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Results for “RNA Stability”

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PubMed1995

Post-transcriptional cosuppression of beta-1,3-glucanase genes does not affect accumulation of transgene nuclear mRNA.

Silencing of a Nicotiana plumbaginifolia beta-1,3-glucanase (gn1) transgene in tobacco line T17 occurs in homozygous and in haploid plants with one transgene locus dosage per chromosome set. We have previously shown that the silent state is manifested by a reduced gn1 steady state mRNA level and results from a post-transcriptional process that is under developmental control in homozygous T17 plants. In this study, we show that specific endogenous beta-1,3-glucanase genes are cosuppressed with gn1 in homozygous T17 plants. We also demonstrate that the developmental timing of cosuppression depends on environmental conditions and that once silencing is established it is stably maintained during plant development. Analyses of additional transgenic tobacco lines revealed that gn1 silencing is not restricted to the T17 line and showed that silencing can also take place in R0 plants containing independent loci in hemizygous states. Furthermore, silencing can also be obtained in progeny plants in which expressing loci have been combined. Importantly, cosuppression occurs via a post-transcriptional mechanism that does not interfere with the accumulation of transgene nuclear mRNA. These results strongly suggest that the silencing mechanism operates at RNA transport and/or RNA stability levels.

Base Sequence↗

PubMed2005

Activity of stabilized short interfering RNA in a mouse model of hepatitis B virus replication.

To develop synthetic short interfering RNA (siRNA) molecules as therapeutic agents for systemic administration in vivo, chemical modifications were introduced into siRNAs targeted to conserved sites in hepatitis B virus (HBV) RNA. These modifications conferred significantly prolonged stability in human serum compared with unmodified siRNAs. Cell culture studies revealed a high degree of gene silencing after treatment with the chemically modified siRNAs. To assess activity of the stabilized siRNAs in vivo initially, an HBV vector-based model was used in which the siRNA and the HBV vector were codelivered via high-volume tail vein injection. More than a 3 log10 decrease in levels of serum HBV DNA and hepatitis B surface antigen, as well as liver HBV RNA, were observed in the siRNA-treated groups compared with the control siRNA-treated and saline groups. Furthermore, the observed decrease in serum HBV DNA was 1.5 log10 more with stabilized siRNA compared with unmodified siRNA, indicating the value of chemical modification in therapeutic applications of siRNA. In subsequent experiments, standard systemic intravenous dosing of stabilized siRNA 72 hours after injection of the HBV vector resulted a 0.9 log10 reduction of serum HBV DNA levels after 2 days of dosing. In conclusion, these experiments establish the strong impact that siRNAs can have on the extent of HBV infection and underscore the importance of stabilization of siRNA against nuclease degradation.

Animals↗

PubMed2001

Stem-loop SL4 of the HIV-1 psi RNA packaging signal exhibits weak affinity for the nucleocapsid protein. structural studies and implications for genome recognition.

Encapsidation of the genome of the human immunodeficiency virus type-1 (HIV-1) during retrovirus assembly is mediated by interactions between the nucleocapsid (NC) domains of assembling Gag polyproteins and a approximately 110 nucleotide segment of the genome known as the Psi-site. The HIV-1 Psi-site contains four stem-loops (SL1 through SL4), all of which are important for genome packaging. Recent isothermal titration calorimetry (ITC) studies have demonstrated that SL2 and SL3 are capable of binding NC with high affinity (K(d) approximately 140 nM), consistent with proposals for protein-interactive functions during packaging. To determine if SL4 may have a similar function, NC-interactive studies were conducted by NMR and gel-shift methods. In contrast to previous reports, we find that SL4 binds weakly to NC (K(d)=(+/-14 microM), suggesting an alternative function. NMR studies indicate that the GAGA tetraloop of SL4 adopts a classical GNRA-type fold (R=purine, N=G, C, A or U), a motif that stabilizes RNA tertiary structures in other systems. In combination with previously reported gel mobility studies of Psi-site deletion mutants, these findings suggest that SL4 functions in genome recognition not by binding to Gag, but by stabilizing the structure of the Psi-site. Differences in the affinities of NC for SL2, SL3 and SL4 stem-loops can now be rationalized in terms of the different structural properties of stem loops that contain GGNG (SL2 and SL3) and GNRA (SL4) sequences.

Amino Acid Sequence↗

PubMed1997

Developmental expression of the murine spliceosome-associated protein mSAP49.

We have isolated the mouse homologue of human spliceosome-associated protein SAP49, mSAP49. mSAP49 contains two RNA recognition motifs (RRM) in the N terminus of the predicted amino acid sequence, and a highly basic C terminus rich in glycine/proline. mSAP49 displayed a plastic of expression in cardiac development. In the adult mouse, mSAP49 is widely distributed, although it was found at relatively lower levels in the heart. In situ hybridization analysis of mSAP49 mRNA distribution in staged mouse embryos showed that mSAP49 onset occurs later in the heart than in other embryonic tissues. While mSAP49 expression was found at day 10.0 postconception (pc) in the optic eminence, optic vesicle, hindbrain, and somites, it was not in cardiac structures. mSAP49 was detected in the ventricles at day 11.5, and at day 13.5 it was also detected in the atria. Northern analysis showed that mSAP49 mRNA displayed a peak of expression in the heart at days 14.0-15.0 pc, and its abundance decayed in the adult. This dynamic pattern of cardiac expression suggests that mSAP49 may be contributing to a change in the ratio of spliceosome components during cardiac growth and development, which may have consequences for tissue-specific splicing, RNA stabilization, or translation.

Amino Acid Sequence↗

PubMed1997

The steady-state level of mRNA from the Ogura cytoplasmic male sterility locus in Brassica cybrids is determined post-transcriptionally by its 3' region.

We have investigated the control of the expression of three different configurations of the mitochondrial gene orf138, whose expression is correlated with Ogura cytoplasmic male-sterility in rapeseed cybrids. These configurations, termed Nco2.5/13S, Nco2.7/13F and Bam4.8/18S, specific to the 13S (sterile), 13F (fertile) and 18S (sterile) cybrids respectively, have the same 5' regions but different 3' regions. The orf138 transcript from Bam4.8/18S is 10-fold more abundant than the one from Nco2.5/13S, while no orf138 transcript from Nco2.7/13F accumulates. However, transcriptional activity measurements show that the rate of transcription is equivalent for the three configurations. These results strongly suggest that the steady-state level of mRNA from the orf138 locus is determined post-transcriptionally, most likely by its 3' region. To determine the role of these 3' regions, we have established an in vitro decay and processing system. In the presence of rapeseed mitochondrial lysate, synthetic RNAs corresponding to the 3' region of the Nco2.7/13F transcript are, as expected, less stable than RNAs corresponding to the 3' regions of the Nco2.5/13S and Bam4.8/18S transcripts. We have also observed in vitro processing of synthetic RNAs at the sites corresponding to the 3' ends of the natural mRNAs from Nco2.5/13S and Bam4.8/18S. Further analysis of the role of these 3' regions in in vitro RNA stability should help us to better understand post-transcriptional control in plant mitochondria.

Base Sequence↗

PubMed2002

Stabilization of hsp70 mRNA on prolonged cell exposure to hypertonicity.

Prolonged exposure of 3T3 cells to 0.5 osM hypertonic medium induced the accumulation of hsp70 mRNAs. This increase in mRNA levels required active protein synthesis. A weak and transient activation of heat shock factor 1 (HSF1) was noted, but it was temporally uncoupled to the accumulation of the hsp70 mRNAs. Nuclear run-on assay and transfection experiments showed that hsp70 gene transcription was not affected by hypertonicity. ActD chase experiments showed that during hypertonic treatment, degradation of hsp70 mRNAs was markedly reduced. This effect did not appear to be a general phenomenon since the increase in mRNA level of another gene induced by hypertonicity (ATA2 transporter) was scarcely due to RNA stabilization. These findings suggest that hypertonic treatment increases the production of hsp70 protein in 3T3 cells via a stabilization of its corresponding mRNA.

3T3 Cells↗

PubMed2003

The parathyroid hormone mRNA 3'-untranslated region AU-rich element is an unstructured functional element.

Parathyroid hormone (PTH) gene expression is regulated post-transcriptionally by hypocalcemia and hypophosphatemia. This regulation is dependent upon binding of protective trans-acting factors to a specific element in the PTH mRNA 3'-untranslated region (UTR). We have previously demonstrated that a 63-nucleotide (nt) AU-rich PTH mRNA element is sufficient to confer regulation of RNA stability by calcium and phosphate in an in vitro degradation assay (IVDA). The 63-nt element consists of a core 26-nt minimal binding sequence and flanking regions. We have now studied the functionality of this element in HEK293 cells using reporter genes and showed that it destabilizes mRNAs for green fluorescent protein (GFP) and growth hormone, similar to its effect in the IVDA. To understand how the cis-element functions as an instability element, we have analyzed its structure by RNase H, primer extension, and computer modeling. The results indicate that the PTH mRNA 3'-UTR and in particular the region of the cis-element are dominated by significant open regions with little folded base pairing. Mutation analysis of the 26-nt core element demonstrated the importance of defined nucleotides for protein-RNA binding. In the GFP reporter system, the same mutations that prevented binding were also ineffective in destabilizing GFP mRNA in HEK293 cells. This is the first study of an AU-rich element that relates function to structure. The PTH mRNA 3'-UTR cis-acting element is an open region that utilizes the distinct sequence pattern to determine mRNA stability by its interaction with trans-acting factors.

3' Untranslated Regions↗

PubMed1988

Fibroblast growth factor stabilizes ribonucleic acid and regulates differentiated functions in a multipeptide-secreting neuroendocrine cell line.

A clonal cell line (44-2C) which synthesizes and secretes somatostatin, neurotensin, calcitonin (CT), and CT gene-related peptide and transiently expresses c-fos was used to characterize the mechanism of action of basic fibroblast growth factor (bFGF). bFGF had two modes of action: 1) short term incubation of 44-2C cells with bFGF increased the cellular content of neurotensin, somatostatin, and CT; and 2) bFGF enhanced the response of the cells to rat hypothalamic GRF-mediated cAMP efflux. The long term action of bFGF was manifested by the permissive effect of the molecule. bFGF had a sustained effect on RNA synthesis, and pretreatment with bFGF for 24 h altered the time course of response of the cells to rat GRF. In this cell line the cellular action of bFGF was not mediated via protein kinase-C action. bFGF was not mitogenic in 44-2C cells. bFGF stimulated uridine incorporation without affecting thymidine incorporation. Results obtained with actinomycin-D and alpha-amanitin suggest that the above effects of bFGF can be correlated with increased RNA stability produced by bFGF.

Amanitins↗

PubMed2000

DNA-damaging aryl hydrocarbons induce Mdm2 expression via p53-independent post-transcriptional mechanisms.

During previous studies, we found that mdm2 mRNA levels were elevated in benzo[a]pyrene (BaP, a polycyclic aryl hydrocarbon)-treated cells under conditions of DNA damage-induced cell cycle arrest (Vaziri, C., and Faller, D. V. (1997) J. Biol. Chem. 272, 2762-2769). We have identified potential aryl-hydrocarbon receptor-binding sites in the mdm2 promoter. However, we show that induction of mdm2 mRNA by BaP is entirely dependent upon aryl-hydrocarbon-induced genotoxicity and does not involve direct aryl-hydrocarbon receptor-mediated transcriptional activation of the mdm2 gene. Heterologous mdm2 promoter-reporter constructs containing p53-response elements were not responsive to BaP treatment. Therefore the p53-response elements in the mdm2 promoter are insufficient to confer DNA damage-dependent expression of mdm2. Furthermore, mdm2 transcripts were induced by BaP in p53 null cells from transgenic mice (although both basal and BaP-induced mdm2 expression levels were reduced in these cells relative to p53(+/+) cultures). These data show that p53-mediated mechanisms cannot account for BaP/DNA damage-induced mdm2 expression. Mdm2 promoter-reporter gene assays and nuclear run-off analyses of nascent mdm2 transcripts showed that transcriptional induction was unable to account for the large changes in mdm2 transcript levels following BaP treatment. However, mdm2 mRNA half-life measurements showed stabilization of the mdm2 transcript (from approximately 1 h to >4 h) in response to BaP. To our knowledge, this is the first report of control of mdm2 at the post-transcriptional level and in a p53-independent manner. Transient ectopic expression of mdm2 strongly augmented aryl-hydrocarbon-induced apoptosis, demonstrating that mdm2 levels can have a profound effect on the cellular response to DNA damage. Overall, our results suggest a potentially important link between DNA damage signaling and RNA stability that may be relevant to cell cycle regulation, tumor suppression, and environmental carcinogenesis.

3T3 Cells↗

PubMed2005

Treatment of advanced hepatitis C with a low accelerating dosage regimen of antiviral therapy.

Patients with advanced hepatitis C virus (HCV) are at risk of death and are candidates for liver transplantation. After transplantation, HCV recurs and may rapidly progress to cirrhosis and graft loss. Treatment is needed to prevent progression of disease and minimize recurrence after liver transplantation. We evaluated the effectiveness, tolerability, and outcome of a low accelerating dose regimen (LADR) of antiviral therapy in the treatment of patients with advanced HCV. One hundred twenty-four patients (male/female ratio 81:43; age range 37-71 years; 70% genotype 1) were treated with LADR. Sixty-three percent had clinical complications of cirrhosis (ascites, spontaneous bacterial peritonitis, varices, variceal hemorrhage, encephalopathy). The mean Child-Turcotte-Pugh (CTP) score was 7.4 +/- 2.3, and the mean MELD score was 11.0 +/- 3.7. Fifty-six patients were CTP class A, 45 were class B, and 23 were class C. Forty-six percent were HCV RNA-negative at end of treatment, and 24% were HCV RNA-negative at last follow-up. Sustained virological response (SVR) was 13% in patients infected with genotype 1 HCV and 50% in patients infected with non-1 genotypes (P < .0001). Non-1 genotype, CTP class A (genotype 1 only), and ability to tolerate full dose and duration of treatment (P < .0001) were predictors of SVR. Twelve of 15 patients who were HCV RNA-negative before transplantation remained HCV RNA-negative 6 months or more after transplantation. In conclusion, in a sizeable proportion of patients with advanced HCV, LADR may render blood free of HCV RNA, stabilize clinical course, and prevent posttransplantation recurrence.

Adult↗

PubMed1988

Role of the intercistronic region in post-transcriptional control of gene expression in the histidine transport operon of Salmonella typhimurium: involvement of REP sequences.

The high-affinity histidine permease of Salmonella typhimurium is encoded by a four-gene operon containing a large intercistronic region located between the first gene (hisJ) and the three distal genes (hisQ, hisM, hisP). The level of expression of hisJ is 30-fold greater than that of hisP. In order to investigate the role of the intercistronic region in intra-operonic control of gene expression, we have isolated MudII-mediated lacZ gene fusions to hisQ, hisM and hisP. We have used these fusions to isolate and analyse mutants that have altered levels of expression of the hisQ gene, the first gene downstream from the intercistronic region. The results indicate that intra-operonic regulation is due to a combination of factors including efficiency of translational initiation, mRNA degradation, and retroregulation of hisJ expression. They also suggest that the REP (Repetitive Extragenic Palindromic) sequences, which are located in the hisJ-hisQ intercistronic region, may interfere with translation of the hisQ gene and affect upstream messenger RNA stability by protecting it from 3' to 5' nuclease degradation (in agreement with data presented by Newbury et al., 1987).

Base Sequence↗

PubMed1995

A nuclear post-transcriptional event responsible for overproduction of argininosuccinate synthetase in a canavanine-resistant variant of a human epithelial cell line.

The Canr1 cell line, a canavanine-resistant variant of the cultured human epithelial cell line, RPMI 2650, overproduces argininosuccinate synthetase more than 200-fold. Run-on transcription assays showed no significant difference in transcription initiation of the argininosuccinate synthetase gene between Canr1 and RPMI 2650 cells. Furthermore, no difference in the relative transcription rate was seen along this 63-kb gene, suggesting that neither transcription initiation nor elongation is responsible for differential expression of argininosuccinate synthetase in these two cell lines. However, when isolated nuclei were labeled for a longer period of time in the transcription assay, precursor RNA of argininosuccinate synthetase in RPMI 2650 cells was found to be very labile. Apparently, a nuclear event affecting precursor RNA stability is responsible for the dramatic difference in argininosuccinate synthetase levels in these two cell lines. Using a microsatellite polymorphic marker, it was demonstrated that argininosuccinate synthetase from both alleles of the gene in Canr1 cells was overexpressed. This suggests that a trans-acting mechanism may be responsible for regulation of overproduction in this cell line. Furthermore, when protein synthesis was blocked by cycloheximide, less precursor RNA was observed in Canr1 cells. These data suggest that a labile protein factor(s) participates in the regulation.

Argininosuccinate Synthase↗

PubMed1991

RNase E cleavage in the atpE leader region of atpE/interferon-beta hybrid transcripts in Escherichia coli causes enhanced rates of mRNA decay.

Chimeric transcripts containing the ribosome binding site of the Escherichia coli atpE gene and variants of the human structural interferon-beta gene are subject to RNase E processing in the 5'-untranslated atpE part of the transcripts. The absence of processing at two sites in the atpE leader-sequence caused by the RNase E deficiency in E. coli host N3431 leads to a considerable stabilization of the mRNA moiety. RNase E has originally been described as a processing enzyme for non-mRNAs such as precursor 5 S rRNA and RNA1, but cleavage mRNA substrates have also been reported. RNase E processing of the atpE gene leader sequence-containing transcripts leads to an increased rate of mRNA breakdown. The two RNase E-dependent processing sites in the atpE part of the mRNA transcripts exhibit some similarity to the other known RNase E processing sites. The influence of RNase E cleavage upon post-transcriptional regulation such as RNA stability and the efficiency of translational initiation is discussed.

Bacteriophage lambda↗

PubMed1981

Stability of poliovirus RNA in cell-free translation systems utilizing two initiation sites.

The stability of purified poliovirus RNA in cell-free translation systems prepared from HeLa cells or rabbit reticulocytes has been examined. Degradation of the RNA occurs with a t1/2 of approximately 35 min at 30 degrees C under conditions used for in vitro translation. Degradation is due in part to activity in the cell lysate, and in part to contaminants in the commercial preparations of creatine phosphokinase used in the energy-regenerating system. Addition of crude preparations of initiation factors significantly slows degradation, presumably as a result of protein-RNA interactions which confer resistance to nuclease action. Prior treatment of RNA with methylmercury hydroxide has no effect on degradation rates. On the other hand, endogenous mRNA, present as a messenger ribonucleoprotein particle in extracts from poliovirus-infected HeLa cells, remains completely intact during in vitro translation. These infected cell extracts synthesize the normal complement of viral proteins and utilize two different initiation sites for translation. Treatment of the infected cell extract with micrococcal nuclease destroys the endogenous mRNA. Subsequent addition of exogenous RNA to the same extract results in the formation of a protein-associated RNA particle with sedimentation properties slightly different from the endogenous messenger ribonucleoprotein, and the added RNA is unstable. We conclude that two initiation sites can be utilized on intact poliovirus mRNA, and fragmentation of the RNA is not prerequisite for generation of a second site in this RNA.

Kinetics↗

PubMed1996

Human type-1 angiotensin II (AT1) receptor gene structure and function.

1. The type-1 angiotensin II (AngII) receptors, designated AT1, mediate most of the biological actions of the peptide hormone AngII. They are the most recent drug target for the treatment of hypertension and cardiac failure and basic research is now focusing on the mechanisms that regulate their expression. 2. In humans there is a single AT1 gene. It encodes a 47 kb pre-mRNA containing five exons, with the previously described AT1 open reading frame (ORF) on exon 5. Alternative splicing results in the production of mature mRNA that are translated at different efficiencies and encode two receptor isoforms. The inclusion of exon 2 markedly inhibits translation of the down-stream ORF, both in vitro and in vivo. Nonetheless, this exon is present in up to one-half of AT1 mRNA in all tissues studied. 3. Transcripts containing exon 3 spliced to exon 5 encode a receptor with an amino-terminal extension of 32 amino acids and represent up to one-third of total AT1 mRNA in each tissue examined. In vitro, these latter transcripts are translated to produce a longer receptor and, in transfected cells, they encode a functional AT1 receptor with ligand-binding and signalling properties similar to those of the short isoform. 4. Exon 4 is of minor significance as it is rarely spliced into AT1 mRNA. 5. These data indicate that, in addition to characterizing factors that modulate AT1 promoter activity and RNA stability, it is important to analyse the splicing patterns of this gene when studying the regulation of its expression.

Alternative Splicing↗

PubMed1981

Regulation or procollagen messenger ribonucleic acid levels in Rous sarcoma virus transformed chick embryo fibroblasts.

Using cloned cDNAs for pro-alpha 1 and pro-alpha 2 collagen messenger ribonucleic acid (mRNA), we have investigated the regulation of collagen mRNA levels in Rous sarcoma virus (RSV) transformed chick embryo fibroblasts (CEF). We find that both pro-alpha 1 and pro-alpha 2 mRNA levels are decreased approximately 10-fold in CEF transformed by either the Bryan high-titer strain or the Schmidt-Ruppin strain of RSV. Using temperature-sensitive mutants in the transforming gene src, we also investigated the rate of change in the levels of the two mRNA species. We employed mutants of both the Bryan high-titre strain (BHTa) and the Schmidt-Ruppin strain (ts68). With both mutants the results were similar. Upon shift from the permissive temperature (35 degrees C) to the non-permissive temperature (41 degrees C), collagen mRNA synthesis, did not increase until more than 5 h had passed, suggesting that action of src on collagen gene expression is indirect. Upon shift from 41 to 35 degrees C, collagen mRNA levels fell with a half-life of 10 h. Whether this fall reflects the half-life of procollagen mRNA or an effect of src on procollagen RNA stability is unclear. Both pro-alpha 1 and pro-alpha 2 mRNA levels were coordinately controlled.

Animals↗

PubMed2000

The sequence and secondary structure of the 3'-UTR affect 3'-end maturation, RNA accumulation, and translation in tobacco chloroplasts.

RNA maturation and modulation of RNA stability play important roles in chloroplast gene expression. In vitro and in vivo studies have shown that both the 5'- and 3'-untranslated regions (UTRs) contain sequence and structural elements that guide these processes, and interact with specific proteins. We have previously characterized the spinach chloroplast petD 3'-UTR in detail by in vitro approaches. This stem-loop forming sequence is a weak terminator but is required for RNA maturation and also exhibits sequence-specific protein binding. To test petD 3'-UTR function in vivo, tobacco chloroplast transformants were generated containing uidA reporter genes flanked by variants of the petD 3'-UTR, including one which does not form an RNA-protein complex in vitro, and one which lacks a stem-loop structure. Analysis of uidA mRNA indicated that a stable secondary structure is required to accumulate a discrete mRNA, and that changes in the 3'-UTR sequence which affect protein binding in vitro can also affect RNA metabolism in vivo. The 3'-UTR also influenced beta-glucuronidase protein accumulation, but not in proportion to RNA levels. These results raise the possibility that in tobacco chloroplasts, the 3'-UTR may influence translational yield.

3' Untranslated Regions↗

PubMed2003

CSP41a, a multifunctional RNA-binding protein, initiates mRNA turnover in tobacco chloroplasts.

Expression of chloroplast stem-loop binding protein (CSP)41a, a highly conserved chloroplast endoribonuclease, was reduced >90% by the expression of antisense RNA in Nicotiana tabacum. The most striking effects of this silencing were two- to sevenfold decreases in the degradation rates of rbcL, psbA, and petD transcripts in lysed chloroplast extracts. These results are consistent with the hypothesis that CSP41a participates in initiating mRNA turnover through endonucleolytic cleavages. Surprisingly, rbcL and psbA mRNAs accumulated to similar levels in wild-type and antisense lines. This suggested that decreased degradation was compensated by reduced transcription, which was confirmed using run-on transcription assays. The collective accumulation of petD-containing mRNAs in antisense plants decreased by 25% compared to wild-type controls. However, the relative levels of petD processing intermediates in wild-type and antisense plants did not differ, and there were no changes in petD 3'-end maturation, suggesting that CSP41a is not required for petD RNA processing. CSP41a is a Mg2+-dependent enzyme; therefore, extracts from antisense plants were tested at different Mg2+ concentrations. These experiments showed that the half-life of rbcL decreased as the Mg2+ concentration was reduced, and at <1 mm free Mg2+, conditions where CSP41a is nearly inactive in vitro, the rbcL degradation rate was similar in wild-type and antisense extracts, suggesting that CSP41a is normally bypassed under these conditions. Mg2+ has been shown to mediate RNA stability during chloroplast biogenesis, and our data suggest that regulation of CSP41a activity by Mg2+ is a component of this process.

Base Sequence↗