PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “RNA Stability”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 289 records · Page 16Linked to original sources
PubMed1996

Cleavage of RNA hairpins mediated by a developmentally regulated CCCH zinc finger protein.

Control of RNA turnover is a major, but poorly understood, aspect of gene regulation. In multicellular organisms, progress toward dissecting RNA turnover pathways has been made by defining some cis-acting sequences that function as either regulatory or cleavage targets (J. G. Belasco and G. Brawerman, Control of Messenger RNA Stability, 1993). However, the identification of genes encoding proteins that regulate or cleave target RNAs has been elusive (C. A. Beelman and R. Parker, Cell 81:79-183, 1995); this gap in knowledge has made it difficult to identify additional components of RNA turnover pathways. We have utilized a modified expression cloning strategy to identify a developmentally regulated gene from Drosophila melanogaster that encodes a RNase that we refer to as Clipper (CLP). Significant sequence matches to open reading frames encoding unknown functions identified from the Caenorhabditis elegans and Saccharomyces cerevisiae genome sequencing projects suggest that all three proteins are members of a new protein family conserved from lower eukaryotes to invertebrates. We demonstrate that a member of this new protein family specifically cleaves RNA hairpins and that this activity resides in a region containing five copies of a previously uncharacterized CCCH zinc finger motif. CLP's endoribonucleolytic activity is distinct from that associated with RNase A (P. Blackburn and S. Moore, p. 317-433, in P. D. Boyer, ed., The Enzymes, vol. XV, part B, 1982) and is unrelated to RNase III processing of rRNAs and tRNAs (J. G. Belasco and G. Brawerman, Control of Messenger RNA Stability, 1993, and S. A. Elela, H. Igel, and M. Ares, Cell 85:115-124, 1995). Our results suggest that CLP may function directly in RNA metabolism.

Amino Acid Sequence↗
PubMed1975

The half-life of polyadenylated polysomal RNA from normal and transformed cells in monolayer culture.

The small genotypic differences between normal and transformed cells are insufficient to account directly for all their wide phenotypic differences, which probably in some cases at least involve alterations in control of gene expression. To ascertain whether such alterations involved changes in mRNA stability, RNA half-lives were estimated in five monolayer cell lines, including two pairs of normal cells and their transformed counterparts. The results for the polyadenylated fractions in all cases fit with those expected from a model in which the whole fraction has a single half-life, of less than one generation time. From both the transformed/untransformed cell pairs, there is evidence that a relationship exists between cell generation time and the half-life of the polyadenylated polysomal RNA fraction, which persists even through the process of transformation. Considerable alteration in the pattern of RNA stability is therefore unlikely to be obligatory in in vitro transformation.

Cell Line↗
PubMed2000

cis- and trans-Acting determinants for translation of psbD mRNA in Chlamydomonas reinhardtii.

Chloroplast translation is mediated by nucleus-encoded factors that interact with distinct cis-acting RNA elements. A U-rich sequence within the 5' untranslated region of the psbD mRNA has previously been shown to be required for its translation in Chlamydomonas reinhardtii. By using UV cross-linking assays, we have identified a 40-kDa RNA binding protein, which binds to the wild-type psbD leader, but is unable to recognize a nonfunctional leader mutant lacking the U-rich motif. RNA binding is restored in a chloroplast cis-acting suppressor. The functions of several site-directed psbD leader mutants were analyzed with transgenic C. reinhardtii chloroplasts and the in vitro RNA binding assay. A clear correlation between photosynthetic activity and the capability to bind RNA by the 40-kDa protein was observed. Furthermore, the data obtained suggest that the poly(U) region serves as a molecular spacer between two previously characterized cis-acting elements, which are involved in RNA stabilization and translation. RNA-protein complex formation depends on the nuclear Nac2 gene product that is part of a protein complex required for the stabilization of the psbD mRNA. The sedimentation properties of the 40-kDa RNA binding protein suggest that it interacts directly with this Nac2 complex and, as a result, links processes of chloroplast RNA metabolism and translation.

5' Untranslated Regions↗
PubMed2001

A comprehensive characterization of a group IB intron and its encoded maturase reveals that protein-assisted splicing requires an almost intact intron RNA.

The group I intron (AnCOB) of the mitochondrial apocytochrome b gene from Aspergillus nidulans encodes a bi-functional maturase protein that is also a DNA endonuclease. Although the AnCOB intron self-splices, the encoded maturase protein greatly facilitates splicing, in part, by stabilizing RNA tertiary structure. To determine their role in self-splicing and in protein-assisted splicing, several peripheral RNA sub-domains in the 313 nucleotide intron were deleted (P2, P9, P9.1) or truncated (P5ab, P6a). The sequence in two helices (P2 and P9) was also inverted. Except for P9, the deleted regions are not highly conserved among group I introns and are often dispensable for catalytic activity. Nevertheless, despite the very tight binding of AnCOB RNA to the maturase and the high activity of the bimolecular complex (the rate of 5' splice-site cleavage was >20 min(-1) with guanosine as the cofactor), the intron was surprisingly sensitive to these modifications. Several mutations inactivated splicing completely and virtually all impaired splicing to varying degrees. Mutants containing comparatively small deletions in various regions of the intron significantly decreased binding affinity (generally >10(4)-fold), indicating that none of the domains that remained constitutes the primary recognition site of the maturase. The data argue that tight binding requires tertiary interactions that can be maintained by only a relatively intact intron RNA, and that the binding mechanism of the maturase differs from those of two other well-characterized group I intron splicing factors, CYT-18 and Cpb2. A model is proposed in which the protein promotes widespread cooperative folding of an RNA lacking extensive initial tertiary structure.

Apoproteins↗
PubMed2006

TaqMan PCR assay in the control of RNA normalization in human post-mortem brain tissue.

The brain tissue obtained after death is subjected to several circumstances that can affect RNA integrity. The present study has been directed to reveal possible pitfalls and to control RNA normalization in post-mortem samples in order to recognize the limitations and minimize errors when using TaqMan PCR technology. This has been carried out in samples of the frontal cortex in a series of control and diseased cases covering Parkinson's disease, dementia with Lewy bodies pure form and common form, and Alzheimer's disease. Special attention has been paid to the value of the agonal state, post-mortem delay and pH of the nervous tissue as approximate predictors of the quality of RNA, as well as to the use of the Bioanalyzer to confirm RNA preservation. In addition, since possible disease-modified mRNAs have to be normalized with ideal unaltered RNAs, TaqMan human endogenous control plates have been used to determine the endogenous control most appropriate for the study. beta-glucuronidase (GUS) and beta-actin were good endogenous controls because their expression levels showed a small variation across a representative number of control and pathological cases. RNA stability was also analysed in a paradigm mimicking cumulative delay in tissue processing. GUS mRNA levels were not modified although beta-actin mRNA levels showed degradation at 22 h. Finally, the control of RNA degradation for the normalization of genes of interest was also tested. mRNA expression levels for superoxide dismutase 1 (SOD1) and metalloproteinase domain 22 (ADAM22) were examined at several artificial post-mortem times, and their expression levels compared with those for putative controls beta-actin and GUS. In our paradigm, the expressions of SOD1 and ADAM22 were apparently not modified when normalized with beta-actin. Yet their expression levels were reduced with post-mortem delay when values were normalized with GUS. Taken together, these observations point to practical consequences in TaqMan PCR studies. Short post-mortem delays and acceptable pH of the brain are not sufficient to rule out RNA degradation. The selection of adequate endogenous controls is pivotal in the study. beta-actin and GUS are found to be good endogenous controls in these pathologies, although GUS but not beta-actin expression levels are preserved in samples with long post-mortem delay.

Aged↗
PubMed2025

Minimal Perturbation Analysis of mRNA Degradation Rates with Tet-Off and RT-qPCR.

Messenger RNA stability is an important variable in gene expression and its dynamics. High stability ensures a constant level of synthesized protein, whereas mRNA instability can be critical for regulatory processes in which protein production needs to be stopped, such as development, inflammation, or adaptation to stress. Accurate measurements of RNA degradation rates are important for understanding how RNA features and RNA binding proteins affect the posttranscriptional life of an mRNA. As an alternative to global transcriptional inhibition methods, the use of a Tet-off repressible promoter has the advantage that cells are minimally perturbed by the addition of doxycyclin during the assay. We illustrate the use of a reporter mRNA expressed from a plasmid in Saccharomyces cerevisiae cells, but similar methods can be applied to other regulated promoters, on plasmids or by genome editing, and in other organisms. RNA levels are measured by reverse transcription followed by quantitative PCR. An exponential decay law is then used to estimate how well the measurements follow this expected trend for the simplest possible mechanism of RNA degradation, where the decay is proportional to the amount of RNA present at any given time.

RNA Stability↗
PubMed2006

Evaluation of methods for duration of preservation of RNA quality in rat liver used for transcriptome analysis.

In The Toxicogenomics Project, about 150 chemicals are administered to rats, and gene expression in the liver analyzed by Affymetrix GeneChip and stored in the database. As the quality of RNA greatly influences the accuracy of gene expression data, conditions of the storage of the sample are very important. Recently, an RNA stabilization solution, RNAlater, has become commercially available. In this study, the new storage method was compared with the traditional storage method (stored in freezer or liquid nitrogen) under various conditions by looking at the degradation of RNA assessed by its total yield, OD260/280 ratio, 28S/18S ratio, and quantity of beta-actin. It was confirmed that RNAlater preserved the liver tissue sample by maintaining the quality of RNA for one year (in liquid N(2) or -80 degrees C), for 3 days (4 degrees C), or for 2 hr (room temperature) without degradation of RNA. Quality of RNA samples dissolved in buffer RLT and stored at -20 degrees C tended to decrease, but samples stored at -80 degrees C were almost equivalent to those stored in liquid nitrogen. In conclusion, we recommend the following procedure for preservation of liver tissue for extraction of RNA: 1) tissues removed should be put into chilled RNAlater as soon as possible; 2) samples in RNAlater must be stored overnight or longer at 4 degrees C and can be left for as long as 2 weeks without freezing; 3) samples in RNAlater can be stored for at least one year under less than -20 degrees C and 4) samples dissolved in buffer RLT can be preserved at least for one year under -80 degrees C.

Actins↗
PubMed1999

Stability of RNA transcripts in post-mortem psychiatric brains.

RNA isolated from frozen human post-mortem brain tissue was used for analysis of five gene products with a recently developed sensitive and competitive RT-PCR technique. Samples varying in post-mortem intervals up to four days from controls, schizophrenics and alcoholics were analyzed. Evaluation of three housekeeping genes, as well as Trk B and Trk C demonstrated that the levels of mRNA transcripts were stable in brain samples at all time periods (one to four days) examined. This observation demonstrates that this RT-PCR protocol is a sensitive and reliable method to study relative amounts of mRNAs. The overall stability of housekeeping transcripts implicates the value of post-mortem brain samples for differential gene expression studies.

Actins↗
PubMed1988

Stability of RNA in developing Xenopus embryos and identification of a destabilizing sequence in TFIIIA messenger RNA.

Synthetic capped RNA transcripts injected into fertilized eggs of Xenopus laevis have a half-life of 3-4 h. Addition of a long (approximately 200 nucleotide) poly(A) tail increases the half-life to 6-8 h which approaches the half-life of natural polyadenylated globin RNA injected into embryos. Since exonucleolytic action alone could account for the degradation of RNA, we tested whether circular RNA is stable after injection and find that circles are exceptionally stable (half-life greater than 40 h). After the midblastula transition, polyadenylated chloramphenicol transferase (CAT) mRNAs transcribed from injected plasmids have a half-life of 2.5 h. Insertion of a 1000 nucleotide 3' untranslated region from the Xhox-36 gene into the transcripts does not affect the half-life. In contrast to the finding that internal sequences do not affect stability, we find that sequences from the TFIIIA message reduce the half-life of CAT mRNA from 2.5 h to less than 30 min. We conclude that most RNAs are degraded exonucleolytically from the 3' end, but specialized internal sequences can greatly destabilize the RNA, possibly by acting as a site for an endonuclease.

Animals↗
PubMed2003

Gene expression in plant mitochondria: transcriptional and post-transcriptional control.

The informational content of the mitochondrial genome in plants is, although small, essential for each cell. Gene expression in these organelles involves a number of distinct transcriptional and post-transcriptional steps. The complex post-transcriptional processes of plant mitochondria such as 5' and 3' RNA processing, intron splicing, RNA editing and controlled RNA stability extensively modify individual steady-state RNA levels and influence the mRNA quantities available for translation. In this overview of the processes in mitochondrial gene expression, we focus on confirmed and potential sites of regulatory interference and discuss the evolutionary origins of the transcriptional and post-transcriptional processes.

Cell Nucleus↗
PubMed2005

Engineering thermal stability in RNA phage capsids via disulphide bonds.

The RNA bacteriophages, a group that includes phages Qbeta and MS2, have a number of potential bionanotechnological applications, including cell specific drug delivery and as substrates for the formation of novel materials. Despite extensive sequence identity between their coat protein subunits, and an almost identical three-dimensional fold, Qbeta and MS2 capsids have dramatically different thermal stabilities. The increased stability of Qbeta has been correlated with the inter-subunit disulphide bonds present in that capsid and not present in MS2. We have tested this hypothesis directly using mass spectrometry. Analysis of the dissociated coat protein subunits suggests that inter-molecular disulphides are formed at the capsid five-fold but may not be at the three-fold axes. This conclusion has been tested by engineering disulphide cross-links into either the five-fold or three-fold positions of the recombinant MS2 capsid. Five-fold cross-linking results in a mutant with stability properties similar to those of Qbeta. Three-fold cross-linking results in a mutant unable to assemble T = 3 shells, implying that five-fold structures are on pathway to capsid assembly in these phages. The results demonstrate how it is possible to redesign the physical properties of phage shells and may be of general relevance to future applications of viruses and virus-like particles.

Allolevivirus↗
PubMed2004

Artemin is an RNA-binding protein with high thermal stability and potential RNA chaperone activity.

Encysted embryos of the crustacean, Artemia franciscana, are among the most stress-resistant of all multicellular eukaryotes, due in part to massive amounts of p26, a small heat shock protein, that acts as a molecular chaperone. These embryos contain equally large amounts of another protein called artemin, of previously unknown function, that we report on here. Its thermal stability allows large-scale purification in about a day, using ammonium sulfate fractionation and incubation at 70 degrees C for 7 min, followed by gel filtration. The latter yields an artemin-RNA complex from which the pure protein, apo-artemin, was obtained by anion-exchange chromatography. We evaluated the possibility that artemin acts as a molecular chaperone for proteins, but obtained no evidence for that in vitro. The association of RNA with apo-artemin occurs at high temperatures and, although it is not yet clear whether artemin has a specific role as an RNA chaperone, it does bind non-polyadenylated RNAs which are then translated in vitro. Artemin-RNA is thermostable, some molecules resisting destruction after 30 min at 90 degrees C. The first order rate constant for denaturation and aggregation of artemin-RNA at 85 degrees C is 8.5 x 10(-3)min(-1), which compares well with other thermostable proteins of similar size ( approximately 500 kDa) such as the ferritins with which artemin has amino acid sequence similarity. The amount of artemin extracted from embryos that had been stored dry, under laboratory conditions, since 1951 is comparable to the amount in contemporary embryos, indicating its stability in situ, and supporting the in vitro heating studies.

Amino Acid Sequence↗
PubMed1994

Utilization of multiple polyadenylation signals in the human RHOA protooncogene.

Little is known regarding the regulation of expression of the RHOA protooncogene, a member of the family of genes encoding Ras-related GTP-binding proteins. We have previously reported that the 3' untranslated region (UTR) of RHOA was contained within a genomic sequence which flanked the 5' end of the human glutathione peroxidase 1-encoding gene [J.A. Moscow et al., J. Biol. Chem. 267 (1992) 5949-5958]. Our previous studies revealed the presence of multiple (1.8 and 1.5 kb) RHOA mRNA species in breast cancer cell lines and of three putative polyadenylation signals in the RHOA 3' UTR. In this report, we have isolated several RHOA cDNAs from a multidrug-resistant MCF-7 human breast cancer cell line. Sequence analyses of these RHOA cDNA clones indicate that multiple polyadenylation signals are used to terminate RHOA transcripts. RNase-protection analysis demonstrated that all three polyadenylation signals are utilized in breast cancer cell lines and RNA stability studies demonstrated that RHOA RNA species with different 3' ends have equivalent stability. Since little is known about the RNA expression of RHOA in human tumors, and since both activated and non-activated RHOA genes possess transformation potential, we analyzed RHOA mRNA in lung and colon tumors by Northern blot and RNase-protection analyses. In all eight lung tumors examined, RHOA RNA levels were decreased relative to the level in normal surrounding tissue, whereas RHOA expression was decreased in only two of six colon tumors. We also found that lovastatin-induced cell cycle arrest resulted in increased RHOA RNA expression in breast cancer cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1991

Structural features that give rise to the unusual stability of RNA hairpins containing GNRA loops.

The most frequently occurring RNA hairpins in 16S and 23S ribosomal RNA contain a tetranucleotide loop that has a GNRA consensus sequence. The solution structures of the GCAA and GAAA hairpins have been determined by nuclear magnetic resonance spectroscopy. Both loops contain an unusual G-A base pair between the first and last residue in the loop, a hydrogen bond between a G base and a phosphate, extensive base stacking, and a hydrogen bond between a sugar 2'-end OH and a base. These interactions explain the high stability of these hairpins and the sequence requirements for the variant and invariant nucleotides in the GNRA tetranucleotide loop family.

Base Sequence↗
PubMed1995

Stabilization of RNA stacking by pseudouridine.

The effect of the modified nucleoside pseudouridine (psi) on RNA structure was compared with uridine. The extent of base stacking in model RNA oligonucleotides was measured by 1H NMR, UV, and CD spectroscopy. The UV and CD results indicate that the model single-stranded oligoribonucleotides AAUA and AA psi A form stacked structures in solution and the CD results for AA psi A are consistent with a general A-form helical conformation. The AA psi A oligomer exhibits a greater degree of UV hypochromicity over the temperature range 5-55 degrees C, consistent with a better stacked, more A-form structure compared with AAUA. The extent of stacking for each nucleotide residue was inferred from the percent 3'-endo sugar conformation as indicated by the H1'-H2' NMR scalar coupling. This indirect indication of stacking was confirmed by sequential NOE experiments. NMR measurements as a function of temperature indicate that pseudouridine forms a more stable base stacking arrangement than uridine, an effect that is propagated throughout the helix to stabilize stacking of neighboring purine nucleosides. The N1-H imino proton in AA psi A exchanges slowly with solvent, suggesting a role for the extra imino proton in stabilizing the conformation of pseudouridine. These results show that the conformational stabilization is an intrinsic property of pseudouridine occurring at the nucleotide level. The characteristics of pseudouridine in these models are consistent with earlier studies on intact rRNA, indicating that pseudouridine probably performs the same stabilizing function in most structural contexts.

Magnetic Resonance Spectroscopy↗
PubMed1998

Stability of RNA hairpins closed by wobble base pairs.

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequences of the type GGXANmAYCC, where XY is the wobble base pair, GU or UG, and the underlined loop sequences are three to eight nucleotides. A nearest-neighbor analysis indicates the free energy of loop formation is dependent upon loop size and closing base pair. Hairpin loops closed by UG base pairs are on average 1.3 kcal/mol less stable than hairpins closed by GU base pairs. The hairpin loops closed by UG have approximately the same stability as hairpin loops closed by AU/UA base pairs, while the loops closed by GU are approximately 0.7 kcal/mol more stable than hairpins loops closed by GC/CG base pairs. These results, combined with the model previously developed [Serra et al. (1997) Biochemistry 36, 4844] to predict the stability for hairpin loops closed by Watson-Crick base pairs, allow for the following model to predict the stability of hairpin loops: delta G degree 37L(n) = delta G degree 37iL(n) + delta G degree 37mm + 0.6 (if closed by AU, UA, or UB) - 0.7 (if closed by GU) - 0.7 (if first mismatch is GA or UU except for loops closed by GU). Here, delta G degree 37iL(n) is the free energy increment for initiating a loop of n nucleotides with a CG or GC pair, and delta G degree 37mm is the free energy for the interaction of the first mismatch with the closing base pair. For hairpin loops of n = 4-9, delta G037iL(n) is 4.9, 5.0, 5.0, 5.0, 4.9, and 5.5 kcal/mol, respectively. For hairpin loops of n = 3, delta G degree 37L(3) = +4.8 + 0.6 (if closed by AU, UA, or UG) kcal/mol. Thermodynamic parameters for hairpin formation in 1 M NaCl for 13 naturally occurring RNA hairpin sequences closed by wobble base pairs are reported. The model provides good agreement for both TM and delta G degree 37 for most hairpins studied. Thermodynamic values for five terminal mismatches adjacent to wobble base pairs are also reported.

Base Composition↗
PubMed1994

Crystal structure of unmodified tRNA(Gln) complexed with glutaminyl-tRNA synthetase and ATP suggests a possible role for pseudo-uridines in stabilization of RNA structure.

tRNA(2Gln) made in vitro by transcription with T7 RNA polymerase does not contain the pseudouridines at positions 38, 39, and 55, the 4-thiouridine at position 8, or any of the methylated bases found in the tRNA(2Gln) made in vivo. Cocrystals of unmodified tRNA(2Gln) complexed with glutaminyl-tRNA synthetase from Escherichia coli are isomorphous with those of the complex with modified tRNA(2Gln). A difference electron density map between the complexes with modified and unmodified tRNAs calculated at 2.5-A resolution shows no differences in the protein or tRNA structures, except for some very small shifts in atoms contacting the thiol at the 4 position of uridine 8 that are required to accommodate the smaller oxygen in the unmodified tRNA. Perhaps the most functionally significant change in the unmodified tRNA is the absence of the specifically bound water molecules that are observed to cross-link the N5 of the pseudo-uridines to their 5' phosphate. This suggests a possible role for pseudouridinylation in stabilization of the tRNA through water-mediated linking of these modified bases to the backbone, which is consistent with the lower thermal stability of the unmodified tRNA. An identical water-bridging structure is possible at four of the five other psuedo-uridines in known tRNA structures.

Adenosine Triphosphate↗