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Results for “RNA Stability”

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At least 55 records · Page 3Linked to original sources
PubMed1999

Assessment of hepatitis C virus RNA stability in serum by the Quantiplex branched DNA assay.

OBJECTIVES: Quantification of hepatitis C virus (HCV) RNA in serum is used to assess the probability of treatment response and to monitor antiviral therapy. Since serum specimens often are shipped to central sites for HCV RNA testing, it is important to define conditions that preserve HCV RNA integrity. METHODS: We evaluated the stability of HCV RNA in 25 previously frozen (PF) and 11 fresh, never previously frozen (NPF) specimens subjected to handling and short-term storage conditions that mimic those encountered during interlaboratory shipping. All sera were separated within 4 h of collection. PF samples covering a approximately 3 log10 HCV RNA dynamic range were thawed, divided into aliquots, incubated at 4, 23, and 37 degrees C (+/- 1.5 degrees C) for 24, 48, 72 and 96 h (+/- 2 h), and then refrozen at -70 degrees C prior to testing with the Quantiplex HCV RNA 2.0 assay. Eleven NPF samples were stored at -70, -20, and 4 degrees C (+/- 1.5 degrees C) for up to 1 month prior to testing. RESULTS: Linear regression analysis showed no HCV RNA degradation in PF specimens kept at 4 degrees C over 4 days. However, HCV RNA levels in PF specimens decreased over 4 days by 20 and 105% at 23 and 37 degrees C, respectively. Three independent statistical methods showed that the probability of specimen failure in PF specimens, defined as a loss of 20% or more of HCV RNA, was lowest at 4 degrees C and increased with increasing temperature. The HCV RNA quantification of the 11 NPF specimens stored at 4 degrees C was similar to their frozen controls. CONCLUSION: HCV RNA in separated serum specimens is stable for at least 4 days at 4 degrees C.

DNA, Viral↗
PubMed2003

Multicenter evaluation of use of dried blood and plasma spot specimens in quantitative assays for human immunodeficiency virus RNA: measurement, precision, and RNA stability.

Eleven laboratories evaluated the use of dried blood and plasma spots for quantitation of human immunodeficiency virus (HIV) RNA by two commercially available RNA assays, the Roche Amplicor HIV-1 Monitor and the bioMerieux NucliSens HIV-1 QT assays. The recovery of HIV RNA was linear over a dynamic range extending from 4,000 to 500,000 HIV type 1 RNA copies/ml. The Monitor assay appeared to have a broader dynamic range and seemed more sensitive at lower concentrations. However, the NucliSens assay gave more consistent results and could be performed without modification of the kit. HIV RNA was stable in dried whole blood or plasma stored at room temperature or at -70 degrees C for up to 1 year. Dried blood and dried plasma spots can be used as an easy and inexpensive means for the collection and storage of specimens under field conditions for the diagnosis of HIV infection and the monitoring of antiretroviral therapy.

Antiviral Agents↗
PubMed2006

Smad4-dependent regulation of urokinase plasminogen activator secretion and RNA stability associated with invasiveness by autocrine and paracrine transforming growth factor-beta.

Metastasis is a primary cause of mortality due to cancer. Early metastatic growth involves both a remodeling of the extracellular matrix surrounding tumors and invasion of tumors across the basement membrane. Up-regulation of extracellular matrix degrading proteases such as urokinase plasminogen activator (uPA) and matrix metalloproteinases has been reported to facilitate tumor cell invasion. Autocrine transforming growth factor-beta (TGF-beta) signaling may play an important role in cancer cell invasion and metastasis; however, the underlying mechanisms remain unclear. In the present study, we report that autocrine TGF-beta supports cancer cell invasion by maintaining uPA levels through protein secretion. Interestingly, treatment of paracrine/exogenous TGF-beta at higher concentrations than autocrine TGF-beta further enhanced uPA expression and cell invasion. The enhanced uPA expression by exogenous TGF-beta is a result of increased uPA mRNA expression due to RNA stabilization. We observed that both autocrine and paracrine TGF-beta-mediated regulation of uPA levels was lost upon depletion of Smad4 protein by RNA interference. Thus, through the Smad pathway, autocrine TGF-beta maintains uPA expression through facilitated protein secretion, thereby supporting tumor cell invasiveness, whereas exogenous TGF-beta further enhances uPA expression through mRNA stabilization leading to even greater invasiveness of the cancer cells.

Autocrine Communication↗
PubMed1993

Direct evidence for selective modulation of psbA, rpoA, rbcL and 16S RNA stability during barley chloroplast development.

The turnover of RNAs encoded by seven different barley chloroplast genes was analyzed after treatment of barley shoots with tagetitoxin, a selective inhibitor of chloroplast transcription. Changes in RNA stability were examined during chloroplast development using basal and apical leaf sections of 4.5-day-old dark-grown seedlings and apical leaf sections of 4.0-day-old dark-grown seedlings which had been illuminated for 12 h. Of the RNAs examined, a 2.6 kb unspliced precursor of tRNA(lys) exhibited the shortest half-life, which was estimated to be 3 h. The 16S rRNA and psbA mRNA had the longest estimated half-lives, which were greater than 40 h. Among mRNAs, half-lives were estimated to range from 6 h for psaA mRNA, to over 40 h for psbA mRNA. Therefore, barley chloroplast mRNAs have long half-lives relative to bacterial mRNAs. The stability of atpB mRNA and the unspliced precursor of tRNA-lys was not altered during chloroplast development, while the stability of psaA mRNA decreased 2-fold. In contrast, the stability of the 16S rRNA and mRNAs for rpoA, psbA and rbcL increased during chloroplast development. The stability of 16S rRNA increased markedly during chloroplast development in the dark and this increase was maintained in illuminated seedlings. The stability of rbcL mRNA increased 2.5-fold during chloroplast development in the dark, and then decreased 2-fold in chloroplasts of light-grown plants. The initial increase in rpoA and psbA mRNA stability was also light-independent, with total increases in stability of at least 5-fold. In the case of rpoA, the stability of 2 of the 13 polycistronic rpoA transcripts that were detected in dark-grown plants was selectively increased during chloroplast development. In conclusion, the stability of some transcripts is selectively increased and further modulated during chloroplast development in barley. We propose that the selective stabilization of chloroplast mRNA, which occurred independent of light, is an indication that non-light regulated developmental signals are involved in barley chloroplast mRNA stability.

Base Sequence↗
PubMed1993

Multicopy STS1 restores both protein transport and ribosomal RNA stability in a new yeast sec23 mutant allele.

A thermosensitive mutant was selected on the basis of its resistance after a heat shock to the lethal effects of lomofungin, a drug that inhibits RNA synthesis. We demonstrate that the single mutation conferring thermosensitivity and lomofungin resistance is heteroallelic to sec23-1. This new allele of sec23 is designated sec23-11. The yeast SEC23 gene has previously been reported to function in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. After a 1-min shift to elevated temperature, a sec23-1 mutant becomes defective in vesicle formation from the ER. We show here that upon a shift to high temperature, both sec23 alleles confer defects in ER-to-Golgi transport, and both mutants stop accumulating nascent RNA after 20 to 30 min. Transcription and early maturation of ribosomal RNA, the major RNA species, are normal in a sec23-11 mutant, but the matured ribosomal RNA is then degraded. This ribosomal RNA instability may reflect improper assembly of ribosomal subunits, due to failure to localize essential factors such as ribosomal proteins to the nucleus. In addition to the sec23 mutants, other ER-to-Golgi secretion mutants exhibit a strong RNA biosynthetic defect, and we conclude that continued post-ER protein transport, not just a functional SEC23 gene product (Sec23p), is required for ribosomal RNA stability. We also identified an extragenic multicopy suppressor of the sec23-11 mutant, the essential STS1 gene ("Sec Twenty-three Suppressor 1"). Besides restoring partial temperature resistance to the sec23-11 mutant, overexpression of STS1 also overcomes both the RNA synthesis and the protein transport blocks in this mutant. In contrast, multicopy STS1 exerts only small effects on the sec23-1 mutant. The predicted STS1 gene product (Sts1p) is a low-abundance protein of 36,500 daltons, with no signal peptide and no hydrophobic stretch of sufficient length to span a membrane. Like Sec23p, Sts1p is found in a cytosolic compartment. These features suggest that Sts1p may play a catalytic or regulatory role in conjunction with the more abundant Sec23p.

ATP-Binding Cassette Transporters↗
PubMed1996

Point mutations in a transcription terminator, lambda tI, that affect both transcription termination and RNA stability.

The terminator tI is located approx. 280 nucleotides beyond the int gene of bacteriophage lambda. Besides its role as a transcription terminator, tI may confer stability to the int message by protecting it from 3' exonucleolytic degradation. In order to study the role of the tI sequence in transcription termination and RNA stability, three different point mutations tI1, tI2, and tI3 were isolated and characterized. All the tI mutations map in the G + C-rich region of dyad symmetry in the terminator and decrease the transcriptional termination of tI in vivo from 99% for the wild type terminator to 81-93% as determined by galactokinase activity and in vitro from 80% for the wild type terminator to 8-12% using the E. coli RNA polymerase. Additionally, the tI mutations cause upstream transcript instability in vivo. This instability defect caused by tI mutations is compensated by the host mutant deficient in polynucleotide phosphorylase resulting in increased steady state levels of these mutant transcripts. The results show that the intact hairpin of tI is essential for efficient transcription termination and for maintaining mRNA stability by blocking the 3' to 5' exonucleolytic activity of polynucleotide phosphorylase.

Bacteriophage lambda↗
PubMed1997

The poly(A) tail inhibits the assembly of a 3'-to-5' exonuclease in an in vitro RNA stability system.

We have developed an in vitro system which faithfully reproduces several aspects of general mRNA stability. Poly(A)- RNAs were rapidly and efficiently degraded in this system with no detectable intermediates by a highly processive 3'-to-5' exonuclease activity. The addition of a poly(A) tail of at least 30 bases, or a 3' histone stem-loop element, specifically stabilized these transcripts. Stabilization by poly(A) required the interaction of proteins with the poly(A) tail but did not apparently require a 3' OH or interaction with the 5' cap structure. Finally, movement of the poly(A) tract internal to the 3' end caused a loss of its ability to stabilize transcripts incubated in the system but did not affect its ability to interact with poly(A) binding proteins. The requirement for the poly(A) tail to be proximal to the 3' end indicates that it mediates RNA stability by blocking the assembly, but not the action, of an exonuclease involved in RNA degradation in vitro.

Exoribonucleases↗
PubMed2000

A role for the Escherichia coli H-NS-like protein StpA in OmpF porin expression through modulation of micF RNA stability.

When a wild-type strain of Escherichia coli and its stpA, hns and stpA hns mutant derivatives were compared by two-dimensional protein gel electrophoresis, the levels of expression of several proteins were found to vary. One of these was identified as the outer membrane porin protein, OmpF. In the stpA hns double mutant, the level of OmpF was downregulated dramatically, whereas in hns or stpA single mutants, it was affected only slightly. Transcription from the ompF promoter was reduced by 64% in the double mutant; however, the level of ompF mRNA was reduced by 96%. This post-transcriptional expression was found to result from a strong reduction in the half-life of ompF message in the double mutant. The micF antisense RNA was shown to be involved in OmpF regulation by StpA using a strain deleted for micF. Moreover, micF antisense RNA accumulated considerably in an stpA hns background. Transcriptional data from a micF-lacZ fusion and measurements of micF RNA half-life confirmed that this was caused by transcriptional derepression of micF as a result of the hns lesion and increased micF RNA stability due to the absence of StpA (a known RNA chaperone). These data suggest a novel facet to the regulation of OmpF expression, namely destabilization of micF RNA by StpA.

Base Sequence↗
PubMed2005

Interaction between Brome mosaic virus proteins and RNAs: effects on RNA replication, protein expression, and RNA stability.

Brome mosaic virus (BMV) RNA replication has been examined in a number of systems, including Saccharomyces cerevisiae. We developed an efficient T-DNA-based gene delivery system using Agrobacterium tumefaciens to transiently express BMV RNAs in Nicotiana benthamiana. The expressed RNAs can systemically infect plants and provide material to extract BMV replicase that can perform template-dependent RNA-dependent RNA synthesis in vitro. We also expressed the four BMV-encoded proteins from nonreplicating RNAs and analyzed their effects on BMV RNA accumulation. The capsid protein that coinfiltrated with constructs expressing RNA1 and RNA2 suppressed minus-strand levels but increased plus-strand RNA accumulation. The replication proteins 1a and 2a could function in trans to replicate and transcribe the BMV RNAs. None of the BMV proteins or RNA could efficiently suppress posttranscriptional silencing. However, 1a expressed in trans will suppress the production of a recombinant green fluorescent protein expressed from the nontranslated portions of BMV RNA1 and RNA2, suggesting that 1a may regulate translation from BMV RNAs. BMV replicase proteins 1a did not affect the accumulation of the BMV RNAs in the absence of RNA replication, unlike the situation reported for S. cerevisiae. This work demonstrates that the Agrobacterium-mediated gene delivery system can be used to study the cis- and trans-acting requirements for BMV RNA replication in plants and that significant differences can exist for BMV RNA replication in different hosts.

Base Sequence↗
PubMed2003

Novel functional role of CA repeats and hnRNP L in RNA stability.

CA dinucleotide repeat sequences are very common in the human genome. We have recently demonstrated that the polymorphic CA repeats in intron 13 of the human endothelial nitric oxide synthase (eNOS) gene function as an unusual, length-dependent splicing enhancer. The CA repeat enhancer requires for its activity specific binding of hnRNP L. Here we show that in the absence of bound hnRNP L, the pre-mRNA is cleaved directly upstream of the CA repeats. The addition of recombinant hnRNP L restores RNA stability. CA repeats are both necessary and sufficient for this specific cleavage in the 5' adjacent RNA sequence. We conclude that-in addition to its role as a splicing activator-hnRNP L can act in vitro as a sequence-specific RNA protection factor. Based on the wide abundance of CA repetitive sequences in the human genome, this may represent a novel, generally important role of this abundant hnRNP protein.

Base Sequence↗
PubMed2003

RNA stability in terminally differentiating fibre cells of the ocular lens.

During terminal differentiation of lens fibre cells all cytoplasmic organelles are degraded abruptly. This process eliminates light-scattering elements from the optical axis of the lens and thereby ensures the transparency of the tissue. With the breakdown of the nucleus, transcription ceases, but the degree to which extant RNA is translated in the anucleated cells is uncertain. Previous studies indicated that fibre cell mRNA is unusually stable. For example, full-length delta-crystallin transcripts have been detected in core fibres months after transcription in these cells ceased. In the present study, we used the embryonic chicken lens as a model to examine the fate of RNA in the period immediately before and after organelle degradation. We mapped the tissue distribution of ribosomal RNA (rRNA) using acridine orange staining, in situ hybridization, and direct visualization of ribosomes by electron microscopy. These experiments suggested that rRNA decayed in the anucleated core fibre cells with a half-life of approximately 2.5 days. Similarly, in situ hybridization analysis of polyadenylated transcripts, beta-actin, or GAPDH mRNA indicated that these sequences were not stable in the core fibre cells. However, in agreement with earlier findings, we detected a strong in situ hybridization signal for delta-crystallin in the lens core, many days after transcription had ceased. We used quantitative PCR to compare the levels of GAPDH, L14 and delta-crystallin transcripts in the core region during development. Surprisingly, all three mRNAs decayed with indistinguishable kinetics. We conclude that the persistent delta-crystallin hybridization signal was not evidence of an unusually stable mRNA but, rather, reflected the extraordinary initial abundance of this transcript. Taken together, our data indicate that the half-life of both mRNA and the protein synthetic machinery in the lens core is only a few days. Given that, in vertebrate lenses, nuclei in this region of the lens are degraded during embryonic development, protein synthesis in central lens fibre cells is probably completed well before birth.

Acridine Orange↗
PubMedDate not supplied

Trypanosome alternative oxidase is regulated post-transcriptionally at the level of RNA stability.

In the bloodstream form of African trypanosomes, trypanosome alternative oxidase (TAO), the non-cytochrome ubiquinol:oxidoreductase, is the only terminal oxidase of the mitochondrial electron transport system. TAO is developmentally regulated during mitochondrial biogenesis in this parasite. During in vitro differentiation of Trypanosoma brucei from the bloodstream to the procyclic form, the overall rate of oxygen consumption decreased about 80%. The mode of respiration changed over a 2- to 3-wk period from a cyanide-insensitive, SHAM-sensitive pathway to a predominantly cyanide-sensitive pathway. The TAO protein level gradually decreased to the level present in the procyclic forms during this 3-wk period. However, within the first week of differentiation, the TAO transcript level decreased about 90% and then in the following weeks it reached the level present in the established procyclic form, that is about 20% of that in bloodstream forms. Like other trypanosomatid genes TAO transcript synthesis remains unaltered in fully differentiated bloodstream and procyclic trypanosomes. The half-life of the TAO mRNA was about 3.2 h in the procyclic trypanosomes, whereas the TAO transcript level remained unaltered even after 4 h of incubation with actinomycin D in bloodstream forms. Inhibition of protein synthesis resulted in about a four-fold accumulation of the TAO transcript in the procyclic trypanosomes, comparable to the level present in the bloodstream forms. Thus, TAO is regulated at the level of mRNA stability and de novo protein synthesis is required for the reduction of the TAO mRNA pool in the procyclic form.

Animals↗
PubMed2000

Base opening in RNA and DNA duplexes: implication for RNA stability.

The energetics of a low-energy single base opening in several RNA duplex crystal structures has been calculated and compared to DNA duplexes. Base opening in RNA appears to have an overall preference towards the major groove, similar to results previously reported for B-DNA. Movement of each of the adenine, uracil, and cytosine bases into the minor groove is blocked by a high-energy barrier due to severe close contact with neighboring bases. Guanine bases are able to open towards both grooves because of the unique orientation of the base that avoids steric clash along the opening pathway. RNA bases are found to have a substantially smaller major groove opening extent than that of their B-DNA counterparts. A comparison with base opening behavior of A-DNA duplexes suggests that this difference results from helix constraint associated with A-form backbone conformation. The reduced opening extent correlates with the RNA duplex stability and is consistent with observed slower imino proton exchange rates in RNA duplexes.

Base Sequence↗
PubMed2002

Evidence for in vivo modulation of chloroplast RNA stability by 3'-UTR homopolymeric tails in Chlamydomonas reinhardtii.

Polyadenylation of synthetic RNAs stimulates rapid degradation in vitro by using either Chlamydomonas or spinach chloroplast extracts. Here, we used Chlamydomonas chloroplast transformation to test the effects of mRNA homopolymer tails in vivo, with either the endogenous atpB gene or a version of green fluorescent protein developed for chloroplast expression as reporters. Strains were created in which, after transcription of atpB or gfp, RNase P cleavage occurred upstream of an ectopic tRNA(Glu) moiety, thereby exposing A(28), U(25)A(3), [A+U](26), or A(3) tails. Analysis of these strains showed that, as expected, polyadenylated transcripts failed to accumulate, with RNA being undetectable either by filter hybridization or reverse transcriptase-PCR. In accordance, neither the ATPase beta-subunit nor green fluorescent protein could be detected. However, a U(25)A(3) tail also strongly reduced RNA accumulation relative to a control, whereas the [A+U] tail did not, which is suggestive of a degradation mechanism that does not specifically recognize poly(A), or that multiple mechanisms exist. With an A(3) tail, RNA levels decreased relative to a control with no added tail, but some RNA and protein accumulation was observed. We took advantage of the fact that the strain carrying a modified atpB gene producing an A(28) tail is an obligate heterotroph to obtain photoautotrophic revertants. Each revertant exhibited restored atpB mRNA accumulation and translation, and seemed to act by preventing poly(A) tail exposure. This suggests that the poly(A) tail is only recognized as an instability determinant when exposed at the 3' end of a message.

3' Untranslated Regions↗
PubMed2003

An expressed pseudogene regulates the messenger-RNA stability of its homologous coding gene.

A pseudogene is a gene copy that does not produce a functional, full-length protein. The human genome is estimated to contain up to 20,000 pseudogenes. Although much effort has been devoted to understanding the function of pseudogenes, their biological roles remain largely unknown. Here we report the role of an expressed pseudogene-regulation of messenger-RNA stability-in a transgene-insertion mouse mutant exhibiting polycystic kidneys and bone deformity. The transgene was integrated into the vicinity of the expressing pseudogene of Makorin1, called Makorin1-p1. This insertion reduced transcription of Makorin1-p1, resulting in destabilization of Makorin1 mRNA in trans by way of a cis-acting RNA decay element within the 5' region of Makorin1 that is homologous between Makorin1 and Makorin1-p1. Either Makorin1 or Makorin1-p1 transgenes could rescue these phenotypes. Our findings demonstrate a specific regulatory role of an expressed pseudogene, and point to the functional significance of non-coding RNAs.

Alleles↗
PubMed2001

Constitutive overexpression of cytosolic glutamine synthetase (GS1) gene in transgenic alfalfa demonstrates that GS1 may be regulated at the level of RNA stability and protein turnover.

Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of NH4+ with glutanate to yield glutamine. Gene constructs consisting of the cauliflower mosaic virus (CaMV) 35S promoter driving a cytosolic isoform of GS (GS1) gene have been introduced into alfalfa (Medicago sativa). Although transcripts for the transgene were shown to accumulate to high levels in the leaves, they were undetectable in the nodules. However, significant amounts of beta-glucuronidase activity could be detected in nodules of plants containing the CaMV 35S promoter-beta-glucuronidase gene construct, suggesting that the transcript for the GS1 transgene is not stable in the root nodules. Leaves of alfalfa plants with the CaMV 35S promoter-GS1 gene showed high levels of accumulation of the transcript for the transgene when grown under low-nitrogen conditions and showed a significant drop in the level of GS1 transcripts when fed with high levels of NO3-. However, no increase in GS activity or polypeptide level was detected in the leaves of transgenic plants. The results suggest that GS1 is regulated at the level of RNA stability and protein turnover.

Cytosol↗