SEARCH · PubMed Health

Results for “RNA Stability”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 343 records · Page 19Linked to original sources

PubMed2001

RNA tertiary interactions in the large ribosomal subunit: the A-minor motif.

Analysis of the 2.4-A resolution crystal structure of the large ribosomal subunit from Haloarcula marismortui reveals the existence of an abundant and ubiquitous structural motif that stabilizes RNA tertiary and quaternary structures. This motif is termed the A-minor motif, because it involves the insertion of the smooth, minor groove edges of adenines into the minor groove of neighboring helices, preferentially at C-G base pairs, where they form hydrogen bonds with one or both of the 2' OHs of those pairs. A-minor motifs stabilize contacts between RNA helices, interactions between loops and helices, and the conformations of junctions and tight turns. The interactions between the 3' terminal adenine of tRNAs bound in either the A site or the P site with 23S rRNA are examples of functionally significant A-minor interactions. The A-minor motif is by far the most abundant tertiary structure interaction in the large ribosomal subunit; 186 adenines in 23S and 5S rRNA participate, 68 of which are conserved. It may prove to be the universally most important long-range interaction in large RNA structures.

Adenosine↗

PubMed2005

A catalog of stability-associated sequence elements in 3' UTRs of yeast mRNAs.

BACKGROUND: In recent years, intensive computational efforts have been directed towards the discovery of promoter motifs that correlate with mRNA expression profiles. Nevertheless, it is still not always possible to predict steady-state mRNA expression levels based on promoter signals alone, suggesting that other factors may be involved. Other genic regions, in particular 3' UTRs, which are known to exert regulatory effects especially through controlling RNA stability and localization, were less comprehensively investigated, and deciphering regulatory motifs within them is thus crucial. RESULTS: By analyzing 3' UTR sequences and mRNA decay profiles of Saccharomyces cerevisiae genes, we derived a catalog of 53 sequence motifs that may be implicated in stabilization or destabilization of mRNAs. Some of the motifs correspond to known RNA-binding protein sites, and one of them may act in destabilization of ribosome biogenesis genes during stress response. In addition, we present for the first time a catalog of 23 motifs associated with subcellular localization. A significant proportion of the 3' UTR motifs is highly conserved in orthologous yeast genes, and some of the motifs are strikingly similar to recently published mammalian 3' UTR motifs. We classified all genes into those regulated only at transcription initiation level, only at degradation level, and those regulated by a combination of both. Interestingly, different biological functionalities and expression patterns correspond to such classification. CONCLUSION: The present motif catalogs are a first step towards the understanding of the regulation of mRNA degradation and subcellular localization, two important processes which--together with transcription regulation--determine the cell transcriptome.

3' Untranslated Regions↗

PubMed2003

A Hox gene mutation that triggers nonsense-mediated RNA decay and affects alternative splicing during Drosophila development.

Nonsense mutations are usually assumed to affect protein function by generating truncated protein products. Nonetheless, it is now clear that these mutations affect not just protein synthesis but also messenger RNA stability. The surveillance mechanism responsible for the detection and degradation of 'nonsense' RNA messages is termed nonsense-mediated RNA decay (NMD). Essential biochemical components of the NMD machinery have been defined in several species. Here we identify the Drosophila orthologue of one of these factors, Upf1, and document its expression during embryogenesis. To test whether NMD acts during Drosophila development, we make use of a mutation that introduces a stop codon into a variably spliced exon of the Hox gene Ultrabithorax (Ubx). Using real-time quantitative RT-PCR we demonstrate that Ubx transcripts containing the premature stop codon are expressed at lower levels than their wild type counterpart. Unexpectedly, we also find that the same mutation significantly increases the levels of a Ubx splicing isoform that lacks the exon containing the premature termination codon. These findings indicate that NMD is operational during Drosophila development and suggest that nonsense mutations may affect development by altering the spectrum of splicing products formed, as well as by reducing or eliminating protein synthesis.

Alternative Splicing↗

PubMed1987

The length but not the sequence of the polyoma virus late leader exon is important for both late RNA splicing and stability.

Polyoma virus late RNA processing provides a convenient model system in which to study the mechanics of splicing in vivo. In order to understand further the role of the untranslated "late leader" unit in late RNA processing we have constructed a group of polyoma viruses with deletions and substitutions in the leader exon. This has allowed us to determine that there is a minimum exon size required for both pre-mRNA splicing and stability in this system. We show here that the non-viability of a mutant (ALM) with a 9 base late leader unit is due to a general defect in late RNA splicing. In addition, ALM-infected cells show at least 40-fold depression in the accumulation of late nuclear RNA (spliced or unspliced). The ALM late promoter, however, functions nearly normally. Substituted leader variants with 51- to 96-base long exons of unrelated sequence are viable (G. Adami and G. Carmichael, J. Virol. 58, 417-425, 1986). We show here that late RNA from one of these substituted leader mutants (containing a 51-base leader exon) is spliced at wild type levels, with virtually no defect in accumulation. Thus, in the polyoma system, splice sites separated by only 9 bases can inhibit each others usage, presumably by steric interference. We suggest that this type of inhibition leads to extreme RNA instability.

Animals↗

PubMed1993

Cholinergic regulation of rat preproenkephalin RNA in the adrenal medulla.

Expression of the rat preproenkephalin (ppENK) gene involves transsynaptic cholinergic mechanisms. We evaluated the effects of cholinergic agonist treatments in vivo on the expression of adrenomedullary ppENK RNA. Cholinergic treatment with nicotinic + muscarinic receptor agonists resulted in a synergistic 100-fold rise in steady-state ppENK messenger RNA levels, but only a 30- to 35-fold rise in initiation of steady-state ppENK RNA transcripts. The levels of initiated ppENK steady-state RNA peaked at two days, whereas mature (1.45 kb) ppENK mRNA levels continued to rise, peaking at four days. This suggested that other transcriptional (attenuation or alternative splicing) or post-transcriptional (RNA stabilization) regulatory mechanisms must be operative. As multiple ppENK RNA start sites exist, we examined how usage of multiple sites was altered by cholinergic treatments. The predominant start site changed from E2 in the basal state, to E4 after primary cholinergic stimulation, to E3 after re-treatment. This represents novel example of differential usage of multiple RNA initiation start sites in vivo. Differences in initiated and mature transcripts are consistent with at least four mechanisms involved in control of cholinergic-induced ppENK RNA expression: (i) simply new initiation of RNA transcripts, (ii) differential usage of the multiple RNA start sites, (iii) stabilization of mRNA transcripts, and (iv) attenuation and/or alternative RNA splicing of RNA transcripts.

Adrenal Medulla↗

PubMed2001

HuD, a neuronal-specific RNA-binding protein, increases the in vivo stability of MYCN RNA.

MYCN amplification and consequent deregulated expression plays a crucial role in determining the clinical behavior of neuroblastoma. Enhanced expression of MYCN confers growth potential to neuroblastoma cells, and a direct link between MYCN expression and the development of neuroblastoma has been demonstrated in transgenic mice studies. Although the molecular pathways underlying the regulation of MYCN have not been fully elucidated, post-transcriptional mechanisms appear to be important. Previously, we reported that an embryonic lethal abnormal vision-like (ELAV) protein binds with high specificity to at least two AU-rich elements within the MYCN 3'-untranslated region. In this study, we characterized the ability of cis-acting elements within the MYCN 3'-untranslated region to destabilize mRNA in cells and examined the functional consequences of its interactions with the ELAV protein HuD. We show that at least 4 cis-acting elements within the MYCN 3'-untranslated region are able to signal the degradation of stable heterologous mRNA. Ectopic overexpression of HuD dramatically inhibits RNA decay mediated by the full-length MYCN 3'-untranslated region and cis-acting destabilizing elements that harbor HuD binding sites in vivo. HuD may contribute to the malignant phenotype of neuroblastoma cells by stabilizing MYCN mRNA, thereby enhancing steady-state levels of expression of this oncogene.

3' Untranslated Regions↗

PubMed2003

Translation efficiency determines differences in cellular infection among dengue virus type 2 strains.

We have investigated the molecular basis for differences in the ability of natural variants of dengue virus type 2 (DEN2) to replicate in primary human cells. The rates of virus binding, virus entry, input strand translation, and RNA stability of low-passage Thai and Nicaraguan and prototype DEN2 strains were compared. All strains exhibited equivalent binding, entry, and uncoating, and displayed comparable stability of positive strand viral RNA over time in primary cells. However, the low-passage Nicaraguan isolates were much less efficient in their ability to translate viral proteins. Sequence analysis of the full-length low-passage Nicaraguan and Thai viral genomes identified specific differences in the 3' untranslated region (3'UTR). Substitution of the different sequences into chimeric RNA reporter constructs demonstrated that the changes in the 3'UTR directly affected the efficiency of viral translation. Thus, differences in infectivity among closely related DEN2 strains correlate with efficiency of translation of input viral RNA.

3' Untranslated Regions↗

PubMed2003

Intragenic HIV-1 env sequences that enhance gag expression.

Expression of HIV-1 genes is regulated at multiple levels including the complex RNA splicing and transport mechanisms. Multiple cis-acting elements involved in these regulations have been previously identified in various regions of HIV-1 genome. Here we show that another cis-acting element was present in HIV-1 env region. This element enhanced the expression of Gag when inserted together with Rev response element (RRE) into a truncated HIV-1 genome in the presence of Rev. The enhancing activity was mapped to a 263-bp fragment in the gp41 region downstream to RRE. RNA analysis showed that it might function by promoting RNA stability and Rev-dependent RNA export. The enhancement was specific to Rev-dependent expression, since it did not enhance Gag expression driven by Sam68, a cellular protein that has been shown to be able to substitute for Rev in RNA export function.

Base Sequence↗

PubMed1995

Vesicular stomatitis virus transcription inhibited by purified MxA protein.

MxA is a GTPase encoded by an interferon-inducible human gene. Its constitutive expression renders transfected mammalian cells resistant to infections with several different RNA viruses, including vesicular stomatitis virus (VSV). Differences in viral RNA levels of VSV-infected cells either expressing or lacking MxA indicated that VSV mRNA synthesis is the principal target of MxA action. We now used purified histidine-tagged MxA (His-MxA) that we produced in Escherichia coli to successfully inhibit VSV in vitro transcription, a reaction catalyzed by VSV ribonucleoprotein complexes isolated from virus-infected cells or from purified virions. MxA was inactive when added to preformed VSV mRNAs, arguing against the possibility that it has a negative effect on viral RNA stability. MxA inhibited both leader RNA and mRNA synthesis of VSV, suggesting that it interfered with transcription initiation. The degree of VSV inhibition correlated directly with the specific GTPase activities of the various wild-type MxA preparations. No inhibition of viral mRNA synthesis was observed when a C-terminally truncated, GTPase-inactive variant of His-MxA was added to the transcription reactions. Purified His-MxA-E645R, a mutant of MxA with normal GTPase activity whose range of antiviral activity in vivo is altered so that it no longer inhibits VSV, showed no inhibitory effect on VSV in vitro transcription. Since MxA inhibited VSV RNA synthesis in the presence of GMP-PNP or GTP gamma S, GTP analogs that are readily accepted by the viral polymerase but cannot be hydrolyzed by MxA, the possibility was excluded that MxA acts by depleting the viral polymerase for its nucleotide substrates. Thus, binding of GTP rather than its hydrolysis seems of importance for the anti-VSV activity of MxA.

Animals↗

PubMed1978

Biochemical and electron microscopic characterization of DNA-RNA complexes from HeLa cell mitochondria.

The previous electron microscopic investigations on the occurrence in HeLa cell mitochondria of transcription complexes of mitochondrial DNA [Aloni, Y., and Attardi, G. (1972a), J. Mol. Biol. 70, 363-373] have been extended with the aim of obtaining these complexes in a reasonably pure form for biochemical analysis. By using conditions designed to minimize losses of such structures and any possible contamination by nuclear DNA, it has been shown that a substantial fraction (40 to 50%) of mitochondrial DNA can be isolated from exponentially growing HeLa cells in the form of fastsedimenting complexes with RNA. These complexes have been characterized with respect to density and sedimentation properties, content in newly synthesized RNA, stability of the association of RNA with DNA, presence of different forms of mitochondrial DNA, and electron microscopic appearance. The properties of these complexes, as well as the results of reconstruction experiments, strongly suggest that the majority of such structures represent true transcriptional intermediates. The occurrence in this fraction of replicating or newly replicated mitochondrial DNA molecules has been observed. Although the presence of single-stranded DNA segments makes the replicative intermediates particularly susceptible to aggregation with free RNA, electron microscopic observations point to the possibility that these intermediates may be recruited for transcription.

DNA, Mitochondrial↗

PubMed1997

Site-specific phosphorylation of the human immunodeficiency virus type-1 Rev protein accelerates formation of an efficient RNA-binding conformation.

Phosphorylation is important in the regulation of many cellular processes, yet the precise role of protein phosphorylation for many RNA-binding protein substrates remains obscure. In this report, we demonstrate that phosphorylation of a recombinant human immunodeficiency virus type-1 Rev protein promotes rapid formation of an efficient RNA-binding state. The apparent dissociation constant for ligand binding is enhanced 7-fold for the protein following phosphorylation; however, phosphate addition leads to a 1. 6-fold decrease in RNA ligand-protein complex stability. RNA ligand binding stimulates slow formation of an equally competent binding state for the unphosphorylated protein, indicating that the addition of phosphate or ligand binding promotes a similar conformational change in Rev. Phosphorylation directly alters the conformation of Rev, as revealed by modification experiments that monitor the solvent accessibility of cysteines in the protein. These biochemical properties are attributed to the addition of phosphate at one of two serine residues (Ser-54 or Ser-56) that lie within the multimerization domain adjacent to the RNA-binding helix. Glutaraldehyde-mediated cross-linking experiments revealed that phosphorylation of Rev does not affect Rev multimerization activity. The Rev protein from the less pathogenic HIV-2 isolate lacks this phosphorylation site in the amino acid sequence; thus, the described biochemical properties of the phosphorylated protein may contribute to Rev activity and possibly to HIV-1 virulence during natural infection.

Amino Acid Sequence↗

PubMed1988

Internal sequences that distinguish yeast from metazoan U2 snRNA are unnecessary for pre-mRNA splicing.

U2 small nuclear RNA is a highly conserved component of the eukaryotic cell nucleus involved in splicing messenger RNA precursors. In the yeast Saccharomyces cerevisiae, U2 RNA interacts with the intron by RNA-RNA pairing between the conserved branchpoint sequence UACUAAC and conserved nucleotides near the 5' end of U2 (ref. 4). Metazoan U2 RNA is less than 200 nucleotides in length, but yeast U2 RNA is 1,175 nucleotides long. The 5' 110 nucleotides of yeast U2 are homologous to the 5' 100 nucleotides of metazoan U2 (ref. 6), and the very 3' end of yeast U2 bears a weak structural resemblance to features near the 3' end of metazoan U2. Internal sequences of yeast U2 share primary sequence homology with metazoan U4, U5 and U6 small nuclear RNA (ref. 6), and have regions of complementarity with yeast U1 (ref. 7). We have investigated the importance of the internal U2 sequences by their deletion. Yeast cells carrying a U2 allele lacking 958 nucleotides of internal U2 sequence produce a U2 small nuclear RNA similar in size to that found in other organisms. Cells carrying only the U2 deletion grow normally, have normal levels of spliced mRNA and do not accumulate unspliced precursor mRNA. We conclude that the internal sequences of yeast U2 carry no essential function. The extra RNA may have a non-essential function in efficient ribonucleoprotein assembly or RNA stability. Variation in amount of RNA in homologous structural RNAs has precedence in ribosomal RNA and RNaseP.

Base Sequence↗

PubMed2002

Molecular characterization of the human La protein.hepatitis B virus RNA.B interaction in vitro.

The La protein was recently identified as a host factor potentially involved in the cytokine-induced post-transcriptional down-regulation of hepatitis B virus (HBV) RNA. The La binding site was mapped to a predicted stem-loop structure within a region shared by all HBV RNAs, and it was concluded that the La protein might be an HBV RNA-stabilizing factor. To characterize the RNA binding mediated by the different RNA recognition motifs (RRMs) of the human La protein, several La deletion mutants were produced and analyzed for HBV RNA binding ability. The data demonstrate that the first RRM is not required for binding, whereas the RNP-1 and RNP-2 consensus sequences of the RRM-2 and RRM-3 are separately required for binding, indicating a cooperative function of these two RRMs. Furthermore, the results suggest that multimeric La disassembles into monomeric La upon binding of HBV RNA.B. By gel retardation assay the affinity of the wild type human La.HBV RNA.B interaction was determined in the nanomolar range, comparable to the affinity determined for the mouse La.HBV RNA.B interaction. This study identified small regions within the human La protein mediating the binding of HBV RNA. Hence, these binding sites might represent targets for novel antiviral strategies based on the disruption of the human La.HBV RNA interaction, thereby leading to HBV RNA degradation.

Amino Acid Motifs↗

PubMed1994

Developmental regulation of DEAD box proteins and cloning of putative RNA helicase genes from Dictyostelium discoideum.

RNA secondary structure is essential for RNA function in pre-mRNA splicing, mRNA translation, ribosome assembly and RNA stability. The involvement of DEAD/H RNA helicases in the regulation of these processes has been demonstrated in some cases. To investigate the repertoire of DEAD box proteins expressed in Dictyostelium discoideum, we used PCR techniques to clone two cDNAs coding for DEAD box proteins with high similarity to known yeast proteins: Dictyostelium Hel2A is about 45% identical to Saccharomyces cerevisiae DBP2 and S. pombe dbp2, the yeast homologues of human p68. Dictyostelium Hel2B is about 43% identical to the S. cerevisiae splicing factor PRP28, but has a different domain at the N-terminus, which is unique for Dictyostelium discoideum. Using a polyclonal antibody directed against a DEAD box peptide we show differential expression of three DEAD box proteins during the developmental cycle of Dictyostelium.

Amino Acid Sequence↗

PubMed2005

Influence of clindamycin on the stability of coa and fnbB transcripts and adherence properties of Staphylococcus aureus Newman.

We investigated whether a subinhibitory concentration of clindamycin (Cli), corresponding to 1/2 the strain-specific minimum inhibitory concentration (MIC), could affect expression and stability of transcripts from genes coding for specific adhesins such as fibronectin binding proteins A (fnbA) and B (fnbB) as well as coagulase (coa) in Staphylococcus aureus strain Newman. Furthermore, the effect of 1/2 MIC of Cli on adherence properties and expression of type 5 capsular polysaccharides (CP5) was investigated. Northern slot blot experiments confirmed that the amount of coa- and fnbB-specific mRNA, in contrast to that of fnbA-specific mRNA, was increased 2-fold after treatment of S. aureus Newman with 1/2 MIC of Cli. Analysis of RNA stability revealed that the increased amounts of transcripts of coa and fnbB were due to stabilization of the respective mRNAs. However, when treated with 1/2 MIC of Cli, S. aureus Newman showed no significant changes neither in its adherence patterns to fibrinogen- or fibronectin-coated micotitre plates, nor to epithelial HEp-2 cells and also not in its CP5 expression. Therefore, we conclude that increased mRNA stability of fnbB and coa by 1/2 MIC Cli, in contrast to the situation seen with the protein biosynthesis inhibiting antibiotic florfenicol, does not result in an increase in adherence of S. aureus Newman.

Adhesins, Bacterial↗

PubMed1999

Mutations in VPS16 and MRT1 stabilize mRNAs by activating an inhibitor of the decapping enzyme.

Decapping is a rate-limiting step in the decay of many yeast mRNAs; the activity of the decapping enzyme therefore plays a significant role in determining RNA stability. Using an in vitro decapping assay, we have identified a factor, Vps16p, that regulates the activity of the yeast decapping enzyme, Dcp1p. Mutations in the VPS16 gene result in a reduction of decapping activity in vitro and in the stabilization of both wild-type and nonsense-codon-containing mRNAs in vivo. The mrt1-3 allele, previously shown to affect the turnover of wild-type mRNAs, results in a similar in vitro phenotype. Extracts from both vps16 and mrt1 mutant strains inhibit the activity of purified Flag-Dcp1p. We have identified a 70-kDa protein which copurifies with Flag-Dcp1p as the abundant Hsp70 family member Ssa1p/2p. Intriguingly, the interaction with Ssa1p/2p is enhanced in strains with mutations in vps16 or mrt1. We propose that Hsp70s may be involved in the regulation of mRNA decapping.

Adenosine Triphosphatases↗

PubMed2026

FMRP promotes gastric cancer progression via m6A-dependent stabilization of DVL2 and activation of the Wnt/PCP-JNK pathway.

BACKGROUND: N6-methyladenosine (m6A) modification has emerged as a critical regulator in gastric cancer progression. Fragile X messenger ribonucleoprotein 1 (FMRP), an RNA-binding protein with tumorigenic potential, remains poorly characterized in gastric cancer. METHODS: FMRP expression in gastric cancer was assessed through bioinformatic analyses and tissue microarray-based immunohistochemistry. Its biological functions were examined through loss-of-function experiments in vitro and in vivo. Mechanistic investigations, including RNA interaction, m6A site mutation, and RNA stability analyses, were performed to identify downstream targets and pathways regulated by FMRP. RESULTS: Bioinformatic analyses and tissue microarray-based immunohistochemistry showed that FMRP was significantly upregulated in gastric cancer tissues. FMRP depletion suppressed tumor growth and metastasis. Subsequently, Dishevelled segment polarity protein 2 (DVL2) was identified as a candidate downstream target of FMRP. DVL2 was also significantly upregulated in gastric cancer tissues, and its expression positively correlated with FMRP expression. Mechanistically, FMRP bound to DVL2 mRNA and enhanced its stability in an m6A-dependent manner. Among the candidate sites tested, mutation of 1271A attenuated FMRP-mediated regulation of DVL2 reporter activity, supporting the involvement of this site. Functionally, FMRP promoted activation of the noncanonical Wnt/planar cell polarity (PCP) signaling pathway and enhanced the proliferative, migratory, and invasive capacities of gastric cancer cells via DVL2. CONCLUSIONS: These results define FMRP as an oncogenic driver in gastric cancer that operates via the DVL2/Wnt/PCP axis. Targeting this pathway may provide a potential therapeutic strategy for gastric cancer.

Stomach Neoplasms↗

PubMed1999

Analysis of mRNA stabilities during pollen development and in BY2 cells

RNA stability is important in post-transcriptional gene expression. However, its role during plant male gametophyte development has not been studied in detail, probably because of technical difficulties and because earlier work suggested that, in general, mRNAs expressed during late pollen development would be stable. We determined mRNA stabilities of 12 pollen-expressed genes. We measured pollen mRNA decay during in planta development and in vitro maturation after transcriptional inhibition with ActinomycinD (ActD). Because five of these genes were also expressed in somatic cells we also measured RNA decay in BY2 cells after transcriptional inhibition with ActD. Ten of the mRNAs were highly stable during pollen development (t1/2 > 30 h), including all seven pollen-specific mRNAs. However, two mRNAs showed a logarithmic decrease as pollen development proceeded, both in planta and in vitro. In fact, for one of these two, GUT8-2b, the mRNA level decreased significantly within 3 h. Our results show that many pollen-expressed mRNAs are indeed long-lived but that at least some mRNAs undergo decay during pollen maturation. Thus, there is no overall cytoplasmic condition in pollen that stabilizes mRNAs and specific mRNA turnover mechanisms must exist. We detected reciprocal mRNA stabilities with two genes that are expressed in both BY2 cells and pollen. GUT15 mRNA, known to be unstable (t1/2 < 3 h) in BY2 cells, was highly stable in pollen. In contrast, GRP2 mRNA decayed in pollen but was highly stable in BY2 cells. Therefore, we conclude that mRNA stability can be tissue-specific.

Journal Article↗