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Results for “RNA Stability”

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At least 793 records · Page 44Linked to original sources

PubMed2004

Wide-range transcriptional modulating effect of ntrR under microaerobiosis in Sinorhizobium meliloti.

A mutation in the second gene in the ntrPR operon results in increased expression of nodulation (nod) and nitrogen fixation (nif) genes in Sinorhizobium meliloti. Since this pleiotropic effect is particularly pronounced in the presence of external combined nitrogen, a nitrogen regulatory function has been suggested for NtrR. To identify the complete set of protein-coding genes influenced by loss of ntrR function, microarray hybridizations were carried out to compare transcript levels in the wild type and mutant strains grown under aerobic and microaerobic conditions. Of the 6207 genes examined, representing the entire genome of S. meliloti, 7% exhibited altered expression: 4.5% of the genes are affected under oxic, 2.5% under microoxic conditions. 0.4% of all the genes are affected under both oxygen concentrations. A microoxic environment is required for the induction of genes related to symbiotic functions but results in the down-regulation of other (e.g. metabolic) functions. When the alterations in transcription levels at low oxygen concentration in the mutant strain were compared to those of the wild type, a modulating effect of the ntrR mutation was observed. For example, symbiotic nif/fix genes were induced in both strains, but the level of induction was higher in the ntrR mutant. In contrast, genes related to transcription/translation functions were down-regulated in both strains, and the effect was greater in the wild-type strain than in the ntrR mutant. A relatively wide range of functions was affected by this modulating influence, suggesting that ntrR is not a nitrogen regulatory gene. Since genes encoding various unrelated functions were affected, we propose that NtrR may either interfere with general regulatory mechanisms, such as phosphorylation/dephosphorylation, or may influence RNA stability.

Aerobiosis↗

PubMed2003

Translation enhancer in the 3'-untranslated region of rotavirus gene 6 mRNA promotes expression of the major capsid protein VP6.

The eleven rotavirus mRNAs contain 5'-cap structures and most end with the 3'-consensus sequence 5'-UGACC-3'. The UGACC functions as a common translation enhancer (3'-TE-con) that upregulates viral protein expression through a process mediated by the nonstructural protein NSP3. To address the possibility that gene-specific enhancers are also contained in the untranslated regions (UTRs) of the rotavirus mRNAs, we used rabbit reticulocyte lysates to investigate the translation efficiencies of analog RNAs containing viral-specific 5'-and 3'-UTRs and the open reading frame for chloramphenicol acetyltransferase. These experiments combined with the analysis of full-length viral RNAs and RNAs containing 3'-truncations showed that a highly active enhancer was present near the 5'-end of the 139-nucleotide 3'-UTR of the gene 6 mRNA (3'-TEg6). The 3'-TEg6 represents a functionally independent enhancer, as no other portion of the gene 6 mRNA was required for its activity. The 3'-TEg6 differs significantly from the 3'-TE-con in that the gene 6-specific enhancer does not require viral protein for activity and is formed by a sequence unique to only one of the eleven viral mRNAs. Together, our findings suggest that the 3'-UTR of the gene 6 mRNA contains two TEs, one is gene-specific (3'-TEg6) and the other is common to nearly all rotavirus genes (3'-TE-con). The activity of the 3'-TEg6 is likely important for directing the efficient translation of the gene 6 mRNA at levels sufficient to provide the 780 copies of VP6 necessary for the assembly of each progeny virion.

3' Untranslated Regions↗

PubMed2005

Characterization of resistance in transgenic Nicotiana benthamiana encoding N-terminal deletion and assembly mutants of the tobacco etch potyvirus coat protein.

The resistance of transgenic Nicotiana benthamiana plants encoding wild type, truncated and point mutants of the tobacco etch virus (TEV) coat protein (CP) was analyzed. After R1 plants from 45 transgenic lines were challenged with TEV, six percent of the lines exhibited high resistance, 38% exhibited low resistance, and the remainder were susceptible. The phenomenon of recovery and delay in symptom development was observed in 65% and 56% of the resistant and susceptible lines, respectively. Plants containing genes that encode sequences of two assembly-deficient mutants of TEV-CPDelta1-63 exhibited resistance to infection, suggesting that self-assembly of the CP is not responsible for resistance. Highly resistant lines accumulated low levels of transgene mRNA and non-detectable amounts of protein, and tissues accumulated lower amounts of transgene mRNA following recovery than before infection. In addition, co-suppression of replication of a recombinant tobamovirus containing the TEV-CPDelta1-63 sequence was observed in several lines, suggesting homology-dependent degradation of RNA, most likely through induction of post-transcriptional gene silencing. Plants not exhibiting high resistance via gene silencing exhibited moderate levels of resistance that is attributed to and/or affected by the CP molecule.

Capsid Proteins↗

PubMed2006

Armored RNA as positive control and standard for quantitative reverse transcription-polymerase chain reaction assay for rubella virus.

RV Armored RNA constructed in the laboratory was shown to be resistant to RNase and DNase digestion. It could remain stable at 4 degrees C for at least 60 days, and was much more stable than RV strain TR23 [corrected] in normal human plasma. Therefore it could be used as stable, well-characterized positive control and standard for quantitative RT-PCR assay.

Base Sequence↗

PubMed2001

Down-regulation of translation driven by hepatitis C virus internal ribosomal entry site by the 3' untranslated region of RNA.

The genome of hepatitis C virus (HCV) is a single-stranded RNA of positive polarity that has a poly(U/C) tract followed by a highly conserved 98-nt sequence, termed the X region, in the 3' untranslated region (UTR). To investigate the effect of the 3'UTR on the HCV translation that depends on the internal ribosomal entry site (IRES), we prepared a deletion HCV RNA, MA delta, that lacked the RNA region from nt 1286 to 8785. A series of MA delta RNAs that differ in the primary structure of their 3'UTR, were generated and examined for their translation efficiencies in reticulocyte lysates. Deletion of the poly(U/C) tract and/or stem-loop structure (SL) 3 region of 3'X resulted in enhancement of the translation efficiency. Translation of MA delta RNA was inhibited by the addition of recombinant polypyrimidine tract-binding protein (PTB). A similar inhibition by PTB, however, was observed when an RNA lacking the poly(U/C) tract or SL3 region was used. The inhibitory effect by PTB was not obvious for MA delta (1041) RNA composed of nt 1 to 1041 but MA delta (8928) RNA composed of nt 1 to 1285 and nt 8786 to 8928. These results suggest that the observed down-regulation of HCV translation by the 3'UTR is mediated by some host factor(s) other than PTB, and that a PTB site for inhibition resides in the coding sequence of nt 1042 to 8928 of MA delta RNA.

3' Untranslated Regions↗

PubMed2006

Identification of a novel non-coding RNA, MIAT, that confers risk of myocardial infarction.

Through a large-scale case-control association study using 52,608 haplotype-based single nucleotide polymorphism (SNP) markers, we identified a susceptible locus for myocardial infarction (MI) on chromosome 22q12.1. Following linkage disequilibrium (LD) mapping, haplotype analyses revealed that six SNPs in this locus, all of which were in complete LD, showed markedly significant association with MI (chi2=25.27, P=0.0000005; comparison of allele frequency, 3,435 affected individuals versus 3,774 controls, in the case of intron 1 5,338 C>T; rs2331291). Within this locus, we isolated a complete cDNA of a novel gene, designated myocardial infarction associated transcript (MIAT). MIAT has five exons, and in vitro translation assay showed that MIAT did not encode any translational product, indicating that this is likely to be a functional RNA. In vitro functional analyses revealed that the minor variant of one SNP in exon 5 increased transcriptional level of the novel gene. Moreover, unidentified nuclear protein(s) bound more intensely to risk allele than non-risk allele. These results indicate that the altered expression of MIAT by the SNP may play some role in the pathogenesis of MI.

Alleles↗

PubMed2005

Degradation of RNA during the autolysis of Saccharomyces cerevisiae produces predominantly ribonucleotides.

Autolytic degradation of yeast RNA occurs in many foods and beverages and can impact on the sensory quality of the product, but the resulting complex mixture of nucleotides, nucleosides and nucleobases has not been properly characterised. In this study, yeast autolysis was induced by incubating cell suspensions of Saccharomyces cerevisiae at 30-60 degrees C (pH 7.0), and at pH 4.0-7.0 (40 degrees C) for 10-14 days, and the RNA degradation products formed during the process were determined by reversed-phase HPLC. Up to 95% of cell RNA was degraded, with consequent leakage into the extracellular environment of mainly 3'-, 5'- and 2'-ribonucleotides, and lesser amounts of polynucleotides, ribonucleosides and nucleobases. The rate of RNA degradation and the composition of the breakdown products varied with temperature and pH. RNA degradation was fastest at 50 degrees C (pH 7.0). Autolysis at lower temperatures (30 degrees C and 40 degrees C) and at pH 5.0 and 6.0 favoured the formation of 3'-nucleotides, whereas autolysis at 40 degrees C and 50 degrees C (pH 7.0) favoured 5'- and 2'-nucleotides. The best conditions for the formation of the two flavour-enhancing nucleotides, 5'-AMP and 5'-GMP, were 50 degrees C (pH 7.0) and pH 4.0 (40 degrees C), respectively.

Adenosine Monophosphate↗

PubMed2006

Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR.

Relative quantification in quantitative real-time RT-PCR is increasingly used to quantify gene expression changes. In general, two different relative mRNA quantification models exist: the delta-delta Ct and the efficiency-corrected Ct model. Both models have their advantages and disadvantages in terms of simplification on the one hand and efficiency correction on the other. The particular problem of RNA integrity and its effect on relative quantification in qRT-PCR performance was tested in different bovine tissues and cell lines (n = 11). Therefore different artificial and standardized RNA degradation levels were used. Currently fully automated capillary electrophoresis systems have become the new standard in RNA quality assessment. RNA quality was rated according the RNA integrity number (RIN). Furthermore, the effect of different length of amplified products and RNA integrity on expression analyses was investigated. We found significant impact of RNA integrity on relative expression results, mainly on cycle threshold (Ct) values and a minor effect on PCR efficiency. To minimize the interference of RNA integrity on relative quantification models, we can recommend to normalize gene expression by an internal reference gene and to perform an efficiency correction. Results demonstrate that innovative new quantification methods and normalization models can improve future mRNA quantification.

Algorithms↗

PubMed2006

Inhibitory cis-element-mediated decay of human papillomavirus type 16 L1-transcript in undifferentiated cells.

Production of human papillomavirus type 16 major capsid protein L1 in undifferentiated cells is negatively regulated by several yet unidentified cis-acting inhibitory RNA elements, among which a major element is located within the first 514 nucleotides of the L1-mRNA. By Northern blotting we examined effect of the major element on the steady-state level of mRNA transiently transcribed in 293T cells from the firefly luciferase (Fluc) gene combined with the L1 DNA fragment encoding the major element. As reported previously, the element down-regulated steady-state level of the mRNA. The most efficient down-regulation was achieved by insertion of the element near the 5' end of mRNA, resulting in an undetectable level of the mRNA. The longer the distance from the 5' end of the mRNA to the element, the weaker the down-regulation. The half-life of the mRNA having the element was similar to that of normal Fluc-mRNA. When the element near the 5' end was removed by splicing, the steady-state level of the resultant mRNA was raised to a readily detectable level. The steady-state level of RNA synthesized by RNA polymerase-I was not influenced by the presence of the element. Taken together, it is suggested that DNA region encoding the major inhibitory element does not disturb transcription and that the pre-mRNA is degraded by an RNA element-mediated mechanism after the splicing step in the course of mRNA maturation.

Capsid Proteins↗

PubMed2006

Production of siRNA targeted against TYLCV coat protein transcripts leads to silencing of its expression and resistance to the virus.

The coat protein (CP) of Tomato yellow leaf curl virus (TYLCV), encoded by the v1 gene, is the only known component of the viral capsid. In addition, the CP plays a role in the virus transport into the host cell nucleus where viral genes are replicated and transcribed. In this study, we analyzed the effect of small interfering double-stranded RNAs (siRNAs), derived from an intron-hairpin RNA (ihpRNA) construct and targeting the v1 gene product, on CP accumulation. Transient assays involving agroinfiltration of the CP-silencing construct followed by infiltration of a fused GFP-CP (green fluorescent protein-coat protein) gene showed down-regulation of GFP expression in Nicotiana benthamiana. Some of the transgenic tomato plants (cv. Micro-Tom), expressing the siRNA targeted against the TYLCV CP gene, did not show disease symptoms 7 weeks post-inoculation with the virus, while non-transgenic control plants were infected within 2 weeks post inoculation. The present study demonstrates, for the first time, that siRNA targeted against the CP of TYLCV can confer resistance to the virus in transgenic tomato plants, thereby enabling flowering and fruit production.

Agrobacterium tumefaciens↗

PubMed2005

The conserved, 5' termini of RNAs 1 and 2 of Tomato aspermy virus are dispensable for infection but affect virulence.

The 5' terminus of each of the three genomic RNAs (RNAs 1, 2 and 3) of Tomato aspermy virus (TAV) begins with the sequence 5'-GUUU, which is also shared by a number of other viruses. Mutagenic analyses showed that the 5'-GUUU sequence of RNAs 1 and 2 of TAV was dispensable for viral infection and did not prevent symptom induction. On the other hand, substitution of U at position 5 for G in RNA 1, but not RNA 2, induced veinal necrosis symptoms in Nicotiana glutinosa. The mutants constructed included insertion of UUU into the 5'-GUUU sequence of TAV RNAs 1 and/or 2. All RNA 2 mutants induced more severe symptoms than viral RNAs containing either mutated RNA 1 or most combinations of mutated RNAs 1 and 2. Some combinations of mutated RNAs 1 and 2 also induced veinal necrosis in N. glutinosa. Virulence was unrelated to the levels of viral RNA accumulation. Sequence analysis of progeny viral RNAs showed that only the mutant viral RNAs with a G to U substitution in RNA 1 and the deletion of the 5'-GUUU in both RNAs 1 and 2 were able to maintain the same sequence as the inoculum. The other mutants either reverted to the wildtype sequence or underwent further deletion or insertion. None of the constructed mutants were able to compete for accumulation with the wildtype virus after co-inoculation to the plant species tested.

Base Sequence↗

PubMed1996

Induction of murine erythroleukemia cell differentiation is associated with methylation and differential stability of poly(A)+ RNA transcripts.

Murine erythroleukemia (MEL) cells exposed to DMSO were assessed for their ability to methylate poly(A)+ RNA and accumulate RNA transcripts of globin and nonglobin genes (c-myc, beta-actin and MER5). Cells were pulse-labeled with L-[methyl-3H]methionine, cytoplasmic RNA was isolated, selected for poly(A)+ RNA and analyzed by HPLC chromatography for methylated nucleosides. When MEL cells were exposed to inhibitors of RNA methylation (neplanocin A, 3-deazaneplanocin A and cycloleucine) and assessed for their ability to differentiate by DMSO, accumulate RNA transcripts, produce hemoglobin, methylate poly(A)+ and poly(A)- RNA and synthesize S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), we observed the following: (a) MEL cells treated with DMSO underwent hypermethylation in poly(A)+ RNA that preferentially occurred at the 5'-cap structures (7-methylguanosine and 2'-O-methylcytidine and 2'-O-methyluridine); (b) inducer-treated MEL cells exhibited a decrease in the intracellular level of SAH that led to a lower ratio of SAH/SAM, an event that favors methylation; and (c) treatment of MEL cells with inhibitors of RNA methylation suppressed methylation of poly(A)- and poly(A)+ RNA, reversed the ratio SAH/SAM seen in differentiated MEL cells and prevented differentiation to occur. Moreover, we observed that treatment of MEL cells with selective inhibitors of RNA methylation caused fragmentation of beta major globin and c-myc mRNAs, two RNA transcripts coded by developmentally regulated genes, while had no detectable effect on the structural integrity of poly(A)+ RNA transcripts transcribed by two housekeeping genes (beta-actin and MER5). These data indicate that induction of erythroid cell differentiation of MEL cells is associated with changes in methylation of poly(A)+ RNA and selective differential stability of RNA transcripts, two events that might be related to each other.

Actins↗

PubMed2005

RNA constitution and estrogen-responsive gene expression in the ovariectomized rat uterus.

Because reproductive tracts in ovariectomized rodents, which are commonly used in endocrinological studies, exhibit drastic changes in response to exogenous estrogens, quantitative evaluation of gene expression requires extra caution. We found that the whole mRNA content of total RNA in the uterus of the ovariectomized rat was dose-dependently reduced by treatment with 17alpha-ethynylestradiol (EE) for 3 consecutive days. Because of the decline in the ratio of mRNA/total RNA, real-time reverse transcriptase-polymerase chain reaction analysis seemingly showed that the relative ratios of the levels of stable RNAs, rRNA18S, rRNA5S, and tRNA-Gly, to whole mRNA were increased by EE. These results indicate that applying a fixed concentration of total RNA and stable RNA as an internal control to uterine mRNA quantification should be reconsidered. On the other hand, the beta-actin gene showed the most stable expression among the housekeeping genes examined. Using beta-actin mRNA as an internal control, we observed that tissue inhibitor of metalloproteinase-3 (TIMP3) mRNA was dose-dependently reduced 24 h after treatment with EE or bisphenol A for 3 days. Continued investigation of TIMP3 with an appropriate internal control may be helpful in elucidating the mechanisms involved in uterine activation.

Animals↗

PubMed2006

The identification of newborns using messenger RNA profiling analysis.

The ability to determine the physical characteristics of an individual depositing a bloodstain at a crime scene would be an invaluable tool to investigators, akin to eyewitness information. One useful biometric that may be amenable to molecular genetic analysis is the biological age of an individual. In theory, it may be possible to determine patterns of gene expression that are age specific, thereby permitting the distinction among tissue samples originating from individuals of different ages (e.g., newborn, adolescent, middle-age, elderly). We have discovered two novel isoforms of gamma hemoglobin messenger RNA, designated HBG1n and HBG2n, which exhibit an extremely restricted pattern of gene expression, being confined to newborn individuals. Multiplex quantitative reverse transcription PCR (qRT-PCR) assays incorporating these novel mRNAs have been designed, tested, and evaluated for their potential forensic use. The results indicate that the assays provide the ability to determine whether a bloodstain originated from a newborn.

Aging↗

PubMed2006

Quantification of RNA damage by reverse transcription polymerase chain reactions.

RNA damages, such as those generated by nucleic acid-modifying agents, occur randomly in RNA and present challenging problems to organisms. It has been unclear how RNA function would be affected by many forms of RNA damage and how cells are protected against the damage. Elucidation of these mechanisms has been hampered by the lack of sensitive and efficient methodologies detecting damages randomly occurring in RNA, especially for the damage of a specific RNA. In this work, we have developed a method using reverse transcription polymerase chain reactions (RT-PCRs) to determine the level of damage of a specific RNA. The level of damage of the Escherichia coli 16S rRNA caused by oxidative stress was examined. When RNA is treated by H(2)O(2) in vitro, the normalized level of long cDNA is inversely dependent on the dosage of H(2)O(2) as determined by gel-based assay or real-time PCR. Long cDNA was also produced at reduced levels using RNA prepared from H(2)O(2)-treated E. coli cultures compared with RNA from control cultures. Remarkably, the level of cDNA reduction caused by H(2)O(2) treatment depends on the length of cDNA examined, suggesting random occurrences of damage in RNA templates. Approximately 40% of the reduction in cDNA can be detected in each kilobase of RNA from E. coli cultures treated with 0.5 mM H(2)O(2). This method is able to detect any type of damage in RNA-causing termination of reverse transcription and works on specific RNA of interest with high sensitivity.

DNA, Complementary↗

PubMed2005

Plant molecular biology in the space station era: utilization of KSC fixation tubes with RNAlater.

Spaceflight experiments involving biological specimens face unique challenges with regard to the on orbit harvest and preservation of material for later ground-based analyses. Preserving plant material for gene expression analyses requires that the tissue be prepared and stored in a manner that maintains the integrity of RNA. The liquid preservative RNAlater (Ambion) provides an effective alternative to conventional freezing strategies, which are limited or unavailable in current spaceflight experiment scenarios. The spaceflight use of RNAlater is enabled by the Kennedy space center fixation tube (KFT), hardware designed to provide the necessary containment of fixatives during the harvest and stowage of biological samples in space. Pairing RNAlater with the KFT system provides a safe and effective strategy for preserving plant material for subsequent molecular analyses, a strategy that has proven effective in several spaceflight experiments. Possible spaceflight scenarios for the use of RNAlater and KFTs are explored and discussed.

Arabidopsis↗

PubMed2026

CNOT1 is a potential YTHDF2 target that orchestrates maternal mRNA decay and zygotic genome activation during goat embryogenesis.

Timely and efficient degradation of maternal mRNA is essential for early embryonic development, which occurs from fertilization through the initiation of zygotic genome activation (ZGA). Yet, the regulatory mechanisms governing this process remain poorly characterized. In the present study, we investigated the function of CCR4-NOT transcription complex subunit 1 (CNOT1) during goat embryogenesis. We found that CNOT1 was upregulated during mammalian ZGA, and that its knockdown led to developmental arrest and a marked reduction in blastocyst formation. Moreover, CNOT1 knockdown impaired nascent RNA activity, resulting in 814 upregulated and 1014 downregulated genes, which were enriched for RNA splicing, regulation of chromosome organization, and RNA localization. RNA splicing analysis revealed differential splicing events in 2959 genes, of which 259 were downregulated following CNOT1 knockdown. Notably, CNOT1 was predicted to crosstalk with the m6A reader YTHDF2. Knockdown of YTHDF2 resulted in CNOT1 downregulation at the 8-cell stage in goats and increased transcription levels around polyadenylation sites during ZGA in mice. Together, these findings indicate that CNOT1 is a potential YTHDF2 target that orchestrates maternal mRNA decay and ZGA during goat embryogenesis. Our work provides new insight into the complex regulatory landscape underlying ZGA and may inform strategies to improve the efficiency of goat embryogenesis.

Animals↗

PubMed2006

Expression of vector-based small interfering RNA against West Nile virus effectively inhibits virus replication.

RNA interference is one of the effective emerging anti-viral strategies to inhibit virus infection in cells. In this study, a small interfering RNA expressing vector (pSilencer-NS5) targeting the NS5 gene of West Nile virus (WNV) was employed to target and destroy WNV transcripts. Real-time PCR revealed drastic reduction in WNV RNA transcripts in pSilencer-NS5-transfected Vero cells. The virus infectious titre was also significantly reduced by 90% as determined by plaque assays. The resulting decrease in virus replication was shown to be specific since both scrambled and nucleotide(s) mismatch siRNA against WNV NS5 gene did not have any effect on WNV productive yields. Furthermore, Western immunoblot analysis on the expression of viral NS5 and envelope (E) proteins showed significant down-regulation on the expression of viral NS5 and envelope (E) proteins in virus-infected cells that were pre-transfected with pSilencer-NS5. These data clearly supported the notion that the expression of vector-based siRNA against WNV NS5 gene is able to exert its silencing effect on WNV-infected cells without inducing cytotoxicity, hence holding promise in therapeutic treatment of this important emerging infectious disease.

Animals↗