animals↗
PubMed2005

PCR detection of dengue virus using dried whole blood spotted on filter paper.

Whole blood dried onto filter paper constitutes a potentially useful material for molecular testing of viruses, including dengue. In order to assess the stability of viral RNA, we carried out dengue-RNA detection in whole blood infected with dengue virus that had been previously spotted onto filter paper. Filter papers were stored at room temperature, 4 and -70 degrees C and processed for PCR assay at intervals of 2, 4, 6 and 9 weeks. Our results demonstrated that dengue-RNA was stable in filter paper for 9 weeks at all tested temperatures. Furthermore, we evaluated these conditions using frozen sera and dried blood samples onto filter paper from 52 patients with confirmed clinical diagnosis of dengue infection. PCR results showed a 100% specificity and 93% sensitivity for dried blood samples. This storage method facilitates the transportation and analysis by nucleic acid amplification techniques even when freezing conditions are not available.

Blood↗
PubMed2002

Regulated alpha-globin mRNA decay is a cytoplasmic event proceeding through 3'-to-5' exosome-dependent decapping.

The alpha-globin mRNA contains a C-rich stability element (CRE) in its 3' untranslated region (3' UTR) which is critical for the stability of this long-lived mRNA. A protein complex, termed the alpha-complex, forms on the CRE and has been shown to contribute to stabilization of the mRNA by at least two mechanisms, first by interacting with the poly(A)-binding protein (PABP) to prevent deadenylation, and second by protecting the mRNA from attack by an erythroid endoribonuclease. In this report, we demonstrate that the alpha-globin 3' UTR can confer stability on a heterologous mRNA in cells, and this stability is dependent on the alpha-complex. Moreover, the stability was exclusively detected with cytoplasmic mRNA, suggesting that the regulation of alpha-globin mRNA stability is a cytoplasmic event. An additional mechanism by which the alpha-complex can confer stability on an RNA in vitro was also identified and shown to involve inhibition of 3' to 5' exonucleolytic degradation. Furthermore, using an in vitro mRNA decay system, we were able to follow the demise of the alpha-globin RNA and demonstrate that the decay was initiated by deadenylation followed by 3'-to-5' decay carried out by the exosome and ultimately hydrolysis of the residual cap structure.

3' Untranslated Regions↗
PubMedDate not supplied

[Computerized analysis of inverted repeats in Mycobacterium tuberculosis genome].

Thermodynamic stable inverted repeats, capable of stabilizing nuclease-influenced mRNA, have been determined for M. tuberculosis slowly growing isolates H37Rv and CDC1551 with the completely sequenced genome. The genome of laboratory strain H37Rv may contain 50 pin structures formed by inverted repeats, whose stem varies in length from 11 to 28 nucleotide pairs (n.p.), the loop size being equal to 4-5 nucleotides and free energy deltaG=-15.2 to -56.2 kcal/mol. The genome of strain CDC1551 (clinical isolate) having a high level of virulence contains 47 pins. Each of the two isolates contains 8 long inverted repeats with a length of 48-62 nucleotides and free energy deltaG=-38.9 to -56.2 kcal/mol; of these, 6 pins are completely similar. At the same time in the genome of strain CDC1551, in contrast to that of strain H37Rv, a highly stable pin 58 nucleotides long is localized at 5'-end. The localization of the highly stable pin with deltaG=-53.9 kcal/mol in the area of 5'-end of isolate CDC1551 may lead to a different degree of stabilization of RNA-transcripts in M. tuberculosis CDC1551 in comparison with isolate H37Rv. This may, in its turn, serve as one of the reasons of differences in the virulence of strains. The possibility of the formation of pins with stems of 11 n.p. and longer has been visually cofirmed with the use of atomic power microscopy. The possibility of the participation of the secondary structures formed by pins in the protection of mRNA of slowly growing pathogenic M. tuberculosis from the degrading activity of RNases is discussed.

DNA, Bacterial↗