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At least 1,225 records · Page 68Linked to original sources
PubMed2006

Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs.

MicroRNAs (miRNAs) comprise 1 to 3% of all vertebrate genes, but their in vivo functions and mechanisms of action remain largely unknown. Zebrafish miR-430 is expressed at the onset of zygotic transcription and regulates morphogenesis during early development. By using a microarray approach and in vivo target validation, we find that miR-430 directly regulates several hundred target messenger RNA molecules (mRNAs). Most targets are maternally expressed mRNAs that accumulate in the absence of miR-430. We also show that miR-430 accelerates the deadenylation of target mRNAs. These results suggest that miR-430 facilitates the deadenylation and clearance of maternal mRNAs during early embryogenesis.

3' Untranslated Regions↗
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

Human catechol-O-methyltransferase haplotypes modulate protein expression by altering mRNA secondary structure.

Catechol-O-methyltransferase (COMT) is a key regulator of pain perception, cognitive function, and affective mood. Three common haplotypes of the human COMT gene, divergent in two synonymous and one nonsynonymous position, code for differences in COMT enzymatic activity and are associated with pain sensitivity. Haplotypes divergent in synonymous changes exhibited the largest difference in COMT enzymatic activity, due to a reduced amount of translated protein. The major COMT haplotypes varied with respect to messenger RNA local stem-loop structures, such that the most stable structure was associated with the lowest protein levels and enzymatic activity. Site-directed mutagenesis that eliminated the stable structure restored the amount of translated protein. These data highlight the functional significance of synonymous variations and suggest the importance of haplotypes over single-nucleotide polymorphisms for analysis of genetic variations.

Alleles↗
PubMed2006

Synthesis-mediated release of a small RNA inhibitor of RNA polymerase.

Noncoding small RNAs regulate gene expression in all organisms, in some cases through direct association with RNA polymerase (RNAP). Here we report that the mechanism of 6S RNA inhibition of transcription is through specific, stable interactions with the active site of Escherichia coli RNAP that exclude promoter DNA binding. In fact, the DNA-dependent RNAP uses bound 6S RNA as a template for RNA synthesis, producing 14-to 20-nucleotide RNA products (pRNA). These results demonstrate that 6S RNA is functionally engaged in the active site of RNAP. Synthesis of pRNA destabilizes 6S RNA-RNAP complexes leading to release of the pRNA-6S RNA hybrid. In vivo, 6S RNA-directed RNA synthesis occurs during outgrowth from the stationary phase and likely is responsible for liberating RNAP from 6S RNA in response to nutrient availability.

Base Sequence↗
PubMed2007

A hexanucleotide element directs microRNA nuclear import.

MicroRNAs (miRNAs) negatively regulate partially complementary target messenger RNAs. Target selection in animals is dictated primarily by sequences at the miRNA 5' end. We demonstrated that despite their small size, specific miRNAs contain additional sequence elements that control their posttranscriptional behavior, including their subcellular localization. We showed that human miR-29b, in contrast to other studied animal miRNAs, is predominantly localized to the nucleus. The distinctive hexanucleotide terminal motif of miR-29b acts as a transferable nuclear localization element that directs nuclear enrichment of miRNAs or small interfering RNAs to which it is attached. Our results indicate that miRNAs sharing common 5' sequences, considered to be largely redundant, might have distinct functions because of the influence of cis-acting regulatory motifs.

Active Transport, Cell Nucleus↗
PubMed2000

A link between RNA interference and nonsense-mediated decay in Caenorhabditis elegans.

Double-stranded RNA (dsRNA) inhibits expression of homologous genes by a process involving messenger RNA degradation. To gain insight into the mechanism of degradation, we examined how RNA interference is affected by mutations in the smg genes, which are required for nonsense-mediated decay. For three of six smg genes tested, mutations resulted in animals that were initially silenced by dsRNA but then recovered; wild-type animals remained silenced. The levels of target messenger RNAs were restored during recovery, and RNA editing and degradation of the dsRNA were identical to those of the wild type. We suggest that persistence of RNA interference relies on a subset of smg genes.

Adenosine Deaminase↗
PubMed2007

How do microRNAs regulate gene expression?

Several thousand human genes, amounting to about one-third of the whole genome, are potential targets for regulation by the several hundred microRNAs (miRNAs) encoded in the genome. The regulation occurs posttranscriptionally and involves the approximately 21-nucleotide miRNA interacting with a target site in the mRNA that generally has imperfect complementarity to the miRNA. The target sites are almost invariably in the 3'-untranslated region of the messenger RNA (mRNA), often in multiple copies. Metazoan miRNAs were previously thought to down-regulate protein expression by inhibiting target mRNA translation at some stage after the translation initiation step, without much effect on mRNA abundance. However, recent studies have questioned these suppositions. With some targets, an increase in the rate of mRNA degradation by the normal decay pathway contributes to the decrease in protein expression. miRNAs can also inhibit translation initiation, specifically the function of the cap-binding initiation factor, eIF4E. Repressed target mRNAs as well as miRNAs themselves accumulate in cytoplasmic foci known as P-bodies, where many enzymes involved in mRNA degradation are concentrated. However, P-bodies may also serve as repositories for the temporary and reversible storage of untranslated mRNA, and reducing the expression (knockdown) of several distinct P-body protein components can alleviate miRNA-mediated repression of gene expression.

Animals↗
PubMed2001

Regulation of the packaging of Bacillus thuringiensis delta-endotoxins into inclusions.

During sporulation, many Bacillus thuringiensis subspecies synthesize several related delta-endotoxins which are packaged into bipyramidal intracellular inclusions. These inclusions are solubilized in the alkaline, reducing conditions of the midguts of susceptible insect larvae and are converted by proteolysis to active toxins. The toxins insert into the membranes of cells lining the midgut and form cation-selective channels, which results in lethality. There are three delta-endotoxins, Cry1Ab3, Cry1Ca1, and Cry1Da1, present in the inclusions produced by a B. thuringiensis subsp. aizawai cell. While the ratio of the steady-state mRNAs for these three protoxins has been shown to differ (cry1Ab3/cry1Ca1/cry1Da1 mRNA ratio, 4:2:1), the half-lives of the cry1Da1 and cry1Ab3 mRNAs were found to be similar, indicating that there were differences in the transcription rates. The relative contents of these delta-endotoxins in purified inclusions from B. thuringiensis subsp. aizawai have been measured previously, and an even greater relative deficiency of the Cry1Da1 protoxin (ratio, 20:12:1) was found. In order to account for this deficiency, other steps which could be involved in inclusion formation, such as translation and packaging, were examined. The three cry genes have the same dual overlapping promoters, but the ribosome binding sequence for the cry1Da1 gene was not the consensus sequence. Translation was enhanced about fourfold by changing to the consensus sequence. In addition, the relative amount of Cry1Da1 protoxin in inclusions was twofold lower when cells were sporulated in Luria-Bertani (LB) medium than when cells were sporulated in a glucose-yeast extract medium. This difference was attributable to packaging since the relative amounts of Cry1Da1 antigen in cells sporulating in the two media were the same. Some factor(s) required for packaging of the Cry1Da1 protoxin in inclusions is apparently limiting in LB medium. Differences in the initial transcription rates, translation efficiencies, and packaging all contribute to the delta-endotoxin composition of an inclusion.

Amino Acid Sequence↗
PubMed2006

In situ and in vitro gene expression by Vibrio vulnificus during entry into, persistence within, and resuscitation from the viable but nonculturable state.

Isolation of Vibrio vulnificus during winter months is difficult due to the entrance of these cells into the viable but nonculturable (VBNC) state. While several studies have investigated in vitro gene expression upon entrance into and persistence within the VBNC state, to our knowledge, no in situ studies have been reported. We incubated clinical and environmental isolates of V. vulnificus in estuarine waters during winter months to monitor the expression of several genes during the VBNC state and compared these to results from in vitro studies. katG (periplasmic catalase) was down-regulated during the VBNC state in vitro and in situ compared to the constitutively expressed gene tufA. Our results indicate that the loss of catalase activity we previously reported is a direct result of katG repression, which likely accounts for the VBNC response of this pathogen. While expression of vvhA (hemolysin) was detectable in environmental strains during in situ incubation, it ceased in all cases by ca. 1 h. These results suggest that the natural role of hemolysin in V. vulnificus may be in osmoprotection and/or the cold shock response. Differences in expression of the capsular genes wza and wzb were observed in the two recently reported genotypes of this species. Expression of rpoS, encoding the stress sigma factor RpoS, was continuous upon entry into the VBNC state during both in situ and in vitro studies. We found the half-life of mRNA to be less than 60 minutes, confirming that mRNA detection in these VBNC cells is a result of de novo RNA synthesis.

Bacterial Proteins↗
PubMed2005

Structural elements in the 5'-untranslated region of giardiavirus transcript essential for internal ribosome entry site-mediated translation initiation.

Translation of uncapped giardiavirus (GLV) mRNA in Giardia lamblia requires the presence of a 5'-untranslated region (5'-UTR) and a viral capsid coding region. We used dicistronic viral constructs to show that the downstream 253 nucleotides (nt) of the 5'-UTR plus the initial 264-nt capsid coding region constitute an internal ribosome entry site (IRES). Predicted secondary structures in the 253-nt 5'-UTR include stem-loops U3, U4a, U4b, U4c, and U5. Chemical and enzymatic probing analysis confirmed the presence of all predicted stem-loops except U4a. Disruption of stem-loop structures U3 and U5 by site-directed mutagenesis resulted in a drastic reduction in translation of a monocistronic viral transcript, which could be restored by compensatory sequence changes. Mutations disrupting stem-loops U4b and U4c do not exert an appreciable effect on translation, but certain sequences in the U4a region and in U4b do appear to play important roles in the IRES. Structural analysis also suggests that an 8-nt U3 loop sequence (nt 147 to 154) pairs with an 8-nt downstream sequence (nt 168 to 175) to form a pseudoknot. Disruption of this pseudoknot by mutagenesis resulted in a drastic reduction in translation, which could be restored by compensatory sequence changes. This study has defined the secondary structure in the 5'-UTR of the IRES. Together with the previous results, we have now completed analysis of the entire structure of GLV IRES and fully defined the functionally essential structural elements in it.

5' Untranslated Regions↗
PubMed2006

Ebs1p, a negative regulator of gene expression controlled by the Upf proteins in the yeast Saccharomyces cerevisiae.

Mutations in EBS1 were identified in Saccharomyces cerevisiae that cosuppress missense, frameshift, and nonsense mutations. Evidence from studies of loss of function and overexpression of EBS1 suggests that Ebs1p affects gene expression by inhibiting translation and that a loss of EBS1 function causes suppression by increasing the rate of translation. Changes in EBS1 expression levels alter the expression of wild-type genes, but, in general, no changes in mRNA abundance were associated with a loss of function or overexpression of EBS1. Translation of a lacZ reporter was increased in strains carrying an ebs1-Delta mutant gene, whereas translation was decreased when EBS1 was overexpressed. The cap binding protein eIF-4E copurifies with Ebs1p in the absence of RNA, suggesting that the two proteins interact in vivo. Although physical and genetic interactions were detected between Ebs1p and Dcp1p, copurification was RNase sensitive, and changes in the expression of Ebs1p had little to no effect on decapping of the MFA2 transcript. The combined results suggest that Ebs1p inhibits translation, most likely through effects on eIF-4E rather than on decapping. Finally, EBS1 transcript levels are under the control of nonsense-mediated mRNA decay (NMD), providing the first example of an NMD-sensitive transcript whose protein product influences a step in gene expression required for NMD.

Amino Acid Sequence↗
PubMed2000

Classical and El Tor biotypes of Vibrio cholerae differ in timing of transcription of tcpPH during growth in inducing conditions.

Two protein pairs in Vibrio cholerae, ToxRS and TcpPH, are necessary for transcription from the toxT promoter and subsequent expression of cholera virulence genes. We have previously shown that transcription of tcpPH in classical strains of V. cholerae is activated at mid-log-phase growth in ToxR-inducing conditions, while transcription of tcpPH in El Tor strains is not. In this study, we showed that while transcription of tcpPH differs at mid-log-phase growth in ToxR-inducing conditions between the biotypes, transcription is equivalently high during growth in AKI conditions. We used tcpPH::gusA transcriptional fusions to quantitate expression of tcpPH in each biotype throughout growth in ToxR-inducing conditions and showed that although transcription of tcpPH is reduced at mid-log-phase growth in an El Tor strain, transcription is turned on later in growth to levels in excess of those in the classical strain (although cholera toxin is not produced). This suggests that the difference in expression of cholera virulence factors in response to ToxR-inducing conditions between the El Tor and classical biotypes of V. cholerae may be related to the timing of transcription of tcpPH rather than the absolute levels of transcription.

Artificial Gene Fusion↗
PubMed2004

The ClpP protease of Streptococcus pneumoniae modulates virulence gene expression and protects against fatal pneumococcal challenge.

Streptococcus pneumoniae usually colonizes the nasopharynx of humans asymptomatically but occasionally translocates from this niche to the lungs, the brain, and the blood, causing potentially fatal infections. Spread to other host tissues requires a significant morphological change and the expression of virulence factors, such as capsular polysaccharide, and virulence proteins, such as pneumolysin (Ply), PspA, and CbpA. Modulation of the expression of pneumococcal virulence genes by heat shock and by heat shock proteins ClpL and ClpP, as well as the attenuation of virulence of a clpP mutant in a murine intraperitoneal infection model, was demonstrated previously. In this study, we further investigated the underlying mechanism of virulence attenuation by the clpP mutation. The half-lives of the mRNAs of ply and of the first gene of the serotype 2 capsule synthesis locus [cps2A] in the clpP mutant were more than twofold longer than those of the parent after heat shock, suggesting that the mRNA species were regulated posttranscriptionally by ClpP. In addition, the clpP mutant was defective in colonization of the nasopharynx and survival in the lungs of mice after intranasal challenge. The mutant was also killed faster than the parent in the murine macrophage RAW264.7 cell line, indicating that ClpP is required for colonization and intracellular survival in the host. Furthermore, fractionation studies demonstrated that ClpP was translocated into the cell wall after heat shock, and immunization of mice with ClpP elicited a protective immune response against fatal systemic challenge with S. pneumoniae D39, making ClpP a potential vaccine candidate for pneumococcal disease.

Adenosine Triphosphatases↗
PubMed2006

Structural organization and functional properties of miniature DNA insertion sequences in yersiniae.

YPALs (Yersinia palindromic sequences) are miniature DNA insertions scattered along the chromosomes of yersiniae. The spread of these intergenic repeats likely occurred via transposition, as suggested by the presence of target site duplications at their termini and the identification of syntenic chromosomal regions which differ in the presence/absence of YPAL DNA among Yersinia strains. YPALs tend to be inserted closely downstream from the stop codon of flanking genes, and many YPAL targets overlap rho-independent transcriptional terminator-like sequences. This peculiar pattern of insertion supports the hypothesis that most of these repeats are cotranscribed with upstream sequences into mRNAs. YPAL RNAs fold into stable hairpins which may modulate mRNA decay. Accordingly, we found that YPAL-positive transcripts accumulate in Yersinia enterocolitica cells at significantly higher levels than homologous transcripts lacking YPAL sequences in their 3' untranslated region.

Base Composition↗
PubMed2000

Restart of exponential growth of cold-shocked Yersinia enterocolitica occurs after down-regulation of cspA1/A2 mRNA.

The cellular content of major cold shock protein (MCSP) mRNA transcribed from the tandem gene duplication cspA1/A2 and growth of Yersinia enterocolitica were compared when exponentially growing cultures of this bacterium were cold shocked from 30 to 20, 15, 10, 5, or 0 degrees C, respectively. A clear correlation between the time point when exponential growth resumes after cold shock and the degradation of cspA1/A2 mRNA was found. A polynucleotide phosphorylase-deficient mutant was unable to degrade cspA1/A2 mRNA properly and showed a delay, as well as a lower rate, of growth after cold shock. For this mutant, a correlation between decreasing cspA1/A2 mRNA and restart of growth after cold shock was also observed. For both wild-type and mutant cells, no correlation of restart of growth with the cellular content of MCSPs was found. We suggest that, after synthesis of cold shock proteins and cold adaptation of the cells, MCSP mRNAs must be degraded; otherwise, they trap ribosomes, prevent translation of bulk mRNA, and thus inhibit growth of this bacterium at low temperatures.

Adaptation, Biological↗