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At least 397 records · Page 22Linked to original sources
PubMed2001

Retroviral RNA elements integrate components of post-transcriptional gene expression.

Retroviruses vary widely in their ability to cause neoplastic transformation or immunodeficiency, and may even lack pathogenicity, but all retroviruses require cytoplasmic expression of intron-containing mRNA. In the cytoplasm, the primary viral transcript has two essential roles as mRNA template for protein synthesis and as genomic RNA for packaging into progeny virions. Cellular proteins are used by the virus to modulate synthesis, processing, and translation of the viral RNA. To subvert the normal RNA processing cascade and achieve nuclear export of intron-containing viral RNA, retroviruses utilize structured RNA elements and viral or cellular protein partners. These nuclear interactions determine the cytoplasmic fate of viral RNAs by facilitating RNA stability, nuclear export, translational efficiency, and even assembly of progeny virions. The HIV Rev responsive element (RRE) and Rev protein have been a informative paradigm for dissection of the process of eukaryotic RNA nuclear export. Rev is an adapter protein that bridges RRE-containing RNA and the CRM1 nuclear export receptor, which delivers intron-containing RNA to a nuclear export pathway typically used for 5s rRNA and protein transport. This review summarizes data indicating that Rev/RRE also targets cytoplasmic transcripts to the cytoskeletal polysomes and activates their translational efficiency. The interesting parallel is discussed that genetically simpler retroviruses lack a Rev-like protein and recruit cellular proteins to distinct RNA elements that modulate post-transcriptional gene expression through different export pathways. These pathways include the global mRNA export pathway mediated by Tap, and Tap- and CRM1-independent pathways. The CRM1-independent nuclear export pathway accessed by the spleen necrosis virus post-transcriptional control element is functionally linked to RU5-mediated translational enhancement in the cytoplasm. The simple retroviral post-transcriptional control elements also modulate RNA splicing efficiency, stability, assembly of virions, and subsequent viral egress from the cell. Thus, multiple layers of post-transcriptional control are executed by these retroviral RNA elements, which serve as a compact platform for interaction with nuclear and possibly cytoplasmic protein partners. Further characterization of the cellular partners and their regulation will be an important step to full understanding of nuclear-cytoplasmic connections that hardwire post-transcriptional gene expression in eukaryotic cells.

Active Transport, Cell Nucleus↗
PubMed2006

Unusual mechanical stability of a minimal RNA kissing complex.

By using optical tweezers, we have investigated the mechanical unfolding of a minimal kissing complex with only two G.C base pairs. The loop-loop interaction is exceptionally stable; it is disrupted at forces ranging from 7 to 30 pN, as compared with 14-20 pN for unfolding hairpins of 7 and 11 bp. By monitoring unfolding/folding trajectories of single molecules, we resolved the intermediates, measured their rate constants, and pinpointed the rate-limiting steps. The two hairpins unfold only after breaking the intramolecular kissing interaction, and the kissing interaction forms only after the folding of the hairpins. At forces that favor the unfolding of the hairpins, the entire RNA structure is kinetically stabilized by the kissing interaction, and extra work is required to unfold the metastable hairpins. The strong mechanical stability of even a minimal kissing complex indicates the importance of such loop-loop interactions in initiating and stabilizing RNA dimers in retroviruses.

Base Sequence↗
PubMed2025

Bundle sheath cell-specific expression of chloroplast genes encoding subunits of the NADH dehydrogenase-like complex in maize.

C4 photosynthesis alleviates the limitation caused by the oxygenase activity of Rubisco by partitioning photosynthetic functions between two distinct cell types: bundle sheath cells (BSCs) and mesophyll cells (MCs). These cell types perform different steps of photosynthesis using specialized machinery, accompanied by differential expression of chloroplast genes. To uncover the underlying molecular mechanisms for this differentiation, we isolated BSCs and MCs and compared their chloroplast transcriptomes, focusing on the chloroplast NADH dehydrogenase-like (NDH) complex, which is enriched in BSCs. To investigate whether RNA stabilization contributes to differential gene expression, we analyzed RNA footprints that reflect the binding of pentatricopeptide repeat (PPR) proteins to their RNA targets. We could not detect cell-type-specific accumulation of footprint RNAs. We then focused on transcriptional regulation, specifically on an operon that starts with the rps15 gene. The operon includes six ndh genes and the psaC gene encoding a photosystem I subunit. Transcript levels of all genes in this operon were higher in BSCs than in MCs, suggesting coordinated regulation as a transcriptional unit. Based on the genomic location of the rps15 gene within inverted repeats near the junctions on both sides of the small single copy region, we demonstrated that rps15, through two distinct promoters, is sufficient to drive preferential accumulation of downstream transcripts in BSCs.

Zea mays↗
PubMed1993

Adenosine and a related carbocyclic nucleoside analogue selectively inhibit tumor necrosis factor-alpha production and protect mice against endotoxin challenge.

Adenosine (ADO) and its structurally related analogues are known to regulate the activities of immune and inflammatory cells, including a number of key functions of mononuclear phagocytes. In this study ADO and the synthetic ADO analogue MDL201112 inhibited TNF-alpha, but not IL-1, production by activated mouse peritoneal macrophages and the macrophage-like cell lines J774 and RAW-264. Northern blot analysis indicated that MDL201112 selectively inhibited the expression of steady-state TNF-alpha RNA in LPS+IFN-gamma-activated J774 and RAW-264 cells. This effect could not be attributed to changes in TNF-alpha RNA stability. In contrast, ADO had no effect on RNA levels for TNF-alpha and IL-1, suggesting that ADO acts at a post-transcriptional biosynthetic step. To determine whether either compound inhibited TNF-alpha-mediated inflammatory responses, mice were treated with ADO or MDL201112 and challenged with a lethal dose of endotoxic LPS and D-galactosamine, an hepatotoxin that sensitizes mice to lethal LPS challenge. A single i.p. injection of MDL201112 (100 mg/kg) protected over 90% of the mice, whether injected 1 h before or at the time of LPS challenge. MDL201112 also inhibited the appearance of TNF-alpha in the serum of LPS-challenged animals. The compound did not block D-galactosamine sensitization nor did it prevent lethality caused by the injection of rTNF-alpha. ADO failed to protect animals against endotoxin lethality, most likely due to the rapid metabolism of the nucleoside in vivo. These results establish ADO and MDL201112 as potent inhibitors of TNF-alpha biosynthesis and suggest that MDL201112 or similar analogues warrant further study as potential agents for the treatment of endotoxin shock and other diseases in which TNF-alpha plays an important pathogenic role.

Adenine↗
PubMed1999

An important base triple anchors the substrate helix recognition surface within the Tetrahymena ribozyme active site.

Key to understanding the structural biology of catalytic RNA is determining the underlying networks of interactions that stabilize RNA folding, substrate binding, and catalysis. Here we demonstrate the existence and functional importance of a Hoogsteen base triple (U300.A97-U277), which anchors the substrate helix recognition surface within the Tetrahymena group I ribozyme active site. Nucleotide analog interference suppression analysis of the interacting functional groups shows that the U300.A97-U277 triple forms part of a network of hydrogen bonds that connect the P3 helix, the J8/7 strand, and the P1 substrate helix. Product binding and substrate cleavage kinetics experiments performed on mutant ribozymes that lack this base triple (C A-U, U G-C) or replace it with the isomorphous C(+).G-C triple show that the A97 Hoogsteen triple contributes to the stabilization of both substrate helix docking and the conformation of the ribozyme's active site. The U300. A97-U277 base triple is not formed in the recently reported crystallographic model of a portion of the group I intron, despite the presence of J8/7 and P3 in the RNA construct [Golden, B. L., Gooding, A. R., Podell, E. R. & Cech, T. R. (1998) Science 282, 259-264]. This, along with other biochemical evidence, suggests that the active site in the crystallized form of the ribozyme is not fully preorganized and that substantial rearrangement may be required for substrate helix docking and catalysis.

Animals↗
PubMed1994

Suppression of mutations in the core of the Tetrahymena ribozyme by spermidine, ethanol and by substrate stabilization.

We have previously described a collection of mutations in conserved residues of the core of the Tetrahymena self-splicing intron. Most of these single base substitutions have less than 10% of the activity of their parental intron derivative [Couture, S., et al., (1990) J. Mol. Biol., 215, 345-358]. We examined the effect of two agents known to stabilize RNA structure, spermidine and ethanol, on the activity of many of these mutant RNAs. In the presence of either 5 mM spermidine or 20% ethanol most substitution mutations were partially or completely suppressed. These conditions also increased the temperature optima of both wild-type and mutant ribozymes. In addition, we find that mutations are also suppressed by a high concentration of GTP, a substrate in the reaction which is bound specifically by the intron. Thus we observe a general suppression of mutations in an RNA enzyme (ribozyme) by spermidine, ethanol and by substrate stabilization. These results are consistent with the idea that most mutations destabilize the folded structure of the ribozyme and can be suppressed by any of a variety of stabilizing influences.

Animals↗
PubMed1995

Differential Transcription of Pea Chloroplast Genes during Light-Induced Leaf Development (Continuous Far-Red Light Activates Chloroplast Transcription).

Plastid gene expression was analyzed in pea (Pisum sativum L.) plants grown in darkness, continuous far-red light, and white light. Responses induced by continuous far-red light were most likely mediated by PHYA. Plastid transcription activity was low in dark-grown plants. In contrast, plastids of plants grown in white or far-red light showed a 10-fold increase in transcription activity between 4 and 6 d postimbibition (dpi) and a decrease between 6 and 9 dpi. Plastid RNAs accumulated in illuminated plants from 5 to 7 dpi. In far-red-light-illuminated plants, plastid mRNA levels remained elevated until 14 dpi. In white-light-grown plants, most plastid RNAs decreased in abundance after 7 dpi to very low levels by 14 dpi. This indicates that white light induces a general decrease in plastid RNA stability compared to far-red-light-illuminated seedlings. PsbA mRNA accumulated in older, dark-grown, far-red, and white-light-illuminated seedlings, consistent with this RNA having high stability. Transcription of genes encoding the plastid's transcription and translation apparatus increased relative to rbcL and other genes encoding proteins of the photosynthetic apparatus from 4 to 5 dpi and then declined 10-fold from 5 to 9 dpi. These data document dynamic modulation of plastid gene transcription and mRNA stability during light-induced chloroplast development in pea.

Journal Article↗
PubMed1995

Comparison of Milli-Q PF Plus water to DEPC-treated water in the preparation and analysis of RNA.

The Milli-Q PF Plus water-polishing system is equipped with high-purity ion and organic removal media and a capillary fiber ultrafiltration device. The system produces ultrapure water practically free of ribonuclease contamination. The necessity for diethyl pyrocarbonate (DEPC)-treated solutions in RNA molecular biological procedures was tested by preparing RNA from a variety of tissues and tissue-cultured cells using either DEPC-treated, autoclaved solutions or pure Milli-Q PF water dispensed directly from the system. Tissue sources included rabbit brain, heart, lung, liver kidney and bladder as well as cultured human corpus cavernosum smooth muscle cells. RNA was prepared by guanidinium isothiocyanate solubilization, phenol/chloroform extraction and isopropanol precipitation followed by Northern blot analysis. Hybridization with fibronectin (ca. 7.6 kb) and glyceraldehyde-3-phosphate dehydrogenase (1.2 kb) revealed that water from a Milli-Q PF water system performed as well as DEPC-treated, autoclaved solutions. RNA stability at 37 degrees C was examined for various times using rabbit lung RNA in either DEPC-treated water for Milli-Q PF water. Intact RNA was detected after 6 hours in total RNA and by Northern blots hybridized with fibronectin. There was no significant difference in RNA degradation between DEPC-treated water and Milli-Q PF water. We conclude that Milli-Q PF water is an acceptable substitute for DEPC-treated water for the preparation of RNA and Northern blot analysis.

Animals↗
PubMed2001

Umbravirus-encoded proteins both stabilize heterologous viral RNA and mediate its systemic movement in some plant species.

The proteins encoded by open reading frame 3 (ORF3) of the umbraviruses pea enation mosaic virus-2 and tobacco mottle virus, like that of groundnut rosette virus, mediated the movement of viral RNA through the phloem of infected Nicotiana benthamiana or N. clevelandii plants when they were expressed from chimeric tobacco mosaic virus in place of the coat protein. However, these chimeras did not move systemically in N. tabacum. In lysates of N. benthamiana or N. tabacum protoplasts, the chimeric RNAs were more stable than was RNA of tobacco mosaic virus lacking the coat protein gene. The chimeric viruses also protected the latter in trans, suggesting that the ORF3 proteins can increase the stability of heterologous viral RNA. Umbraviral ORF3 proteins contain a conserved arginine-rich domain, and the possible roles of this motif in the functions of the proteins are discussed.

Amino Acid Sequence↗
PubMed2006

Calculation of pKas in RNA: on the structural origins and functional roles of protonated nucleotides.

pK(a) calculations based on the Poisson-Boltzmann equation have been widely used to study proteins and, more recently, DNA. However, much less attention has been paid to the calculation of pK(a) shifts in RNA. There is accumulating evidence that protonated nucleotides can stabilize RNA structure and participate in enzyme catalysis within ribozymes. Here, we calculate the pK(a) shifts of nucleotides in RNA structures using numerical solutions to the Poisson-Boltzmann equation. We find that significant shifts are predicted for several nucleotides in two catalytic RNAs, the hairpin ribozyme and the hepatitis delta virus ribozyme, and that the shifts are likely to be related to their functions. We explore how different structural environments shift the pK(a)s of nucleotides from their solution values. RNA structures appear to use two basic strategies to shift pK(a)s: (a) the formation of compact structural motifs with structurally-conserved, electrostatic interactions; and (b) the arrangement of the phosphodiester backbone to focus negative electrostatic potential in specific regions.

Catalysis↗
PubMed1992

Ternary complex formation by vaccinia virus RNA polymerase at an early viral promoter: analysis by native gel electrophoresis.

We have resolved, by native gel electrophoresis, two intermediates in the transcription of a vaccinia virus early gene by the virus-encoded RNA polymerase. Polymerase holoenzyme containing the vaccinia virus early transcription factor (VETF) forms a complex of VETF bound to the promoter as the first step in a pathway leading to establishment of a committed ternary elongation complex. Formation of the VETF-DNA complex is stimulated by magnesium but is uninfluenced by nucleoside triphosphates. A stable binary complex of RNA polymerase bound to DNA is not detected. Assembly of a gel-stable polymerase-DNA complex depends on conditions permissive for RNA synthesis. Nucleotide omission experiments suggest that at least a tetrameric RNA must be made before a ternary complex is stabilized. RNA analysis indicates that complexes containing nascent transcripts 20 nucleotides long are stable and active. Ternary complex formation requires hydrolyzable ATP. This is consistent with an essential role for the ATPase activity of VETF at a step subsequent to DNA binding, as proposed by Broyles (S. S. Broyles, J. Biol. Chem. 266:15545-15548, 1991). The ternary complex, once formed, is resistant to dissociation by competitor DNA, as well as by salt, Sarkosyl, and heparin. The effects of these inhibitory agents on transcription complex formation suggest that they target different steps in the assembly pathway.

Base Sequence↗
PubMed2002

Increased yield of total RNA from fine-needle aspirates for use in expression microarray analysis.

Fine-needle aspirate samples hold the potential for gaining valuable insight into the molecular details and prognostic indicators for certain types of cancer in a limited volume of relatively pure tumor cells. Although limited, such clinical samples can be used with high efficiency when analyzed in conjunction with gene-dense expression microarrays. For this reason, it is essential to retrieve as much high-quality genetic material as possible from each fine-needle aspirate sample. We have conducted a study to improve the efficiency of extracting high quality total RNA to use in microarray analysis from single ex vivo fine-needle aspirate samples of 11 breast cancers added to RNAlater RNA Stabilization Reagent immediately upon collection. Approximately half the total RNA from fine-needle aspirate samples of breast cancers was isolated from the supernatant, and that RNA had similar quality and gene expression profile to the RNA that was isolated from the corresponding cell pellet. We recommend that the supernatant not be discarded when extracting RNA from fine-needle aspirate samples stored in RNAlater.

Biopsy, Needle↗
PubMed2000

The 8-nucleotide-long RNA:DNA hybrid is a primary stability determinant of the RNA polymerase II elongation complex.

The sliding clamp model of transcription processivity, based on extensive studies of Escherichia coli RNA polymerase, suggests that formation of a stable elongation complex requires two distinct nucleic acid components: an 8-9-nt transcript-template hybrid, and a DNA duplex immediately downstream from the hybrid. Here, we address the minimal composition of the processive elongation complex in the eukaryotes by developing a method for promoter-independent assembly of functional elongation complex of S. cerevisiae RNA polymerase II from synthetic DNA and RNA oligonucleotides. We show that only one of the nucleic acid components, the 8-nt RNA:DNA hybrid, is necessary for the formation of a stable elongation complex with RNA polymerase II. The double-strand DNA upstream and downstream of the hybrid does not affect stability of the elongation complex. This finding reveals a significant difference in processivity determinants of RNA polymerase II and E. coli RNA polymerase. In addition, using the imperfect RNA:DNA hybrid disturbed by the mismatches in the RNA, we show that nontemplate DNA strand may reduce the elongation complex stability via the reduction of the RNA:DNA hybrid length. The structure of a "minimal stable" elongation complex suggests a key role of the RNA:DNA hybrid in RNA polymerase II processivity.

Amino Acid Sequence↗
PubMed1997

Stabilization of Xist RNA mediates initiation of X chromosome inactivation.

The onset of X inactivation is preceded by a marked increase in the level of Xist RNA. Here we demonstrate that increased stability of Xist RNA is the primary determinant of developmental up-regulation. Unstable transcript is produced by both alleles in XX ES cells and in XX embryos prior to the onset of random X inactivation. Following differentiation, transcription of unstable RNA from the active X chromosome allele continues for a period following stabilization and accumulation of transcript on the inactive X allele. We discuss the implications of these findings in terms of models for the initiation of random and imprinted X inactivation.

Alleles↗
PubMed1992

Characterization of an internal element in turnip crinkle virus RNA involved in both coat protein binding and replication.

The major coat-protein-binding element of turnip crinkle virus RNA was previously mapped in the region of the UAG termination codon in the viral polymerase gene. This region encompasses two of the high-affinity coat-protein-binding sites (Fa and Ff) that we suggested were physically associated in a stem-loop in a ribonucleoprotein complex involved in assembly initiation (Wei, Heaton, Morris, and Harrison, J. Mol. Biol. 214, 85-95, 1990). We have also demonstrated that this RNA element was capable of specific coat protein binding in vitro (Wei and Morris, J. Mol. Biol. 222, 437-443, 1991). We now provide physical evidence, by in vitro chemical and enzymatic probing of the viral RNA, that support the suggestion that the two coat-protein-binding sites base pair to form a stem structure (A/F stem) surrounding the UAG terminator in wild-type RNA. We have shown here that a mutant with seven conservative nucleotide substitutions in Fa does not accumulate to detectable levels in plants or protoplasts and that the A/F stem structure is drastically altered in this mutant. We suggest that the primary effect of this mutation is on replication rather than on a reduction in RNA stability resulting from a defect in encapsidation of the virion RNA because previous results have shown that encapsidation-deficient mutants have little or no effect on viral RNA replication (Hacker, Petty, Wei, and Morris, Virology 186, 1-8, 1992). The analysis of the A/F stem was extended by construction and characterization of a series of mutants and revertants that displayed variable levels of replication deficiency but minimal concomitant defect in encapsidation efficiency. The extent of the replication defect correlated with the predicted destabilization of the A/F stem structure. We conclude from these results that this RNA element is involved in viral replication, and we tentatively suggest that the A/F stem structure may be functionally involved in the readthrough translation of the viral polymerase.

Base Sequence↗
PubMed2002

Stage-specific gene expression during sexual development in Phytophthora infestans.

Eight genes that are upregulated during sexual development in the heterothallic oomycete, Phytophthora infestans, were identified by suppression subtractive hybridization. Two genes showed very low but detectable expression in vegetative hyphae and became induced about 40- to >100-fold early in mating, before gametangial initials appeared. The remaining six loci were not induced until later in mating, coincident with the formation of gametangia and oospores, with induction levels ranging from 60- to >100-fold. Five genes were single copy, and three were members of families. Sequence analysis revealed that the predicted products of three of the genes had similarity to proteins that influence RNA stability, namely a ribonuclease activator, the pumilio family of RNA-binding proteins and RNase H. The products of two other mating-induced genes resembled two types of Phytophthora proteins previously shown to elicit plant defence responses. Each mating-induced gene was also expressed in a self-fertile strain, which was shown to be a heterokaryon. However, quantitative and qualitative differences existed in their expression in normal matings and in the self-fertile heterokaryon. Besides the mating-induced genes, two extrachromosomal RNA elements were identified.

Amino Acid Sequence↗
PubMed2006

Identification of eukaryotic open reading frames in metagenomic cDNA libraries made from environmental samples.

Here we describe the application of metagenomic technologies to construct cDNA libraries from RNA isolated from environmental samples. RNAlater (Ambion) was shown to stabilize RNA in environmental samples for periods of at least 3 months at -20 degrees C. Protocols for library construction were established on total RNA extracted from Acanthamoeba polyphaga trophozoites. The methodology was then used on algal mats from geothermal hot springs in Tengchong county, Yunnan Province, People's Republic of China, and activated sludge from a sewage treatment plant in Leicestershire, United Kingdom. The Tenchong libraries were dominated by RNA from prokaryotes, reflecting the mainly prokaryote microbial composition. The majority of these clones resulted from rRNA; only a few appeared to be derived from mRNA. In contrast, many clones from the activated sludge library had significant similarity to eukaryote mRNA-encoded protein sequences. A library was also made using polyadenylated RNA isolated from total RNA from activated sludge; many more clones in this library were related to eukaryotic mRNA sequences and proteins. Open reading frames (ORFs) up to 378 amino acids in size could be identified. Some resembled known proteins over their full length, e.g., 36% match to cystatin, 49% match to ribosomal protein L32, 63% match to ribosomal protein S16, 70% to CPC2 protein. The methodology described here permits the polyadenylated transcriptome to be isolated from environmental samples with no knowledge of the identity of the microorganisms in the sample or the necessity to culture them. It has many uses, including the identification of novel eukaryotic ORFs encoding proteins and enzymes.

Acanthamoeba↗