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At least 775 records · Page 43Linked to original sources
PubMed2003

Vector-based in vivo RNA interference: dose- and time-dependent suppression of transgene expression.

RNA interference (RNAi) induced by delivery of a small-interfering RNA (siRNA)-expressing vector was characterized in mice. siRNA-expressing plasmid DNA (pDNA) was injected by a hydrodynamics-based procedure along with pDNA encoding an exogenous target luciferase gene. A comparative study showed that stem-loop-type siRNA-expressing pDNA was superior, in terms of the transgene suppressive efficacy, to the tandem-type in the liver following systemic delivery of these pDNAs. Transgene suppression occurred in the liver, kidney, and lung as well as muscle. The degree of suppression was dependent on the dose of siRNA-expressing pDNA and the time at which transgene expression was determined following simultaneous injection of siRNA-expressing and target pDNAs. A reduction in transgene expression became apparent at 1 day after injection, whereas a lower degree of inhibition was obtained before this, as early as 6 h even in mice treated with an excess of siRNA-expressing pDNA. These results suggest that delivery of siRNA-expressing pDNA requires a period of time for induction of RNAi. A study of sequential injections revealed that prior injection of siRNA-expressing pDNA produced a significant suppression for at least 1 day, which disappeared within 4 days. Confocal microscopic studies indicated that the localization of the cells with successful delivery of transgene was different between primary and secondary hydrodynamics-based injections, accounting for the less effective inhibition following the sequential injections. Taken together, these results demonstrate that vector-based in vivo RNAi is a dose- and time-dependent process and offers the possibility of suppressing endogenous targets in a variety of somatic cells.

Animals↗
PubMed2004

A link between mRNA turnover and RNA interference in Arabidopsis.

In RNA interference (RNAi), double-stranded RNA (dsRNA) triggers degradation of homologous messenger RNA. In many organisms, RNA-dependent RNA polymerase (RdRp) is required to initiate or amplify RNAi, but the substrate for dsRNA synthesis in vivo is not known. Here, we show that RdRp-dependent transgene silencing in Arabidopsis was caused by mutation of XRN4, which is a ribonuclease (RNase) implicated in mRNA turnover by means of decapping and 5'-3' exonucleolysis. When both XRN4 and the RdRp were mutated, the plants accumulated decapped transgene mRNA. We propose that mRNAs lacking a cap structure become exposed to RdRp to initiate or maintain RNAi.

Animals↗
PubMed2006

Decay of endoplasmic reticulum-localized mRNAs during the unfolded protein response.

The unfolded protein response (UPR) allows the endoplasmic reticulum (ER) to recover from the accumulation of misfolded proteins, in part by increasing its folding capacity. Inositol-requiring enzyme-1 (IRE1) promotes this remodeling by detecting misfolded ER proteins and activating a transcription factor, X-box-binding protein 1, through endonucleolytic cleavage of its messenger RNA (mRNA). Here, we report that IRE1 independently mediates the rapid degradation of a specific subset of mRNAs, based both on their localization to the ER membrane and on the amino acid sequence they encode. This response is well suited to complement other UPR mechanisms because it could selectively halt production of proteins that challenge the ER and clear the translocation and folding machinery for the subsequent remodeling process.

Amino Acid Sequence↗
PubMed2006

Untemplated oligoadenylation promotes degradation of RISC-cleaved transcripts.

Double-stranded RNA, processed to small interfering RNAs (siRNAs) by Dicer and incorporated into the RNA-induced silencing complex (RISC), triggers gene silencing by a variety of pathways in eukaryotes. RNA interference involving the degradation of homologous transcripts is the best-characterized mechanism. However, the fate of the RNA fragments resulting from siRNA-directed cleavage is poorly understood. We have identified a gene (MUT68) in the unicellular green alga Chlamydomonas reinhardtii that is required for the efficient decay of siRNA-targeted transcripts. MUT68 encodes a noncanonical polyadenylate polymerase that adds untemplated adenines to the 5' RNA fragments after siRNA-mediated cleavage and appears to stimulate their exosome-dependent degradation.

Adenine Nucleotides↗
PubMed1982

Application of halophilic nuclease H of Micrococcus varians subsp. halophilus to commercial production of flavoring agent 5'-GMP.

RNA was degraded at 60 degrees C for 24 h by halophilic nuclease H in supernatants from broth cultures of Micrococcus varians subsp. halophilus containing 12% NaCl. Since contaminating 5'-nucleotidase exhibited almost no activity under these conditions, the 5'-GMP formed could be recovered from the reaction mixture, and the yield was 805 mg from 5 g of RNA.

Exoribonucleases↗
PubMed2005

Candida albicans biofilm-defective mutants.

Biofilm formation plays a key role in the life cycles and subsistence of many microorganisms. For the human fungal pathogen Candida albicans, biofilm development is arguably a virulence trait, because medical implants that serve as biofilm substrates are significant risk factors for infection. The development of C. albicans biofilms in vitro proceeds through an early phase, in which yeast cells populate a substrate, an intermediate phase, in which pseudohyphal and hyphal cell types are produced, and a maturation phase, in which continued cell growth is accompanied by accumulation of an extracellular matrix. Here we report the results of a screen for C. albicans biofilm-defective mutants, in which homozygous insertions in NUP85, MDS3, KEM1, and SUV3 were found to block biofilm development. Confocal microscopic examination suggests that nup85, suv3, and mds3 mutations cause early-phase arrest, whereas the kem1 mutation causes intermediate-phase arrest. All of the mutants are defective in hypha production in several media. Analysis of mixed-biofilm development indicates that all of the mutants are defective in the production of hyphae in the context of a biofilm. Because all of the mutants are defective in the retention of cells in the biofilm, we infer that hyphae provide an adherent scaffold that stabilizes the biofilm structure.

Alleles↗
PubMed1988

Stabilization of discrete mRNA breakdown products in ams pnp rnb multiple mutants of Escherichia coli K-12.

The degradation of mRNA in Escherichia coli is thought to occur through a series of endonucleolytic and exonucleolytic steps. By constructing a series of multiple mutants containing the pnp-7 (polynucleotide phosphorylase), rnb-500 (RNase II), and ams-1 (altered message stability) alleles, it was possible to study general mRNA turnover as well as the degradation of specific mRNAs. Of most interest was the ams-1 pnp-7 rnb-500 triple mutant in which the half-life of total pulse-labeled RNA increased three- to fourfold at the nonpermissive temperature. RNA-DNA hybridization analysis of several specific mRNAs such as trxA (thioredoxin), ssb (single-stranded-DNA-binding protein), uvrD (DNA helicase II), cat (chloramphenicol acetyltransferase), nusA (N utilization substance), and pnp (polynucleotide phosphorylase) demonstrated two- to fourfold increases in their chemical half-lives. A new method for high-resolution Northern (RNA) analysis showed that the trxA and cat mRNAs are degraded into discrete fragments which are significantly stabilized only in the triple mutant. A model for mRNA turnover is discussed.

Blotting, Northern↗
PubMed1989

Localization of the Escherichia coli rnt gene encoding RNase T by using a combination of physical and genetic mapping.

The rnt gene encoding RNase T was cloned on a 13-kilobase BamHI fragment. Restriction analysis of the fragment and comparison of it with the Escherichia coli restriction map localized rnt to kilobase coordinates 1733 to 1746, corresponding to about 36 min on the genetic map. The map location was confirmed by cotransduction with the nearby zdg-229::Tn10 and ksgB1 markers.

Escherichia coli↗
PubMed1991

Escherichia coli orfE (upstream of pyrE) encodes RNase PH.

RNase PH from extracts of Escherichia coli was purified to homogeneity and subjected to NH2-terminal sequencing. Comparison of this sequence with all open reading frames in the GenBank data base revealed at least 95% identity to an unidentified open reading frame (orfE) upstream of pyrE at 81.7 min on the E. coli chromosome. Clones of orfE overexpress RNase PH activity, verifying that orfE encodes this ribonuclease. We suggest that orfE be renamed rph.

Amino Acid Sequence↗
PubMed1992

vacB, a novel chromosomal gene required for expression of virulence genes on the large plasmid of Shigella flexneri.

Shigellae, the causative agents of bacillary dysentery, are capable of adhering to and invading epithelial cells and spreading into adjacent cells. A chromosomal mutant of Shigella flexneri 2a YSH6000 with reduced invasive capacity was isolated by Tn5 insertion mutagenesis. The linkage of the mutant phenotype to the Tn5 insertion was determined by P1 phage transduction. The site of the Tn5 insertion was assigned to a NotI chromosomal restriction map, confirming that the virulence-associated locus, designated vacB, is a new locus on the chromosome. In the vacB mutant, production of the four plasmid-encoded virulence antigens, IpaB, -C, and -D and VirG, decreased to a low level compared with that in the wild type. In contrast, levels of transcription of the operons for virG, ipa, region-3.4, region-5, virF, and virB on the large plasmid, as determined by Northern dot blotting, were unaffected in the vacB mutant. Furthermore, transcriptional activation of the ipa operon by exploiting a tac promoter could not restore the vacB mutant to production of the same levels of the IpaB, -C, and -D proteins as those in the wild type, indicating that the vacB locus is involved in expression of the vir genes on the large plasmid at the posttranscriptional level. Cloning followed by nucleotide sequencing of the vacB region showed it to contain a 2,280-bp open reading frame encoding an 86.9-kDa protein located 669 bp downstream from the 3' end of the open reading frame for the purA gene. Disruption of the vacB gene of other serotypes of Shigella spp. and enteroinvasive Escherichia coli (EIEC) resulted in reduced expression of virulence phenotypes, indicating that the vacB gene encodes a novel type of virulence-associated gene required for the full expression of the virulence phenotype of Shigella spp. and EIEC.

Amino Acid Sequence↗
PubMed1992

Role of the heat shock response in stability of mRNA in Escherichia coli K-12.

The heat shock response in Escherichia coli involves extensive induction of the heat shock proteins, with the concomitant suppression of the synthesis of the non-heat shock proteins. While the induction of the heat shock proteins has been shown to occur primarily at the transcriptional level, the suppression of non-heat shock proteins is poorly understood. We have investigated the possibility that an increased decay of non-heat shock mRNAs is a means of decreasing the synthesis of non-heat shock proteins during the heat shock response. Heat shock response-defective strains were compared with wild-type controls by several criteria to evaluate both mRNA stability and the induction of enzymes known to be involved in mRNA turnover. Our results indicate that increased mRNA decay is not a mechanism used to regulate the synthesis of non-heat shock proteins.

Bacterial Proteins↗
PubMed1993

Analysis of mRNA decay and rRNA processing in Escherichia coli multiple mutants carrying a deletion in RNase III.

RNase III is an endonuclease involved in processing both rRNA and certain mRNAs. To help determine whether RNase III (rnc) is required for general mRNA turnover in Escherichia coli, we have created a deletion-insertion mutation (delta rnc-38) in the structural gene. In addition, a series of multiple mutant strains containing deficiencies in RNase II (rnb-500), polynucleotide phosphorylase (pnp-7 or pnp-200), RNase E (rne-1 or rne-3071), and RNase III (delta rnc-38) were constructed. The delta rnc-38 single mutant was viable and led to the accumulation of 30S rRNA precursors, as has been previously observed with the rnc-105 allele (P. Gegenheimer, N. Watson, and D. Apirion, J. Biol. Chem. 252:3064-3073, 1977). In the multiple mutant strains, the presence of the delta rnc-38 allele resulted in the more rapid decay of pulse-labeled RNA but did not suppress conditional lethality, suggesting that the lethality associated with altered mRNA turnover may be due to the stabilization of specific mRNAs. In addition, these results indicate that RNase III is probably not required for general mRNA decay. Of particular interest was the observation that the delta rnc-38 rne-1 double mutant did not accumulate 30S rRNA precursors at 30 degrees C, while the delta rnc-38 rne-3071 double mutant did. Possible explanations of these results are discussed.

DNA Mutational Analysis↗
PubMed1993

The Escherichia coli K-12 "wild types" W3110 and MG1655 have an rph frameshift mutation that leads to pyrimidine starvation due to low pyrE expression levels.

The widely used and closely related Escherichia coli "wild types" W3110 and MG1655, as well as their common ancestor W1485, starve for pyrimidine in minimal medium because of a suboptimal content of orotate phosphoribosyltransferase, which is encoded by the pyrE gene. This conclusion was based on the findings that (i) the strains grew 10 to 15% more slowly in pyrimidine-free medium than in medium containing uracil; (ii) their levels of aspartate transcarbamylase were highly derepressed, as is characteristic for pyrimidine starvation conditions; and (iii) their levels of orotate phosphoribosyltransferase were low. After introduction of a plasmid carrying the rph-pyrE operon from strain HfrH, the growth rates were no longer stimulated by uracil and the levels of aspartate transcarbamylase were low and similar to the levels observed for other strains of E. coli K-12, E. coli B, and Salmonella typhimurium. To identify the mutation responsible for these phenotypes, the rph-pyrE operon of W3110 was cloned in pBR322 from Kohara bacteriophage lambda 2A6. DNA sequencing revealed that a GC base pair was missing near the end of the rph gene of W3110. This one-base-pair deletion results in a frame shift of translation over the last 15 codons and reduces the size of the rph gene product by 10 amino acid residues relative to the size of RNase PH of other E. coli strains, as confirmed by analysis of protein synthesis in minicells. The truncated protein lacks RNase PH activity, and the premature translation stop in the rph cistron explains the low levels of orotate phosphoribosyltransferase in W3110, since close coupling between transcription and translation is needed to support optimal levels of transcription past the intercistronic pyrE attenuator.

Amino Acid Sequence↗
PubMed1995

The gene for the longest known Escherichia coli protein is a member of helicase superfamily II.

The Escherichia coli rnt gene, which encodes the RNA-processing enzyme RNase T, is cotranscribed with a downstream gene. Complete sequencing of this gene indicates that its coding region encompasses 1,538 amino acids, making it the longest known protein in E. coli. The gene (tentatively termed lhr for long helicase related) contains the seven conserved motifs of the DNA and RNA helicase superfamily II. An approximately 170-kDa protein is observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 35S-labeled extracts prepared from cells in which lhr is under the control of an induced T7 promoter. This protein is absent when lhr is interrupted or when no plasmid is present. Downstream of lhr is the C-terminal region of a convergent gene with homology to glutaredoxin. Interruptions of chromosomal lhr at two different positions within the gene do not affect the growth of E. coli at various temperatures in rich or minimal medium, indicating that lhr is not essential for usual laboratory growth. lhr interruption also has no effect on anaerobic growth. In addition, cells lacking Lhr recover normally from starvation, plate phage normally, and display normal sensitivities to UV irradiation and H2O2. Southern analysis showed that no other gene closely related to lhr is present on the E. coli chromosome. These data expand the known size range of E. coli proteins and suggest that very large helicases are present in this organism.

Amino Acid Sequence↗
PubMed1996

The nucleotide sequence of the Pseudomonas aeruginosa pyrE-crc-rph region and the purification of the crc gene product.

The gene (crc) responsible for catabolite repression control in Pseudomonas aeruginosa has been cloned and sequenced. Flanking the crc gene are genes encoding orotate phosphoribosyl transferase (pyrE) and RNase PH (rph). New crc mutants were constructed by disruption of the wild-type crc gene. The crc gene encodes an open reading frame of 259 amino acids with homology to the apurinic/apyrimidinic endonuclease family of DNA repair enzymes. However, crc mutants do not have a DNA repair phenotype, nor can the crc gene complement Escherichia coli DNA repair-deficient strains. The crc gene product was overexpressed in both P. aeruginosa and in E. coli, and the Crc protein was purified from both. The purified Crc proteins show neither apurinic/apyrimidinic endonuclease nor exonuclease activity. Antibody to the purified Crc protein reacted with proteins of similar size in crude extracts from Pseudomonas putida and Pseudomonas fluorescens, suggesting a common mechanism of catabolite repression in these three species.

Amino Acid Sequence↗
PubMed1996

Identification and characterization of the Escherichia coli rbn gene encoding the tRNA processing enzyme RNase BN.

The gene encoding RNase BN was localized to 88 min on the Escherichia coli chromosome by a novel suppressor assay and conjugational and transductional analysis. Assay of subclones derived from lambda phage 543 of the Kohara library, which encompasses this region of the chromosome, for elevated RNase BN activity identified o290, a previously reported open reading frame, as the gene encoding RNase BN. Interruption of this gene with a Kan(r) cassette and introduction into the chromosome eliminated cellular RNase BN activity but had no effect on cell growth. On the basis of these data, we suggest that o290 be renamed rbn. Potential homologs of rbn in other organisms also were identified.

Bacterial Proteins↗
PubMed1996

RNases involved in ribozyme degradation in Escherichia coli.

Hammerhead ribozymes are small catalytic RNA molecules that can be designed to specifically cleave other RNAs. These ribozymes have exhibited low efficiency when examined inside cells, perhaps in part because of their sensitivity to intracellular RNases. In an effort to better understand intracellular degradation of small, foreign RNAs and to develop more stable ribozymes, the ability of Escherichia coli RNase mutants to digest ribozymes was examined. In soluble extracts, most (80 to 90%) of the endonucleolytic activity was due to RNases I and I*, since degradative activity was inhibited by Mg2+ and by the rna-2 mutation. Degradation by exonucleolytic activities was temperature sensitive in extracts from an rna pnp rnb(Ts) triple mutant but not in extracts from an rna rnb(Ts) double mutant. Thus, the products of rnb and pnp, RNase II and polynucleotide phosphorylase, respectively, appear to be the major exonucleases that degrade hammerhead ribozymes. Examination of intracellular degradation revealed that RNases I and I* contributed to about half of the degradative activity as judged by comparison of the rate of ribozyme decay in wild-type and rna-2 mutant cells. Little additional effect was observed in rne(RNase E) and rnc (RNaseIII) mutants. Taken together, these data indicate that hammerhead ribozymes are digested largely by the degradative class of RNase (RNases I, I* and II and polynucleotide phosphorylase).

Base Sequence↗