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PubMed2004

A phylogenetically based secondary structure for the yeast telomerase RNA.

BACKGROUND: Telomerase is a ribonucleoprotein complex whose RNA moiety dictates the addition of specific simple sequences onto chromosomes ends. While relevant for certain human genetic diseases, the contribution of the essential telomerase RNA to RNP assembly still remains unclear. Phylogenetic analyses of vertebrate and ciliate telomerase RNAs revealed conserved elements that potentially organize protein subunits for RNP function. In contrast, the yeast telomerase RNA could not be fitted to any known structural model, and the limited number of known sequences from Saccharomyces species did not permit the prediction of a yeast specific conserved structure. RESULTS: We cloned and analyzed the complete telomerase RNA loci (TLC1) from all known Saccharomyces species belonging to the "sensu stricto" group. Complementation analyses in S. cerevisiae and end mappings of mature RNAs ensured the relevance of the cloned sequences. By using phylogenetic comparative analysis coupled with in vitro enzymatic probing, we derived a secondary structure prediction of the Saccharomyces cerevisiae TLC1 RNA. This conserved secondary structure prediction includes a central domain that is likely to orchestrate DNA synthesis and at least two accessory domains important for RNA stability and telomerase recruitment. The structure also reveals a potential tertiary interaction between two loops in the central core. CONCLUSIONS: The predicted secondary structure of the TLC1 RNA of S. cerevisiae reveals a distinct folding pattern featuring well-separated but conserved functional elements. The predicted structure now allows for a detailed and rationally designed study to the structure-function relationships within the telomerase RNP-complex in a genetically tractable system.

Base Sequence↗

PubMed2004

Tris-borate is a poor counterion for RNA: a cautionary tale for RNA folding studies.

Native polyacrylamide gel electrophoresis is a powerful approach for visualizing RNA folding states and folding intermediates. Tris-borate has a high-buffering capacity and is therefore widely used in electrophoresis-based investigations of RNA structure and folding. However, the effectiveness of Tris-borate as a counterion for RNA has not been systematically investigated. In a recirculated Hepes/KCl buffer, the catalytic core of the bI5 group I intron RNA undergoes a conformational collapse characterized by a bulk transition midpoint, or Mg1/2, of approximately 3 mM, consistent with extensive independent biochemical experiments. In contrast, in Tris-borate, RNA collapse has a much smaller apparent Mg1/2, equal to 0.1 mM, because in this buffer the RNA undergoes a different, large amplitude, folding transition at low Mg2+ concentrations. Analysis of structural neighbors using a short-lived, RNA-tethered, photocrosslinker indicates that the global RNA structure eventually converges in the two buffer systems, as the divalent ion concentration approaches approximately 1 mM Mg2+. The weak capacity of Tris-borate to stabilize RNA folding may reflect relatively unfavorable interactions between the bulky Tris-borate ion and RNA or partial coordination of RNA functional groups by borate. Under some conditions, Tris-borate is a poor counterion for RNA and its use merits careful evaluation in RNA folding studies.

Base Sequence↗

PubMed1999

Post-transcriptional regulation of the parathyroid hormone gene by calcium and phosphate.

Parathyroid hormone messenger RNA levels are regulated by calcium, phosphate and 1,25-dihydroxyvitamin D3. Dietary induced hypocalcaemia increases and hypophosphataemia decreases parathyroid hormone mRNA levels post-transcriptionally. This regulation is mediated by binding of parathyroid cytosolic proteins to the parathyroid hormone mRNA 3'-untranslated region, and in particular the terminal 60 nucleotides of the 3'-untranslated region, thereby determining RNA stability.

3' Untranslated Regions↗

PubMed1993

Transcriptional and posttranscriptional regulation of human androgen receptor expression by androgen.

Autoregulation is a control mechanism common to several proteins of the steroid/thyroid hormone receptor superfamily. In this work, the effect of androgens and antiandrogens on the expression of the human androgen receptor (hAR) in prostate and breast cancer cell lines was studied. Northern blot analysis revealed a decrease in hAR steady state RNA levels in LNCaP cells by 3.3 nM of the synthetic androgen mibolerone. Maximal down-regulation of hAR RNA to 30% of control levels occurred 48 h after hormone addition. T47D breast cancer cells showed a similar effect with mibolerone, while hAR expression in normal skin fibroblasts did not respond to androgen treatment. As shown by nuclease S1 analysis, hAR transcripts initiate at three principal start sites, all of which are equally sensitive to androgen. Steroidal as well as nonsteroidal antiandrogens were capable of partially antagonizing androgen-mediated hAR RNA down-regulation in LNCaP and T47D cells, while not exerting a significant effect when administered alone. While hAR RNA stability was increased by hormone, nuclear run-on analysis revealed a 4-fold reduction of hAR gene transcription 96 h after androgen treatment. Although decreased hAR RNA levels did not coincide with a parallel decrease in AR protein levels, analysis of androgen-inducible reporter constructs demonstrated that prolonged androgen administration to cells results in a progressively impaired sensitivity of the intracellular androgen response mechanism. These results show that prolonged androgen exposure leads, besides its effect on hAR RNA levels, to functional inactivation of the AR. Thus, in vivo, posttranslational control of AR activity appears to be a novel mechanism of negative autoregulation of androgen effects on gene expression.

Androgens↗

PubMed2003

An intranuclear frame for chromatin compartmentalization and higher-order folding.

Recent ultrastructural, immunoelectron, and confocal microscopy observations done in our laboratory [Barboro et al. [2002] Exp. Cell. Res. 279:202-218] have confirmed that lamins and the nuclear mitotic apparatus protein (NuMA) are localized inside the interphase nucleus in a polymerized form. This provided evidence of the existence of a RNA stabilized lamin/NuMA frame, consisting of a web of thin ( approximately 3 and approximately 5 nm) lamin filaments to which NuMA is anchored mainly in the form of discrete islands, which might correspond to the minilattices described by Harborth et al. [1999] (EMBO. J. 18:1689-1700). In this article we propose that this scaffold is involved in the compartmentalization of both chromatin and functional domains and further determines the higher-order nuclear organization. This hypothesis is strongly supported by the scrutiny of different structural transitions which occur inside the nucleus, such as chromatin displacement and rearrangements, the collapse of the internal nuclear matrix after RNA digestion and the disruption of chromosome territories induced by RNase A and high salt treatment. All of these destructive events directly depend on the loss of the stabilizing effect exerted on the different levels of structural organization by the interaction of RNA with lamins and/or NuMA. Therefore, the integrity of nuclear RNA must be safeguarded as far as possible to isolate the matrix in the native form. This material will allow for the first time the unambiguous ultrastructural localization inside the INM of the components of the functional domains, so opening new avenues of investigation on the mechanisms of gene expression in eukaryotes.

Animals↗

PubMed1990

Stability of messenger RNA in postmortem human brains and construction of human brain cDNA libraries.

We studied stabilities of poly(A)(+)-RNA in postmortem mouse and human brains for up to 12 hours. The yields of total RNA were not changed significantly during postmortem periods either in mouse brains or human brains. Cell-specific cDNA probes were used to evaluate postmortem stability of poly(A)(+)-RNA in each cell type in the central nervous system. We used neuron-specific enolase (NSE), S-100 beta (S-100), and myelin-associated glycoprotein (MAG) for molecular markers of neuron, astrocyte, and oligodendrocyte, respectively. There was no detectable degradation of mRNAs coding for NSE, S-100, and MAG during the postmortem periods on Northern blot hybridization analyses. These results indicate that intact mRNAs expressed in neuron, astrocyte, or oligodendrocyte can be isolated from postmortem brains for up to 12 hours after death. Using poly(A)(+)-RNA thus isolated from two postmortem human brains, we constructed directional cDNA libraries and demonstrated the presence of full-length cDNAs for NSE, S-100, and MAG on Southern blot hybridization analysis. The present data should encourage studies on altered gene expressions in human brain in various neurologic diseases.

Aged↗

PubMed1994

An RNA-binding peptide from bovine immunodeficiency virus Tat protein recognizes an unusual RNA structure.

The human immunodeficiency virus (HIV) Tat protein binds specifically to an RNA hairpin, TAR, located at the 5' end of its mRNA. Tat uses a single arginine residue within a short region of basic amino acids to recognize a bulge region in TAR. Here we show that a 17 amino acid arginine-rich peptide from the bovine immunodeficiency virus (BIV) Tat protein also binds to an RNA hairpin at the 5' end of its mRNA (BIV TAR), but recognizes different structural features of the RNA. Mutagenesis, RNase mapping, and chemical interference experiments indicate that bulge and stem regions of BIV TAR are recognized simultaneously by the BIV peptide and that the RNA adopts an unusual structure. BIV Tat binds to its TAR site with high affinity and specificity and, unlike HIV Tat, does not appear to use cellular proteins to stabilize RNA binding in vivo. Thus, two related viral activators have evolved rather distinct ways to recognize their RNA targets.

Amino Acid Sequence↗

PubMed2003

Functional redundancy within roX1, a noncoding RNA involved in dosage compensation in Drosophila melanogaster.

Drosophila melanogaster males dosage compensate by twofold upregulation of the expression of genes on their single X chromosome. This process requires at least five proteins and two noncoding RNAs, roX1 and roX2, which paint the male X chromosome. We used a deletion analysis to search for functional RNA domains within roX1, assaying RNA stability, targeting of the MSL proteins to the X, and rescue of male viability in a roX1(-) roX2(-) mutant background. We found that deletion of 10% segments of the RNA did not dramatically reduce function in most cases, suggesting extensive internal redundancy. The 3' 600 nt of roX1 were most sensitive to mutations, affecting proper localization and 3' processing of the RNA. Disruption of an inverted repeat predicted to form a stem-loop structure was found partially responsible for the defects observed.

Animals↗

PubMed2001

A nucleus-encoded suppressor defines a new factor which can promote petD mRNA stability in the chloroplast of Chlamydomonas reinhardtii.

Mutations in the Chlamydomonas reinhardtii nuclear gene MCD1 specifically destabilize the chloroplast petD mRNA, which encodes subunit IV of the cytochrome b6/f complex. The MCD1 gene product is thought to interact with the mRNA 5' end to protect it from degradation by a 5' --> 3' exoribonuclease and may also have a role in translation initiation. Here we report the isolation and characterization of a semidominant, allele-specific, nucleus-encoded suppressor of the mcd1-2 mutation. The suppressor mutation, which defines a new locus MCD2, allows accumulation of 10% of the wild-type level of petD mRNA and as much as 50% of the wild-type subunit IV level. Taken together, these results suggest the suppressor mutation restores photosynthetic growth by stabilizing petD mRNA. In addition, it may promote increased translational efficiency, an inference supported by direct measurements of the subunit IV synthesis rate. Thus, both MCD1 and MCD2 may participate in both chloroplast RNA stability and translation initiation.

Animals↗

PubMed2002

The conformationally flexible S9-S10 linker region in the core domain of p53 contains a novel MDM2 binding site whose mutation increases ubiquitination of p53 in vivo.

Although the N-terminal BOX-I domain of the tumor suppressor protein p53 contains the primary docking site for MDM2, previous studies demonstrated that RNA stabilizes the MDM2.p53 complex using a p53 mutant lacking the BOX-I motif. In vitro assays measuring the specific activity of MDM2 in the ligand-free and RNA-bound state identified a novel MDM2 interaction site in the core domain of p53. As defined using phage-peptide display, the RNA.MDM2 isoform exhibited a notable switch in peptide binding specificity, with enhanced affinity for novel peptide sequences in either p53 or small nuclear ribonucleoprotein-U (snRNP-U) and substantially reduced affinity for the primary p53 binding site in the BOX-I domain. The consensus binding site for the RNA.MDM2 complex within p53 is SGXLLGESXF, which links the S9-S10 beta-sheets flanking the BOX-IV and BOX-V motifs in the core domain and which is a site of reversible conformational flexibility in p53. Mutation of conserved amino acids in the linker at Ser(261) and Leu(264), which bridges the S9-S10 beta-sheets, stimulated p53 activity from reporter templates and increased MDM2-dependent ubiquitination of p53. Furthermore, mutation of the conserved Phe(270) within the S10 beta-sheet resulted in a mutant p53, which binds more stably to RNA.MDM2 complexes in vitro and which is strikingly hyper-ubiquitinated in vivo. Introducing an Ala(19) mutation into the p53(F270A) protein abolished both RNA.MDM2 complex binding and hyper-ubiquitination in vivo, thus indicating that p53(F270A) protein hyper-ubiquitination depends upon MDM2 binding to its primary site in the BOX-I domain. Together, these data identify a novel MDM2 binding interface within the S9-S10 beta-sheet region of p53 that plays a regulatory role in modulating the rate of MDM2-dependent ubiquitination of p53 in cells.

Alleles↗

PubMed2003

mRNA capping enzyme requirement for Caenorhabditis elegans viability.

Capping of the initiated 5' ends of RNA polymerase II products is evolutionarily and functionally conserved from yeasts to humans. The m(7)GpppN cap promotes RNA stability, processing, transport, and translation. Deletion of capping enzymes in yeasts was shown to be lethal due to rapid exonucleolytic degradation of uncapped transcripts or failure of capped but unmethylated RNA to initiate protein synthesis. Using RNA interference and Caenorhabditis elegans we have found that RNA capping is also essential for metazoan viability. C. elegans bifunctional capping enzyme was cloned, and capping activity by the expressed protein as well as growth complementation of yeast deletion strains missing either RNA triphosphatase or guanylyltransferase required terminal sequences not present in the previously isolated cel-1 clone. By RNA interference analysis we show that cel-1 is required for embryogenesis. cel-1(RNAi) embryos formed cytoplasmic granules characteristic of a phenocluster of RNA processing genes and died early in development.

Acid Anhydride Hydrolases↗

PubMed1984

Transcription of intracisternal A-particle genes in mouse myeloma and Ltk- cells.

In vivo- and in vitro-synthesized RNA from the murine plasmacytoma MOPC-315 was found to hybridize to all regions of a 7.2-kilobase-pair intracisternal A-particle (IAP) gene. IAP-specific transcripts were also detected in mouse Ltk- cells, but not in cells derived from normal tissues (kidney, liver, spleen) of 6-week-old BALB/c mice. Three RNA species of 7.2, 5.3, and 3.8 kilobases were identified by Northern blot analysis of MOPC-315 polyadenylated RNA. The 7.2- and 5.3-kilobase transcripts were found in greater levels in nuclear as compared with whole cell RNA, suggesting the involvement of one or more of the following mechanisms: RNA processing, preferential nuclear transport, or differential RNA stability. We show that the primary IAP transcript is initiated within the long terminal repeat by hybridization analysis with restriction digests of cloned IAP DNA and [gamma-S]pppApNp ... RNA synthesized in nuclei with [gamma-S]ATP as the RNA initiating probe. Low concentrations of alpha-amanitin (2 micrograms/ml) inhibited IAP RNA synthesis by greater than 90%, suggesting that RNA polymerase II is responsible for IAP transcription.

Animals↗

PubMed1997

Efficient hammerhead ribozymes targeted to the polycistronic Sendai virus P/C mRNA. Structure-function relationships.

The Sendai virus polycistronic P/C mRNA encodes the P and C proteins from alternate overlapping reading frames. To determine the functions of these proteins in virus replication, hammerhead ribozymes were targeted to cleave the 5'-untranslated region of the P/C mRNA. Both cell-free and intracellular assays were employed to determine ribozyme efficacy. To appropriately compare activities between cell-free and intracellular assays, identical ribozymes were synthesized in vitro as well as expressed in cells. Ribozyme parameters, namely hybridization arm length (HAL) and nonhybridizing extraneous sequences (NES), were found to have rate-determining properties. In cell-free reactions, ribozymes with 13-mer HAL were up to 10-fold more efficient than those with 9-mer HAL. Ribozymes with 9-mer HAL were relatively ineffective in transfected cells. Minimizing the number of NES increased ribozyme efficiency in vitro. However, ribozymes with minimal NES were essentially inert intracellularly. The NES at the termini of the most effective intracellular ribozyme, Rz13st ( approximately 95% inhibition of the p gene expression), were predicted to fold into stem-loop structures. These structures most likely increase ribozyme stability as evidenced by the 8-fold higher resistance to ribonuclease T2 digestion of Rz13st compared with Rz13B. Our results suggest that when designing effective intracellular ribozymes, parameters that enhance formation of productive ribozyme:substrate duplexes and that increase RNA stability should be optimized.

Base Sequence↗

PubMed2002

The Chlamydomonas reinhardtii organellar genomes respond transcriptionally and post-transcriptionally to abiotic stimuli.

The Chlamydomonas reinhardtii plastid and mitochondrial transcriptomes were surveyed for changes in RNA profiles resulting from growth in 12 culture conditions representing 8 abiotic stimuli. Organellar RNA abundance exhibited marked changes during nutrient stress and exposure to UV light, as revealed by both RNA gel blot and DNA microarray analyses. Of particular note were large increases in tufA and clpP transcript abundance during nutrient limitation. Phosphate and sulfur limitation resulted in the most global, yet opposite, effects on organellar RNA abundance, changes that were dissected further using run-on transcription assays. Removal of sulfate from the culture medium, which is known to reduce photosynthesis, resulted in 2-fold to 10-fold decreases in transcription rates, which were reflected in lower RNA abundance. The decrease in transcriptional activity was completely reversible and recovered to twice the control level after sulfate replenishment. Conversely, phosphate limitation resulted in a twofold to threefold increase in RNA abundance that was found to be a post-transcriptional effect, because it could be accounted for by increased RNA stability. This finding is consistent with the known metabolic slowdown under phosphate stress. Additionally, inhibitor studies suggested that unlike those in higher plants, Chlamydomonas chloroplasts lack a nucleus-encoded plastid RNA polymerase. The apparently single type of polymerase could contribute to the rapid and genome-wide transcriptional responses observed within the chloroplast.

Animals↗

PubMed1995

The sequence and context of the 5' splice site govern the nuclear stability of polyoma virus late RNAs.

We have examined the influence of splicing signals on the stability of polyoma virus late RNAs in the nucleus. Late primary transcripts contain a single 5' splice site and three alternative 3' splice sites. In earlier work we showed that the presence of introns was not required for late RNA accumulation, however, the 5' splice site was essential, as removal of only the 5' splice site was sufficient to destabilize late RNAs up to 100-fold when compared with early RNAs. A complementary clone which retained the 5' splice site but which carried small deletions of all late region 3' splice sites produced wild-type levels of unspliced late RNA. In order to extend this work we have constructed additional types of mutants. Point mutations in the 5' splice site confirmed its importance for RNA stability. Other mutants included constructs in which the spacing between the 5' splice site and the late promoter was altered and 5' splice site insertion mutants where a 58 bp fragment containing the 5' splice site sequence was inserted separately at various restriction sites in the late region. Both types of mutants lacked all of the late 3' splice sites and had only a single 5' splice site. RNase protection analyses of late and early RNAs from these constructs revealed that moving the 5' splice site away from the late promoter (or from its normal context) destabilized late RNAs > 10-fold relative to the wild-type. We conclude that both 5' splice site integrity and its proximity to the late promoter play important roles in the nuclear stability of polyoma virus late RNAs.

3T3 Cells↗

PubMed1999

Derivation of RNA aptamer inhibitors of human complement C5.

Specific aptamer inhibitors of the human complement C5 component were produced by the SELEX methodology of directed evolution of nucleic acid ligands. The SELEX procedure started with a pool of random-sequence, 2'F-pyrimidine-modified nuclease-stabilized RNA, and after twelve rounds of iterative C5 binding and nucleic acid amplification an evolved RNA pool was obtained which contained the highest affinity binders to the C5 protein. The evolved RNA pool was then cloned and sequenced, and individual clones were analyzed for binding and function. Twenty-eight clones (out of sixty) were identified which bound C5 (termed aptamers). Seven of these aptamers formed a closely related sequence homology family; these aptamers bound C5 with a Kd 20-40 nM and also inhibited human serum hemolytic activity. In addition, these aptamers inhibited zymosan-induced generation of C5a. Aptamer inhibition of both C5b and C5a suggests that aptamer binding inhibits cleavage of C5 by the C5 convertase of both pathways. One of the inhibitory aptamer sequences was truncated to yield a 38-mer 2'F RNA aptamer which retained C5 binding and inhibitory activity. The structure of this aptamer is predicted to be a stem-loop containing thirteen base pairs, and also containing two bulges. The affinity of this aptamer was improved by performing a second biased SELEX experiment, where the randomized starting RNA pool uses a template where the individual base compositions are biased toward a specific sequence. This second SELEX experiment produced an aptamer with a Kd of 2-5 nM which retained functional activity. Another SELEX to rat C5 produced an aptamer with binding and inhibitory properties virtually identical with the human aptamer. The human and rat aptamers are being evaluated for complement inhibition in vitro and in vivo as potential therapeutics for treatment of human disease.

Animals↗

PubMed2006

Fine-needle aspiration biopsy of non-Hodgkin lymphoma for use in expression microarray analysis.

BACKGROUND: The majority of patients with lymphoma undergo a single biopsy for diagnosis, and there are few opportunities to acquire posttreatment material. Fine-needle aspiration (FNA) biopsy is a minimally invasive procedure, and acquiring routine posttreatment material would require minimal effort and provide needed material for gene expression profiling. METHODS: Ex vivo FNA biopsies were performed using a standard clinical technique with 21-gauge, 22-gauge, 23-gauge, and 25-gauge needles for 8 lymph node specimens and were collected in either RNA stabilization reagent (RNAlater) or Trizol. Eight patients with known or suspected Non-Hodgkin lymphoma (NHL) underwent interoperative (in vivo) FNA biopsies based on the best technique derived from the ex vivo aspirates. RNA derived from the in vivo FNA biopsies and the matched, snap-frozen surgical lymph node biopsy remnant was used for gene expression analysis with proprietary U133A chips. RESULTS: The results confirmed the authors' experience, that RNA isolated from FNA biopsies of lymph nodes collected in Trizol is superior both quantitatively and qualitatively to RNA collected in RNAlater. Gene expression profiles of NHL derived from in vivo FNA biopsies and matched, frozen surgical specimens showed good overall correlation. CONCLUSIONS: High-throughput gene expression analysis in patients with NHL derived from material acquired by FNA biopsy is a feasible approach to developing a platform for real-time analysis of treatment responses in this group of patients.

Biopsy, Fine-Needle↗

PubMed1994

Effect of 5-fluoro- and 5-bromouracil substitution on the translation of human thymidylate synthase mRNA.

The synthesis of thymidylate synthase (TS) from 5-fluorouracil (FUra)- and 5-bromouracil (BrUra)-substituted mRNAs was examined to investigate the effect of incorporation of uracil (Ura) analogs on translation. Human TS cDNA was transcribed in the presence of Ura-, FUra-, or BrUTP to obtain 100% substituted mRNA. The mRNAs were translated in a rabbit reticulocyte lysate system. The TS protein that was formed from each of the templates reacted identically with TS antibody in Western blots. Time courses of TS formation revealed a characteristic peak which occurred at 45 min for the Ura- and FUra-RNAs and at 2 h for the BrUra-RNA. Substitution of Ura with FUra did not alter the rate of translation, while substitution of BrU for Ura decreased the rate of translation. Substitution of Ura with FUra or BrUra enhanced the stability of the mRNAs in the rabbit reticulocyte lysate by 3- and 10-fold, respectively. Incorporation of BrUra influenced the binding and catalysis on the ribosome, resulting in a 3.5-fold greater rate of activation (Kact) and 6-fold lower Vmax than the equivalent values for the Ura- and FUra-substituted mRNAs. Nondenaturing gel electrophoresis revealed that different conformations exist among the mRNAs. These data show that translation can be influenced by the incorporation of fraudulent bases into mRNA and those bases that stabilize RNA secondary structure will have the greatest inhibitory effect on translation.

Base Sequence↗