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Results for “RNA Stability”

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At least 505 records · Page 28Linked to original sources

PubMed1993

Initiation of mRNA decay in Bacillus subtilis.

Ribosome stalling in the leader region of ermC mRNA results in a 10-15-fold increase in ermC mRNA half-life in Bacillus subtilis. Fusion of the ermC 5' regulatory region to several B. subtilis coding sequences resulted in induced stability of the fusion RNAs, showing that the ermC 5' region acts as a general '5' stabilizer'. RNA products of an ermC-lacZ transcriptional fusion were inducibly stable in the complete absence of translation and included a small RNA that is likely to be a decay product arising by blockage of a 3'-to-5' exoribonuclease activity. Insertion of sequences that encode endonucleolytic cleavage sites into the ermC coding sequence resulted in cleavage products whose stability depended on the nature of their 5' and 3' ends. It can be concluded from this study that initiation of mRNA decay in B. subtilis generally occurs at or near the 5' terminus.

Bacillus subtilis↗

PubMed1988

Fibroblast growth factor is a permissive factor mediating the cellular action of rat hypothalamic growth hormone-releasing factor.

A clonal multipeptide-secreting cell line (44-2C) was used to study the interaction of basic fibroblast growth factor (bFGF) and GRF. The experiments were carried out in cells maintained under serum-free conditions in the absence of growth factors and hormonal supplements. We have shown that in 44-2C cells, rat hypothalamic GRF (rGRF) stimulates neurotensin, calcitonin, and somatostatin secretion. bFGF is not a mitogen in the 44-2C cells, but does regulate differentiated function by a mechanism involving regulation of RNA stabilization. The results presented here show for the first time that FGF acts as a competence factor mediating the cellular action of rGRF. In FGF-pretreated cells, 4-h exposure to rGRF stimulated RNA and protein synthesis. In this cell line rGRF was biologically active at 10(-14) M, and the ED50 for rGRF-mediated RNA and protein synthesis ranged from 10(-11) to 10(-12) M. We conclude that the interaction of FGF, acting as a permissive factor, and rGRF results in a novel mechanism of action for rGRF. These findings suggest that hypophysiotropic factors affect the cellular milieu in concert with growth factors, such as bFGF.

Amino Acids↗

PubMed1990

A potential role for RNA turnover in the light regulation of plant gene expression: ribulose-1,5-bisphosphate carboxylase small subunit in soybean.

Post-transcriptional regulation of the genes encoding the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase was examined in soybean seedlings. Substantial discrepancies were detected between relative in vitro transcription rates and steady-state RNA levels in light- and dark-grown seedling leaves, indicating that rbcS RNA may be degraded more rapidly in light than in darkness. Additional data imply that the turnover mechanism is rapidly induced by light, maintained for some time in darkness, and that it may be negatively controlled by far-red light. The proposed RNA turnover system does not affect all RNAs equally since a soybean actin gene showed equivalent in vitro transcription rates and RNA levels in light and darkness. Soybean rbcS genes may be subject to a novel mode of control in which light-induced expression is accompanied by an increased rate of RNA degradation. Models for the specific regulation of rbcS RNA stability in response to light are presented.

Blotting, Western↗

PubMed1995

RNA structure at high resolution.

Studies of RNA structural motifs at high resolution by NMR and X-ray crystallographic methods have provided many insights into the fundamental forces that give rise to the unique structural characteristics of RNA. Non-Watson-Crick purine-pyrimidine, purine-purine, and pyrimidine-pyrimidine base pairing, as well as base-phosphate and base-ribose hydrogen bonding, are important forces for folding and stabilizing RNA structures. Base stacking is as important in determining RNA conformations as hydrogen bonding interactions. With the noncanonical interactions, many single-stranded loop regions such as hairpin loops, bulge loops, and internal loops fold into well-defined secondary structures. Loop-loop and loop-helix interactions can produce tertiary structures such as pseudoknots. Also, single strands adjacent to helical regions can form tertiary contacts with base-paired nucleotides of the helices. As we learn more about the structures of the important motifs we can ask more specific questions about the mechanisms of RNA-mediated functions. Conformational flexibility rather than a specific shape of the RNA may be important for some biological reactions. However, knowledge of the structures and the ease of conformational change of the molecules involved in any process are essential for understanding and eventually controlling the process.

Base Sequence↗

PubMed1996

Structural features and stability of an RNA triple helix in solution.

A 30 nt RNA with a sequence designed to form an intramolecular triple helix was analyzed by one-and two-dimensional NMR spectroscopy and UV absorption measurements. NMR data show that the RNA contains seven pyrimidine-purine-pyrimidine base triples stabilized by Watson-Crick and Hoogsteen interactions. The temperature dependence of the imino proton resonances, as well as UV absorption data, indicate that the triple helix is highly stable at acidic pH, melting in a single sharp transition centered at 62 degrees C at pH 4.3. The Watson-Crick and Hoogsteen pairings are disrupted simultaneously upon melting. The NMR data are consistent with a structural model where the Watson-Crick paired strands form an A-helix. Results of model building, guided by NMR data, suggest a possible hydrogen bond between the 2' hydroxyl proton of the Hoogsteen strand and a phosphate oxygen of the purine strand. The structural model is discussed in terms of its ability to account for some of the differences in stability reported for RNA and DNA triple helices and provides insight into features that are likely to be important in the design of RNA binding compounds.

Base Composition↗

PubMed1998

The 3'-untranslated region of hepatitis C virus RNA enhances translation from an internal ribosomal entry site.

Translation of most eukaryotic mRNAs and many viral RNAs is enhanced by their poly(A) tails. Hepatitis C virus (HCV) contains a positive-stranded RNA genome which does not have a poly(A) tail but has a stretch of 98 nucleotides (X region) at the 3'-untranslated region (UTR), which assumes a highly conserved stem-loop structure. This X region binds a polypyrimidine tract-binding protein (PTB), which also binds to the internal ribosome entry site (IRES) in HCV 5'-UTR. These RNA-protein interactions may regulate its translation. We generated a set of HCV RNAs differing only in their 3'-UTRs and compared their translation efficiencies. HCV RNA containing the X region was translated three- to fivefold more than the corresponding RNAs without this region. Mutations that abolished PTB binding in the X region reduced, but did not completely abolish, enhancement in translation. The X region also enhanced translation from another unrelated IRES (from encephalomyocarditis virus RNA), but did not affect the 5'-end-dependent translation of globin mRNA in either monocistronic or bicistronic RNAs. It did not appear to affect RNA stability. The free X region added in trans, however, did not enhance translation, indicating that the translational enhancement by the X region occurs only in cis. These results demonstrate that the highly conserved 3' end of HCV RNA provides a novel mechanism for enhancement of HCV translation and may offer a target for antiviral agents.

3' Untranslated Regions↗

PubMed1998

The Epstein-Barr virus nuclear protein SM is both a post-transcriptional inhibitor and activator of gene expression.

The Epstein-Barr virus (EBV) nuclear protein BS-MLF1 (SM) is expressed early after entry of EBV into the lytic cycle. SM transactivates reporter gene constructs driven by a wide variety of promoters, but the mechanism of SM action is poorly understood. In this study, we demonstrate that the SM protein inhibits expression of intron-containing genes and activates expression of intron-less genes. We demonstrate that SM has the predicted inhibitory effect on expression of a spliced EBV gene but activates an unspliced early EBV gene. SM inhibited gene expression at the post-transcriptional level by preventing the accumulation of nuclear and cytoplasmic RNA transcripts. Conversely, SM led to increased accumulation of nuclear mRNA from intron-less genes without affecting the rate of transcription, indicating that SM enhances nuclear RNA stability. The ratio of cytoplasmic to nuclear polyadenylated mRNA was increased in the presence of SM, suggesting that SM also enhances nucleo-cytoplasmic mRNA transport. The degree of transactivation by SM was dependent on the sequence of the 3'-untranslated region of the target mRNA. Finally, we demonstrate that the amino-terminal portion of SM fused to glutathione-S-transferase binds radioactively labeled RNA in vitro, indicating that SM is a single-stranded RNA binding protein. Importantly, the latent and immediate-early genes of EBV contain introns whereas many early and late genes do not. Thus, SM may down-regulate synthesis of host cell proteins and latent EBV proteins while simultaneously enhancing expression of specific lytic EBV genes by binding to mRNA and modulating its stability and transport.

Cell Line↗

PubMed1992

Cloning of cDNAs encoding human S-100 alpha and beta subunits and their differential expression in human tumor cell lines.

We isolated nearly full-length clones of S-100 alpha and beta subunit cDNAs from a human brain cDNA library. The alpha subunit cDNA was 579 bp long and contained 99 bp of 5'-noncoding region, 282 bp of coding region, and 198 bp of 3'-noncoding region. The beta subunit cDNA was 743 bp long and contained 57 bp of 5'-noncoding region, 276 bp of coding region, and 410 bp of 3'-noncoding region. An amino acid sequence comparison between human and bovine alpha subunits and between human and rat beta subunits showed that both subunits were nearly entirely conserved. The amino acid sequences of human alpha and beta subunits were conserved at both Ca(2+)-binding domains. Northern blot analysis of brain RNA showed that human alpha and beta subunit cDNA probes discriminated between alpha and beta subunit mRNAs. By using these subunit-specific cDNA probes, it was demonstrated that alpha and beta subunit mRNAs were expressed in different manners among tumor cell lines: beta was detected in melanoma and some glioma cell lines, while alpha was detected only in a melanoma cell line. Southern blot analysis showed that there were no major deletions and rearrangements of alpha and beta subunit genes in these cell lines, regardless of the level of alpha and beta subunit expression, suggesting that the expression of these subunits may be regulated at the transcriptional or RNA stability level.

Amino Acid Sequence↗

PubMed2005

SRprises along a messenger's journey.

SR (serine/arginine-rich) proteins were originally identified as essential splicing factors. Recent work, however, has implicated these proteins in numerous additional steps of mRNA metabolism, including nuclear export, RNA stability, mRNA quality control, and translation.

Animals↗

PubMed2002

Regulation of the NPT gene by a naturally occurring antisense transcript.

The epithelial Na/Pi cotransporter (NaPi-II) is instrumental in maintaining phosphate (Pi) homeostasis in vertebrates. Hormones and metabolic factors (PTH, Pi availability) that acutely influence renal Pi excretion have been demonstrated to target NaPi-II expression. Upon stimulation, newly synthesized transporter molecules become integrated into the brush-border membrane to increase the Vmax of Pi uptake; reduction of Pi reabsorption is achieved by endocytosis of NaPi-II followed by lysosomal degradation of the protein. The long-term regulation of the protein is less well studied. Only recently, regulatory elements for vitamin D3 and Pi have been identified in the promoter region of the npt gene. However, signaling pathways leading to the activation of these regulatory sequences need to be established. Other reports suggested messenger RNA stability to play a role in the medium range regulation of NaPi-II expression. Recent findings in our laboratory added to the complex picture of npt gene regulation. We have identified npt-related endogenous antisense transcripts from mouse, zebrafish, and winter flounder. The two fish transcripts have been cloned and characterized; the mouse homolog has only very recently been detected. The transcripts are devoid of an open reading frame and appear in different splice forms. The evolutionary conservation of bidirectional transcription of the npt gene implies a regulatory function for the antisense transcript. In order to test the functional consequences of bidirectional transcription, we coexpressed sense and the antisense transcripts from zebrafish in Xenopus oocytes. Pi transport activity was reduced as a result of the presence of antisense RNA. Re-extraction of the RNA from injected oocytes followed by Northern blot revealed that the coexpression had no significant effect on the stability of either transcript. We concluded that the antisense mRNA interfered with the translation of the transporter if coexpressed in the Xenopus system. However, the regulatory mechanism(s) involving the npt-related antisense transcript is expected to be much more complicated in vivo, (i.e., requiring supplementary factors like double-stranded RNA recognizing proteins or specific RNases). It is planned to test this hypothesis by a transgenic zebrafish approach and/or knockout mice.

Animals↗

PubMed1995

Evidence for post-transcriptional regulation of cyclin B1 mRNA in the cell cycle and following irradiation in HeLa cells.

Cyclin B1 mRNA expression varies markedly through the cell cycle with its peak in G2/M and lowest level in G1. Cyclin B1 mRNA levels are also transiently reduced in HeLa cells after gamma-irradiation, coincident with the radiation-induced G2 block. In order to understand the mechanisms underlying these variations, we have measured cyclin B1 mRNA stability in HeLa cells during different phases of the cell cycle. The half-life of the mRNA measured after actinomycin D administration is 1.1-1.8 h in both early and late G1, 8 h in S and 13 h in G2/M. We therefore conclude that altered RNA stability is important in modulating cyclin B1 mRNA levels through the HeLa cell cycle. Furthermore, 3 h after irradiation of HeLa cells in S phase with 10 Gy, the half-life of cyclin B1 mRNA is reduced to 5 h; it is further reduced to 2-3 h at 14 h after irradiation. Thus, decreased stability contributes to the reduction in cyclin B1 mRNA following irradiation.

Blotting, Northern↗

PubMed1996

Regulation of gene expression in chloroplasts of higher plants.

Chloroplasts contain their own genetic system which has a number of prokaryotic as well as some eukaryotic features. Most chloroplast genes of higher plants are organized in clusters and are cotranscribed as polycistronic pre-RNAs which are generally processes into many shorter overlapping RNA species, each of which accumulates of steady-state RNA levels. This indicates that posttranscriptional RNA processing of primary transcripts is an important step in the control of chloroplast gene expression. Chloroplast RNA processing steps include RNA cleavage/trimming, RNA splicing, ENA editing and RNA stabilization. Several chloroplast genes are interrupted by introns and therefore require processing for gene function. In tobacco chloroplasts, 18 genes contain introns, six for tRNA genes and 12 for protein-encoding genes. A number of specific proteins and RNA factors are believed to be involved in splicing and maturation of pre-RNAs in chloroplasts. Processing enzymes and RNA-binding proteins which could be involved in posttranscriptional steps have been identified in the last several years. Our current knowledge of the regulation of gene expression in chloroplasts of higher plants is overviewed and further studies on this matter are also considered.

Chloroplasts↗

PubMed1992

Differential regulation of hepatic apolipoprotein A-I and A-II gene expression by thyroid hormone in rat liver.

Apolipoproteins A-I and A-II (apo A-I, apo A-II) are major protein components of high density lipoproteins. Thyroid hormone has a differential effect on the expression of the apo A-I and apo A-II genes in rat liver. Apo A-I gene expression is stimulated by thyroid hormone, whereas apo A-II mRNA abundance is decreased in chronic hyperthyroidism. To determine the regulatory steps involved in this differential effect of thyroid hormone on hepatic apo A-I and apo A-II gene expression, we studied the effect of short term and chronic hyperthyroidism on apo A-I and apo A-II gene transcription rates, nuclear RNA abundance and total cellular mRNA levels. After a single receptor saturating dose of L-triiodothyronine (T3) apo A-II gene transcription was transiently increased to 164% +/- 13% of basal values (P < 0.05) without affecting nuclear apo A-II RNA abundance. Apo A-I gene transcription, however, increased to 158% +/- 8% of baseline levels (P < 0.05) and remained elevated for at least 24 h. Nuclear and total cellular apo A-I mRNA increased more than expected from the increased transcription rate suggesting nuclear RNA stabilization and/or more efficient processing of the primary transcripts. In chronic hyperthyroidism, total cellular apo A-II mRNA abundance decreased to 62% +/- 18% (P < 0.05) and apo A-II gene transcription and apo A-II nuclear RNA were moderately reduced. By contrast, apo A-I nuclear and total cellular RNA were increased several fold by post-transcriptional mechanisms, whereas apo A-I gene transcription was drastically decreased. We conclude that the apo A-I and apo A-II genes in rat liver respond differently to both acute and chronic hyperthyroidism and that their expression is regulated at transcriptional and posttranscriptional levels.

Animals↗

PubMed2000

Unique structural and stabilizing roles for the individual pseudouridine residues in the 1920 region of Escherichia coli 23S rRNA.

The synthesis of a 5'-O-BzH-2'- O -ACE-protected pseudouridine phosphoramidite is reported [BzH, benzhydryloxy-bis(trimethylsilyloxy)silyl; ACE, bis(2-acetoxyethoxy)methyl]. The availability of the phosphoramidite allows for reliable and efficient syntheses of hairpin RNAs containing single or multiple pseudouridine modifications in the stem or loop regions. Five 19-nt hairpin RNAs representing the 1920-loop region (G(1906)-C(1924)) of Escherichia coli 23S rRNA were synthesized with pseudouridine residues located at positions 1911, 1915 and 1917. Thermodynamic parameters, circular dichroism spectra and NMR data are presented for all five RNAs. Overall, three different structural contexts for the pseudouridine residues were examined and compared with the unmodified RNA. Our main findings are that pseudouridine modifications exhibit a range of effects on RNA stability and structure, depending on their locations. More specifically, pseudouridines in the single-stranded loop regions of the model RNAs are slightly destabilizing, whereas a pseudo-uridine at the stem-loop junction is stabilizing. Furthermore, the observed effects on stability are approximately additive when multiple pseudouridine residues are present. The possible relationship of these results to RNA function is discussed.

Base Sequence↗

PubMed2007

Specification of primordial germ cells in medaka (Oryzias latipes).

BACKGROUND: Primordial germ cells (PGCs) give rise to gametes that are responsible for the development of a new organism in the next generation. Two modes of germ line specification have been described: the inheritance of asymmetrically-localized maternally provided cytoplasmic determinants and the induction of the PGC fate by other cell types. PGCs specification in zebrafish appears to depend on inheritance of germ plasm in which several RNA molecules such as vasa and nanos reside. Whether the specification mode of PGCs found in zebrafish is general for other fish species was brought into question upon analysis of olvas expression--the vasa homologue in another teleost, medaka (Oryzias latipes). Here, in contrast to the findings in zebrafish, the PGCs are found in a predictable position relative to a somatic structure, the embryonic shield. This finding, coupled with the fact that vasa mRNA, which is localized to the germ plasm of zebrafish but does not label a similar structure in medaka opened the possibility of fundamentally different mechanisms governing PGC specification in these two fish species. RESULTS: In this study we addressed the question concerning the mode of PGC specification in medaka using embryological experiments, analysis of RNA stability in the PGCs and electron microscopy observations. Dramatic alterations in the somatic environment, i.e. induction of a secondary axis or mesoderm formation alteration, did not affect the PGC number. Furthermore, the PGCs of medaka are capable of protecting specific RNA molecules from degradation and could therefore exhibit a specific mRNA expression pattern controlled by posttrancriptional mechanisms. Subsequent analysis of 4-cell stage medaka embryos using electron microscopy revealed germ plasm-like structures located at a region corresponding to that of zebrafish germ plasm. CONCLUSION: Taken together, these results are consistent with the idea that in medaka the inheritance of maternally provided asymmetrically-localized cytoplasmic determinants directs cells to assume the germ line fate similar to zebrafish PGCs.

Animals↗

PubMed1990

Triple post-transcriptional control.

The ermC gene confers resistance to MLS antibiotics in a Bacillus subtilis host. Synthesis of the ermC gene product, a ribosomal RNA methylase, is inducible by the addition of subinhibitory concentrations of erythromycin. Regulation of ermC gene expression occurs at the post-transcriptional level in three ways: translational attenuation, translational autoregulation, and messenger RNA stabilization.

Bacillus subtilis↗

PubMed1992

Transcriptional and posttranscriptional regulation of the proopiomelanocortin gene in the pars intermedia of the pituitary gland of Xenopus laevis.

In the melanotrope cells of the intermediate pituitary gland of the amphibian Xenopus laevis, expression of the POMC gene is under physiological control, i.e. it is 20- to 30-fold higher in animals adapted to a black background compared to animals adapted to a white background. To investigate whether changes in POMC messenger RNA stability contribute to this difference in expression, a steady state kinetic model for mRNA degradation was used to determine the half-life of POMC mRNA during the induction and deinduction of POMC gene expression in melanotrope cells. During induction of the POMC gene the half-life of POMC mRNA was 3- to 4-fold longer than during deinduction. This difference in mRNA stability is, however, not sufficient to account for the 20- to 30-fold difference in the steady state levels of POMC mRNA between the two physiological conditions. Results from experiments with the protein synthesis inhibitor cycloheximide and the mRNA synthesis inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzidimidazole suggest 1) that during induction of POMC gene expression no de novo protein synthesis is required and, 2) that deinduction of POMC gene expression requires transcriptional activation of an mRNA-degradation system. Pulse-labeling experiments with [3H]uridine showed that in neurointermediate lobes of white-adapted animals there is an 8-fold higher amount of newly synthesized POMC mRNA than in lobes of black animals. This suggests a fast in vitro induction of POMC gene transcription. The dopamine D2-receptor agonist apomorphine decreased POMC mRNA biosynthesis about 5-fold, which confirms that regulation of POMC gene expression includes a transcriptional component. In conclusion, our results demonstrate that during the physiological process of background adaptation the regulation of POMC gene expression in Xenopus melanotrope cells is exerted by alterations in POMC gene transcription as well as in POMC mRNA stability.

Animals↗

PubMed2006

The role of a zinc finger-containing glycine-rich RNA-binding protein during the cold adaptation process in Arabidopsis thaliana.

The mechanistic role of a glycine-rich RNA-binding protein designated atRZ-1a that contributes to enhance cold tolerance in Arabidopsis was investigated. Overexpression of atRZ-1a did not affect the expression of various cold-responsive genes such as COR6.6, COR15a, COR47, RD29A, RD29B and LTI29. Proteome analyses revealed that overexpression of atRZ-1a modulated the expression of several stress-responsive genes, and the transcript levels and RNA stability of these target genes were not affected by atRZ-1a. atRZ-1a successfully complements the cold sensitivity of Escherichia coli lacking four cold shock proteins. These results strongly suggest that atRZ-1a plays a role as an RNA chaperone during the cold adaptation process.

Acclimatization↗