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Results for “RNA Stability”

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At least 433 records · Page 24Linked to original sources

PubMed2000

Paper-based archiving of mammalian and plant samples for RNA analysis.

The ability to archive biological samples for subsequent nucleic acid analysis is essential for tissue specimens and forensic samples. FTA Card is a chemically treated filter paper designed for the collection and room temperature storage of biological samples for subsequent DNA analysis. Its usefulness for the preservation of biological samples for subsequent RNA analysis was tested. Here, we demonstrate that RNA in biological samples stored on FTA Cards is stable and can be used successfully for RT-PCR and northern blot analysis. RNA stability depends on the storage temperature and the type of biological specimen. RNA in mammalian cells stored on FTA Cards is stable for over one year at temperatures at or below -20 degrees C and for two to three months in samples stored at room temperature. For plant leaf, longer storage times (> 5 days) require temperatures at or below -70 degrees C following sample application. FTA Cards may constitute a method not only for convenient collection and storage of biological samples but also for rapid RT-PCR analysis of tissue and cell samples.

Animals↗

PubMed1993

Sodium butyrate causes an increase in the block to transcriptional elongation in the c-myc gene in SW837 rectal carcinoma cells.

Elevated expression of the c-myc oncogene is a frequent finding in tumors and cell lines derived from carcinomas of the colon and rectum. In a previous study we demonstrated that the differentiation agent sodium butyrate causes a rapid reduction in the expression of c-myc RNA in the rectal carcinoma cell line SW837. This effect was blocked by inhibitors of protein synthesis, suggesting that butyrate causes the induction of an activity that has a negative effect on c-myc expression. In the present work we demonstrate that the rapid decrease in the level of c-myc RNA, upon treatment of SW837 cells with 2 mM butyrate, is followed by a slower decrease in the level of p53 RNA and an increase in the RNA levels for fibronectin and a placental type alkaline phosphatase. Using in vitro elongation of nascent transcripts to measure transcription and actinomycin D chase experiments to measure RNA stability, we show that the reduction in expression of c-myc RNA is due to an increase in the block to transcriptional elongation, rather than a decrease in transcriptional initiation or an increase in degradation of the RNA. We conclude that sodium butyrate induces an activity that increases the transcriptional block in SW837 cells, and that regulation of transcriptional elongation is an important mechanism for regulating c-myc expression in this cell type. A shift in relative usage of the two major promoters in the c-myc gene accompanies the reduction in expression. The potential significance of this finding with respect to transcriptional elongation is discussed. Mutations in the exon 1/intron 1 boundary region of the c-myc gene cause an increase in transcriptional elongation in Burkitt lymphoma. We sequenced this region in a series of cell lines derived from colorectal carcinomas, all of which had an elevated level of c-myc expression, to determine if a similar mutational mechanism is at work in this disease. All of the lines examined had a normal c-myc DNA sequence, suggesting that the deregulation of c-myc expression in colon cancer is not due to a cis mutation in this region.

Antisense Elements (Genetics)↗

PubMed1981

Regulation of nitrogenase messenger RNA synthesis and stability in Klebsiella pneumoniae.

Nitrogenase messenger RNA synthesis in Klebsiella pneumoniae was determined by labelling cells with (3H)uracil and isolating total RNA, which was then hybridized to filter-bound recombinant plasmid pSA30 DNA carrying the nitrogenase structural genes nifH, D, and K, Derepression of nitrogenase mRNA starts 1.5 h before the onset of nitrogenase activity (as measured by acetylene reduction). Exposure of nif-derepressed cultures to either NH4+, air, or high temperatures (39 degree C) results in a rapid decrease of the synthesis rates both of nitrogenase mRNA and nitrogenase polypeptides. Nitrogenase mRNA is remarkably stable. After blocking transcription with rifampicin, hybridizable and actively translatable nitrogenase mRNA survives with an average half-life of 18 min. Half-lives are considerably shorter when rifampicin-inhibited cultures are simultaneously shifted to conditions which are non-permissive for nitrogenase synthesis, pointing to some posttranscriptional influence on nitrogenase mRNA stability. In all experiments performed there was no evidence for uncoupling of nitrogenase mRNA synthesis from nitrogenase mRNA translation, indicating that nitrogenase synthesis is regulated solely by transcriptional control.

Genes, Bacterial↗

PubMed1990

Transcriptional regulation of gene expression in Tetrahymena thermophila.

The only well-characterized study of gene expression in Tetrahymena thermophila (1) demonstrates that the temperature dependent expression of the Ser H3 gene is regulated at the level of mRNA stability. A run-on transcription assay was developed to determine if regulation of RNA stability was a major mechanism regulating gene expression in Tetrahymena or if transcriptional regulation dominates. The relative transcriptional activities of 14 Tetrahymena genes were determined in different physiological/developmental states (growing, starved and conjugating) in which many of the genes showed striking differences in RNA abundance. In every case except Ser H3, changes in transcription accompanied changes in RNA abundance. Thus differential transcription, not differential RNA degradation, is the major mechanism regulating RNA abundance in Tetrahymena.

Animals↗

PubMed1967

Physiology of rat-liver polysomes. The stability of messenger ribonucleic acid and ribosomes.

Starvation of rats for several days led to marked decrease in cytoplasmic polysomes and accumulation of breakdown products having S values less than 200s. Re-feeding of the starved animals induced a rapid reassembly of polysomes. These newly formed polysomes, in the presence of actinomycin D, decayed in a biphasic fashion: about two-thirds decayed with an apparent half-life of 3-3(1/2)hr. but the other one-third were much more stable. Evidence that polysome decay is an accurate reflexion of messenger RNA stability is presented, and it is concluded that in the presence of large doses of actinomycin D, rat-liver cytoplasm contains messenger RNA classes of widely varying stability, the more stable class having a half-life of at least 80hr. The half-life of liver ribosomes was also determined and was found to be 110-127hr.

Animals↗

PubMed1992

Requirement for ICR-like sequences in the replication of brome mosaic virus genomic RNA.

Previous studies using a brome mosaic virus (BMV) RNA-2 deletion mutant (pRNA-2 M/S) and additional derivatives as reporters established that viral sequences resembling internal control regions (ICRs) 1 and 2 of tRNA gene promoters are vital to (+)-strand replication in protoplasts. Transfer of these mutations to genomic RNA-2 and functional analysis in protoplast, local lesion, and systemic infections revealed a sequence-specific requirement for bases within the ICR2-like motif. Despite the low (generally less than 20% of wild-type) and sometimes undetectable levels of replication of these RNA-2 mutants, sufficient p2a protein was produced to support at least modest levels of RNA-1, -3 and -4 replication in protoplasts. However, only those RNA-2 ICR2 mutants supporting substantial replication of the viral genome in protoplasts were capable of establishing local lesions in Chenopodium hybridum and systemic infections in barley, further establishing the essential role of the ICR-like sequences in viral infectivity. Upon passage through a second set of barley plants, accumulation patterns for progeny from inocula containing certain RNA-2 mutants paralleled those from wild-type inocula, indicating repair of the introduced mutations. RNA stability and translatability were shown to be unaffected by the introduced mutations. BMV RNA-3 contains several ICR-like sequences, each of which was individually deleted. Whereas deletion of the 5'-terminal ICR2-like motif had little effect on RNA-3 accumulation in protoplasts or local lesion formation, it debilitated systemic spread in barley. Deletion of an internal ICR2-like motif at position 1100 decreased (+):(-) strand asymmetry from greater than 100:1 to 14:1, reduced RNA-3 replication in protoplasts to less than 15% of wild-type, and abolished local lesion and systemic infectivity.

Base Sequence↗

PubMed2006

Mg2+ dependency of HIV-1 reverse transcription, inhibition by nucleoside analogues and resistance.

Metal ions are essential for DNA polymerase and RNase H activities of HIV-1 reverse transcriptase (RT). RT studies are routinely performed at 6-8 mM Mg2+, despite the fact that the in vivo concentration might be as low as 0.2 mM. We studied the influence of MgCl2 and ATP, which likely binds a significant fraction of the magnesium pool in vivo, on the DNA polymerase and RNase H activities of HIV-1 RT, its inhibition by nucleoside RT inhibitors (NRTIs) and primer unblocking by AZT-resistant RT. At low Mg2+ concentration, reverse transcription of a natural template strongly increased despite a dramatically reduced intrinsic polymerase activity under such conditions. Low Mg2+ concentrations affected the RNA stability and indirectly decreased its degradation by the RNase H activity. The reduced RNA degradation prevented premature dissociation of the template and primer strands that otherwise generated dead-end DNA products. In addition, low Mg2+ dramatically decreased the incorporation of NRTIs into DNA and increased nucleotide excision by AZT-resistant RT. The latter effect is also most likely owing to the diminished cleavage of the RNA template. Thus, differences in the free Mg2+ concentration between different cell types or during the cell cycle might strongly affect HIV-1 replication and its inhibition.

Adenosine Triphosphate↗

PubMed2005

Enterobacterial repetitive intergenic consensus sequence repeats in yersiniae: genomic organization and functional properties.

Genome-wide analyses carried out in silico revealed that the DNA repeats called enterobacterial repetitive intergenic consensus sequences (ERICs), which are present in several Enterobacteriaceae, are overrepresented in yersiniae. From the alignment of DNA regions from the wholly sequenced Yersinia enterocolitica 8081 and Yersinia pestis CO92 strains, we could establish that ERICs are miniature mobile elements whose insertion leads to duplication of the dinucleotide TA. ERICs feature long terminal inverted repeats (TIRs) and can fold as RNA into hairpin structures. The proximity to coding regions suggests that most Y. enterocolitica ERICs are cotranscribed with flanking genes. Elements which either overlap or are located next to stop codons are preferentially inserted in the same (or B) orientation. In contrast, ERICs located far apart from open reading frames are inserted in the opposite (or A) orientation. The expression of genes cotranscribed with A- and B-oriented ERICs has been monitored in vivo. In mRNAs spanning B-oriented ERICs, upstream gene transcripts accumulated at lower levels than downstream gene transcripts. This difference was abolished by treating cells with chloramphenicol. We hypothesize that folding of B-oriented elements is impeded by translating ribosomes. Consequently, upstream RNA degradation is triggered by the unmasking of a site for the RNase E located in the right-hand TIR of ERIC. A-oriented ERICs may act in contrast as upstream RNA stabilizers or may have other functions. The hypothesis that ERICs act as regulatory RNA elements is supported by analyses carried out in Yersinia strains which either lack ERIC sequences or carry alternatively oriented ERICs at specific loci.

Base Sequence↗

PubMed2002

Genetic analysis of sequences in the 3' nontranslated region of hepatitis C virus that are important for RNA replication.

The genome of the hepatitis C virus (HCV) is a plus-strand RNA molecule that carries a single long open reading frame. It is flanked at either end by highly conserved nontranslated regions (NTRs) that mediate crucial steps in the viral life cycle. The 3' NTR of HCV has a tripartite structure composed of an about 40-nucleotide variable region, a poly(U/UC) tract that has a heterogeneous length, and a highly conserved 98-nucleotide 3'-terminal sequence designated the X tail or 3'X. Conflicting data as to the role the sequences in the 3' NTR play in RNA replication have been reported. By using the HCV replicon system, which is based on the self-replication of subgenomic HCV RNAs in human hepatoma cell line Huh-7, we mapped in this study the sequences in the 3' NTR required for RNA replication. We found that a mutant with a complete deletion of the variable region is viable but that replication is reduced significantly. Only replicons in which the poly(U/UC) tract was replaced by a homouridine stretch of at least 26 nucleotides were able to replicate, whereas RNAs with homopolymeric guanine, adenine, or cytosine sequences were inactive. Deletions of individual or all stem-loop structures in 3'X were not tolerated, demonstrating that this region is most crucial for efficient RNA replication. Finally, we found that none of these deletions or substitutions within the 3' NTR affected RNA stability or translation, demonstrating that the primary effect of the mutations was on RNA replication. These data represent the first detailed mapping of sequences in the 3' NTR assumed to act as a promoter for initiation of minus-strand RNA synthesis.

3' Untranslated Regions↗

PubMed1995

Overexpression of activation transcriptional factor 1 in lymphomas and in activated lymphocytes.

cAMP-regulated gene expression always involves a conserved cAMP-responsive element (CRE) present in the promoter of cAMP-inducible genes. Two of the highly related proteins, cyclic AMP-responsive element binding protein (CREB) and activation transcriptional factor 1 (ATF-1), have been shown to activate transcription in response to cAMP by interacting with CRE. However, ATF-1 is a much weaker mediator of cAMP response, and its functional role in vivo remains unclear. Here we report a significant enhancement of ATF-1 expression in most transformed lymphocytes. Little variation in CREB level was observed, however. The activation of normal T lymphocytes induced a transient increase of ATF-1 expression to a level comparable to that of T lymphomas. Activation had no effect on the ATF-1 level of transformed T lymphocytes. The induction of ATF-1 required the costimulation of normal T lymphocytes with TPA and A23187. TPA, Ca2+ ionophore, or cAMP alone did not stimulate ATF-1 expression in normal lymphocytes. Nuclear run-on assay indicates that the increased ATF-1 expression in T cell lymphomas and in activated splenic T lymphocytes was not due to an enhanced transcription. Instead, an increase in ATF-1 mRNA stability was found in these lymphocytes. The regulation of ATF-1 expression through RNA stability in cells of different states suggests that ATF-1 may play an active role in cell growth and differentiation.

Activating Transcription Factor 1↗

PubMed1996

FGF-2, BMP-2, and BMP-4 regulate retinoid binding proteins and receptors in 3T3 cells.

An Important part of intercellular signalling is the ability of responding cells to regulate multiple signal transduction pathways. Just as retinoic acid exposure alters expression of many peptide growth factors and their receptors, we have found that peptide growth factors alter the expression of cellular retinoic acid binding proteins (CRABPs) and retinoic acid receptors (RARs). FGF-2 (basic fibroblast growth factor) treatment of BALB 3T3 fibroblasts increased the level of CRABP I RNA, whereas bone morphogenetic protein (BMP)-2 and BMP-4 reduced this level as well as the levels of CRABP II and RAR beta 1/beta 3 transcripts. Regulation of the CRABP I gene by FGF-2 occurred posttranscriptionally by increasing RNA stability. However, BMP-2 down-regulated CRABP I message without affecting message stability. Neither of these mechanisms was dominant, with intermediate levels of CRABP I RNA occurring in the presence of both FGF-2 and BMP-2 or BMP-4. These two different modes of regulation thus allow different levels of CRABP I RNA accumulation in the presence of different ratios of FGF-2 and BMP-2 or BMP-4.

3T3 Cells↗

PubMed1994

Sequence and structural elements critical for U8 snRNP function in Xenopus oocytes are evolutionarily conserved.

We have generated mutants in Xenopus U8 RNA, a nucleolar snRNA required for the maturation of 5.8S and 28S rRNAs, to identify sequences and structural domains essential for RNA stability, particle assembly, and function of the U8 RNP. Activity of the mutants was assayed by microinjection of in vitro-synthesized U8 RNAs into the cytoplasm of Xenopus oocytes. Most of the mutant RNAs were stable, bound fibrillarin, a protein common to several of the nucleolar-specific snRNPs, and became hypermethylated. Although hypermethylation of the 5' cap of U8 RNA and fibrillarin binding can occur in either the cytoplasmic or nuclear compartment of Xenopus oocytes, neither is required for nuclear import. We find that the trimethylguanosine cap, although present on the endogenous U8 RNA, is not essential for stability, particle assembly, or functioning of U8 in the coordinate processing of pre-rRNA at sites 3' of 28S and 5' of 5.8S RNA. Several conserved single- and double-stranded sequences within the 5' domain of U8 RNA are essential for function.

Animals↗

PubMed2002

RNA binding properties of the AU-rich element-binding recombinant Nup475/TIS11/tristetraprolin protein.

Regulation of messenger RNA stability by AU-rich elements is an important means of regulating genes induced by growth factors and cytokines. Nup475 (also known as tristetraprolin, or TIS11) is the prototype for a family of zinc-binding Cys(3)His motif proteins required for proper regulation of tumor necrosis factor mRNA stability in macrophages. We developed an Escherichia coli expression system to produce soluble Nup475 protein in quantity to study its RNA binding properties. Nup475 protein bound a tumor necrosis factor AU-rich element over a broad range of pH and salt concentrations by RNA gel shift. This binding was inhibited by excess zinc metal, providing a potential mechanism for previous reports of zinc stabilization of AU-rich element (ARE) containing messenger RNAs. Immobilized Nup475 protein was used to select its optimal binding site by RNA SELEX and revealed a strong preference for the extended sequence UUAUUUAUU, rather than a simple AUUUA motif. These findings were confirmed by site-directed mutagenesis of the tumor necrosis factor ARE and RNA gel shifts on c-fos, interferon-gamma, and interferon-beta ARE fragments. A weaker binding activity toward adenine-rich sites, such as a poly(A) tail RNA fragment, can partially disrupt the Nup475-tumor necrosis factor AU-rich element complex.

Base Sequence↗

PubMed1990

Intervening sequences increase efficiency of RNA 3' processing and accumulation of cytoplasmic RNA.

Two expression vectors were constructed that differ only in the presence (+) or absence (-) of an intervening sequence (IVS) in their 5'-untranslated leaders. Transient transfection into four mammalian cell lines resulted in higher levels of the indicator protein (CAT) from the IVS(+) vector (6 to 50-fold). Cytoplasmic RNA concentrations in 293s and HeLa cell lines corresponded directly to resultant protein levels; measurements in 293s cells of transcription initiation and elongation, steady-state total nuclear RNA, and cytoplasmic RNA stability, were equivalent for the two vectors. Surprisingly, the amount of poly(A)+ nuclear RNA was greater from the IVS(+) vector. Since this difference matches the ratio seen with polyadenylated cytoplasmic RNA, our results imply that splicing is coupled to a polyadenylation/transport pathway.

Animals↗

PubMed1993

Coupled translation and replication of poliovirus RNA in vitro: synthesis of functional 3D polymerase and infectious virus.

Poliovirus RNA polymerase and infectious virus particles were synthesized by translation of virion RNA in vitro in HeLa S10 extracts. The in vitro translation reactions were optimized for the synthesis of the viral proteins found in infected cells and in particular the synthesis of the viral polymerase 3Dpol. There was a linear increase in the amount of labeled protein synthesized during the first 6 h of the reaction. The appearance of 3Dpol in the translation products was delayed because of the additional time required for the proteolytic processing of precursor proteins. 3Dpol was first observed at 1 h in polyacrylamide gels, with significant amounts being detected at 6 h and later. Initial attempts to assay for polymerase activity directly in the translation reaction were not successful. Polymerase activity, however, was easily detected by adding a small amount (3 microliters) of translation products to a standard polymerase assay containing poliovirion RNA. Full-length minus-strand RNA was synthesized in the presence of an oligo(U) primer. In the absence of oligo(U), product RNA about twice the size of virion RNA was synthesized in these reactions. RNA stability studies and plaque assays indicated that a significant fraction of the input virion RNA in the translation reactions was very stable and remained intact for 20 h or more. Plaque assays indicated that infectious virus was synthesized in the in vitro translation reactions. Under optimal conditions, the titer of infectious virus produced in the in vitro translation reactions was greater than 100,000 PFU/ml. Virus was first detected at 6 h and increased to maximum levels by 12 h. Overall, the kinetics of poliovirus replication (protein synthesis, polymerase activity, and virus production) observed in the HeLa S10-initiation factor in vitro translation reactions were similar to those observed in infected cells.

Cell-Free System↗

PubMed2002

Mechanisms of regulation of G(11)alpha protein by dexamethasone in osteoblastic UMR 106-01 cells.

We have previously demonstrated that glucocorticoids increased G(q/11)alpha protein expression and phospholipase C activity in the rat osteosarcoma cell line UMR 106-01. In this study, we demonstrated that G(11)alpha is the primary G(q)-subtype family member expressed in UMR cells. Dexamethasone treatment increased the expression of G(11)alpha protein in both a time- and a dose-dependent manner. Glucocorticoid treatment significantly increased the half-life of G(11)alpha protein from 20.3 to 63 h. Steady-state G(11)alpha mRNA level was also increased by glucocorticoid treatment by approximately 70%. This change was not the result of changes in RNA stability but rather the result of increased transcription, because the glucocorticoid-mediated upregulation of G(11)alpha mRNA was blocked by the transcription inhibitor actinomycin D. The dexamethasone induction of G(11)alpha mRNA occurred after a time lag of 12-24 h and was blocked by the protein synthesis inhibitor cycloheximide. These results suggest that the dexamethasone-induced rise in G(11)alpha protein results primarily from changes in the degradation rate of the protein, whereas changes in G(11)alpha mRNA play a smaller role and require de novo synthesis of regulatory protein(s).

Animals↗

PubMed1988

CUUCGG hairpins: extraordinarily stable RNA secondary structures associated with various biochemical processes.

The mRNA of bacteriophage T4 contains a strikingly abundant intercistronic hairpin. Within the 55 kilobases of known T4 sequence, the hexanucleotide sequence CTTCGG is found 13 times in the DNA strand equivalent to mRNA sequences. In 12 of those occurrences, the sequence is flanked by inverted repeats predictive of RNA hairpins with UUCG in the loop. Avian myeloblastosis virus reverse transcriptase, which can traverse hairpins of larger calculated stability, terminates efficiently at these CUUCGG hairpins. Thermal denaturation studies of model hairpins show that the loop sequence UUCG dramatically stabilizes RNA hairpins when compared to a control sequence. These data, when combined with previously described parameters of helix stability, suggest that T4 has utilized this loop sequence to optimize the stability of intercistronic hairpins. The stability of CUUCGG hairpins is also utilized in the RNAs of many organisms besides T4.

Base Sequence↗

PubMed1989

Transcriptional analysis of the F plasmid surface exclusion region: mapping of traS, traT, and traD transcripts.

Transcription of the surface exclusion genes, traS and traT, of the F plasmid of Escherichia coli K-12 has been analyzed using S1 nuclease protection experiments. The results show that both genes possess functional promoters in vivo and that a strong transcriptional termination signal lies immediately downstream of traT. Moreover, the adjacent downstream gene, traD, is expressed from its own promoter and appears not to lie within the major transfer operon. RNA stability experiments indicate that the products of traS and traT are translated from a stable RNA message. We propose that the traS and traT promoters serve to supplement the expression of these genes from the major tra promoter, PY.

Amino Acid Sequence↗