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PubMed2004

Polyamine derivatives as selective RNaseA mimics.

Site-selective scission of ribonucleic acids (RNAs) has attracted considerable interest, since RNA is an intermediate in gene expression and the genetic material of many pathogenic viruses. Polyamine-imidazole conjugates for site-selective RNA scission, without free imidazole, were synthesized and tested on yeast phenylalanine transfer RNA. These molecules catalyze RNA hydrolysis non-randomly. Within the polyamine chain, the location of the imidazole residue, the numbers of nitrogen atoms and their relative distances have notable influence on cleavage selectivity. A norspermine derivative reduces the cleavage sites to a unique location, in the anticodon loop of the tRNA, in the absence of complementary sequence. Experimental results are consistent with a cooperative participation of an ammonium group of the polyamine moiety, in addition to it's binding to the negatively charged ribose-phosphate backbone, as proton source, and the imidazole moiety as a base. There is correlation between the location of the magnesium binding sites and the RNA cleavage sites, suggesting that the protonated nitrogens of the polycationic chain compete with some of the magnesium ions for RNA binding. Therefore, the cleavage pattern is specific of the RNA structure. These compounds cleave at physiological pH, representing novel reactive groups for antisense oligonucleotide derivatives or to enhance ribozyme activity.

Anticodon↗
PubMed1997

Structure and aminoacylation capacities of tRNA transcripts containing deoxyribonucleotides.

The contribution of the ribose 2'-hydroxyls to RNA structure and function has been analyzed, but still remains controversial. In this work, we report the use of a mutant T7 RNA polymerase as a tool in RNA studies, applied to the aspartate and methionine tRNA aminoacylation systems from yeast. Our approach consists of determining the effect of substituting natural ribonucleotides by deoxyribonucleotides in RNA and, thereby, defining the subset of important 2'-hydroxyl groups. We show that deoxyribose-containing RNA can be folded in a global conformation similar to that of natural RNA. Melting curves of tRNAs, obtained by temperature-gradient gel electrophoresis, indicate that in deoxyribo-containing molecules, the thermal stability of the tertiary network drops down, whereas the stability of the secondary structure remains unaltered. Nuclease footprinting reveals a significant increase in the accessibility of both single- and double-stranded regions. As to the functionality of the deoxyribose-containing tRNAs, their in vitro aminoacylation efficiency indicates striking differential effects depending upon the nature of the substituted ribonucleotides. Strongest decrease in charging occurs for yeast initiator tRNA(Met) transcripts containing dG or dC residues and for yeast tRNA(Asp) transcripts with dU or dG. In the aspartate system, the decreased aminoacylation capacities can be correlated with the substitution of the ribose moieties of U11 and G27, disrupting two hydrogen bond contacts with the synthetase. Altogether, this suggests that specific 2'-hydroxyl groups in tRNAs can act as determinants specifying aminoacylation identity.

Base Sequence↗
PubMed2006

Strategies to identify potential therapeutic target sites in RNA.

Antisense agents are powerful tools to inhibit gene expression in a sequence-specific manner. They are used for functional genomics, as diagnostic tools and for therapeutic purposes. Three classes of antisense agents can be distinguished by their mode of action: single-stranded antisense oligodeoxynucleotides; catalytic active RNA/DNA such as ribozymes, DNA- or locked nucleic acid (LNA)zymes; and small interfering RNA molecules known as siRNA. The selection of target sites in highly structured RNA molecules is crucial for their successful application. This is a difficult task, since RNA is assembled into nucleoprotein complexes and forms stable secondary structures in vivo, rendering most of the molecule inaccessible to intermolecular base pairing with complementary nucleic acids. In this review, we discuss several selection strategies to identify potential target sites in RNA molecules. In particular, we focus on combinatorial library approaches that allow high throughput screening of sequences for the design of antisense agents.

Animals↗
PubMed1995

Stabilization of RNA stacking by pseudouridine.

The effect of the modified nucleoside pseudouridine (psi) on RNA structure was compared with uridine. The extent of base stacking in model RNA oligonucleotides was measured by 1H NMR, UV, and CD spectroscopy. The UV and CD results indicate that the model single-stranded oligoribonucleotides AAUA and AA psi A form stacked structures in solution and the CD results for AA psi A are consistent with a general A-form helical conformation. The AA psi A oligomer exhibits a greater degree of UV hypochromicity over the temperature range 5-55 degrees C, consistent with a better stacked, more A-form structure compared with AAUA. The extent of stacking for each nucleotide residue was inferred from the percent 3'-endo sugar conformation as indicated by the H1'-H2' NMR scalar coupling. This indirect indication of stacking was confirmed by sequential NOE experiments. NMR measurements as a function of temperature indicate that pseudouridine forms a more stable base stacking arrangement than uridine, an effect that is propagated throughout the helix to stabilize stacking of neighboring purine nucleosides. The N1-H imino proton in AA psi A exchanges slowly with solvent, suggesting a role for the extra imino proton in stabilizing the conformation of pseudouridine. These results show that the conformational stabilization is an intrinsic property of pseudouridine occurring at the nucleotide level. The characteristics of pseudouridine in these models are consistent with earlier studies on intact rRNA, indicating that pseudouridine probably performs the same stabilizing function in most structural contexts.

Magnetic Resonance Spectroscopy↗
PubMed1995

The crystal structure of an all-RNA hammerhead ribozyme.

We have solved the crystal structure of an all-RNA hammerhead ribozyme by isomorphous replacement which contains a single 2'-O-methyl-cytosine at the active site to prevent cleavage. We describe the hammerhead RNA structure from a point of view which shows how the structural elements are disposed to bring the active site nucleotide into the catalytic pocket of the ribozyme. Five potential Mg(II) binding sites can be identified in the hammerhead RNA electron density maps. Of these, one is a newly identified metal site positioned near the ribozyme catalytic pocket, and another site corresponds to a Mn(II) site identified in the previous hammerhead RNA structure. We propose a mechanism for RNA catalytic cleavage on the basis of this new metal-binding site, as well as upon comparisons between the catalytic pocket of the hammerhead RNA and the metal-binding sites in the structurally homologous uridine turn of the anticodon loop in tRNAPhe.

Base Sequence↗
PubMed2006

Reconstitution, activities, and structure of the eukaryotic RNA exosome.

The RNA exosome is a multisubunit 3' to 5' exoribonuclease complex that participates in degradation and processing of cellular RNA. To determine the activities and structure of the eukaryotic exosome, we report the reconstitution of 9-subunit exosomes from yeast and human and reconstitution of 10- and 11-subunit exosomes from yeast. Comparative biochemical analysis between purified subunits and reconstituted exosomes using AU-rich, polyadenylated (poly[A]), generic, and structured RNA substrates reveals processive phosphorolytic activities for human Rrp41/Rrp45 and the 9-subunit human exosome, processive hydrolytic activities for yeast Rrp44 and the yeast 10-subunit exosome, distributive hydrolytic activities for Rrp6, and processive and distributive hydrolytic activities for the yeast 11-subunit exosome. To elucidate the architecture of a eukaryotic exosome, its conserved surfaces, and the structural basis for RNA decay, we report the X-ray structure determination for the 286 kDa nine-subunit human exosome at 3.35 A.

Amino Acid Sequence↗
PubMed2005

Metabolic monitoring by bacterial mRNAs.

There is growing appreciation for diversity in the strategies that bacteria utilize in regulating gene expression. Bacteria must be able to respond in different ways to different stresses and thus require unique regulatory solutions for the physiological challenges they encounter. Recent data indicate that bacteria commonly employ a variety of posttranscriptional regulatory mechanisms to coordinate expression of their genes. In many instances, RNA structures embedded at the 5' ends of mRNAs are utilized to sense particular metabolic cues and regulate the encoded genes. These RNA elements are likely to range in structural sophistication, from short sequences recognized by RNA-binding proteins to complex shapes that fold into high-affinity receptors for small organic molecules. Enough examples of RNA-mediated genetic strategies have been found that it is becoming useful to view this overall mode of regulatory control at a genomic level. Eventually, a complete picture of bacterial gene regulation within a single bacterium, from control at transcription initiation to control of mRNA stability, will emerge. But for now, this article seeks to provide a brief overview of the known categories of RNA-mediated genetic mechanisms within the bacterium Bacillus subtilis, with the expectation that it is representative of bacteria as a whole.

Adaptation, Physiological↗
PubMed1996

Multiple substrate binding sites in the ribozyme from Bacillus subtilis RNase P.

The ribozyme from Bacillus subtilis RNase P (P RNA) recognizes an RNA structure consisting of the acceptor stem and the T stem-loop of tRNA substrates. An in vitro selection experiment was carried out to obtain potential RNA substrates that may interact with the P RNA differently from the tRNA substrate. Using a P RNA-derived ribozyme that contains most, if not all, of the structural elements thought to be involved in active site formation of P RNA, but lacks the putative binding site for the T stem-loop of tRNA, a single RNA substrate was isolated after nine rounds of selection. This RNA is a competent substrate for the ribozyme used in selection as well as for the full-length P RNA. Biochemical characterization shows that this selected substrate interacts at a different site compared with the tRNA substrate. The selection experiment also identified a self-cleaving RNA seemingly different from other known ribozymes. These results indicate that a biological ribozyme can contain different binding sites for different RNA substrates. This alternate binding site model suggests a simple mechanism for evolving existing ribozymes to recognize RNA substrates of diverse structures.

Bacillus subtilis↗
PubMed2004

Structures of two RNA octamers containing tandem G.A base pairs.

The crystal structures of two RNA octamers, 5'-GGC(GA)GCC-3' and 5'-GIC(GA)GCC-3', have been determined from X-ray diffraction data to 2.8 and 2.7 A resolution, respectively. The RNA octamers crystallize in isomorphous unit cells containing two mispairs arranged in a self-complementary manner and one single strand in the asymmetric unit. The single strand pairs with another single strand related by crystallographic symmetry to form a third unique double helix. Tandem non-Watson-Crick G.A/A.G base pairs of the sheared type comprise an internal loop in the middle of each duplex. The NMR structure of this octameric RNA sequence is also known, allowing comparison of the variation between the six crystallographic duplexes and the solution structure. In the symmetric duplex of the octamer containing inosine, the sheared G.A pairs incorporate a bound water molecule. This duplex also binds one water molecule per strand in the minor groove adjacent to the G.A pairs.

Base Pairing↗
PubMed2006

A subset of cell clusters with malignant features in morphologically normal-appearing and hyperplastic tissues.

BACKGROUND: The development of human breast cancer may be a multistep process, sequentially undergoing normal, hyperplastic, in situ, invasive, and metastatic stages. METHODS: Our previous and current studies have revealed that a subset of morphologically normal-appearing and hyperplastic breast tissues adjacent to or distant from malignant breast lesions contained cell clusters that showed malignancy-associated immunohistochemical and cytological alterations. RESULTS: Compared to their morphologically similar counterparts within the same lesion, these cell clusters exhibited several unique features: (1) a significantly increased frequency of focal disruptions in surrounding myoepithelial cell layers and the loss of estrogen receptor expression, (2) signs of stromal and vascular invasion, (3) distinct alterations in the cytoplasmic-nuclear ratio and nuclear shape, size, and polarity, (4) the expression of multiple malignancy-associated biomarkers, and (5) malignancy-associated nuclear changes in benign-appearing cells. CONCLUSIONS: These changes are likely to reflect DNA structural abnormalities resulting from the disposition of DNA and RNA structural defects or cancer susceptibility genes that facilitate progression and invasion, or to suggest that breast tumor progression and invasion may not always follow the hypothesized sequences. Thus, an integrated immunohistochemical and cytological assessment might facilitate the differential diagnosis, early detection and prevention of malignant breast lesions.

Actins↗
PubMed1983

Poly(A) RNA of the egg cytoplasm: structural resemblance to the nuclear RNA of somatic cells.

This paper concerns the structural characteristics of the poly(A) RNA stored in unfertilized amphibian and echinoderm eggs. Though located in the egg cytoplasm, at least two-thirds of these maternal transcripts display an interspersed sequence organization similar to that of nuclear RNA. In Xenopus laevis interspersed poly(A) RNA molecules are synthesized and deposited in the oocyte cytoplasm throughout the main growth phase of oogenesis. Regions of the sea urchin genome that are represented by interspersed maternal transcripts have been recovered from recombinant clone libraries. In one case the same single-copy sequence is found both in an abundant message-sized 1.6 kilobase (kb) maternal transcript and in a 7.5 kb maternal transcript that structurally resembles a precursor form and is not found in embryonic polysomes. In a second example considered, a 9.5 kb transcript was identified in embryo nuclear RNA that may be identical in structure with an interspersed maternal poly(A) RNA derived from the same transcription unit. Transcription of this sequence appears to be constitutive in somatic cell nuclei, though no homologous cytoplasmic RNAs are found after early cleavage. This may be a widespread form of regulation for transcription units expressed in female germ cells, and represented in the maternal poly(A) RNA pools of unfertilized eggs.

Animals↗
PubMed2006

Thermodynamics of the fragile X mental retardation protein RGG box interactions with G quartet forming RNA.

Fragile X syndrome, the most common form of inherited mental retardation, is the result of an unstable expansion of a CGG trinucleotide repeat in the 5' UTR of the fragile X mental retardation-1 (FMR1) gene. The abnormal hypermethylation of the expanded CGG repeats causes the transcriptional silencing of the FMR1 gene and, consequently, the loss of the fragile X mental retardation protein (FMRP). FMRP is an RNA binding protein that binds to G quartet forming RNA using its RGG box motif. In this study we have performed a thermodynamic analysis of the interactions between the FMRP RGG box domain and Sc1, an RNA molecule which had been previously shown to be bound with high affinity by both the full-length FMRP and by its RGG box domain. We have determined that the association between the FMRP RGG box and Sc1 RNA is dominated by hydrophobic and hydrogen bond interactions, with minor contributions from electrostatic interactions, and that the FMRP RGG box binding increases the stability of the G quartet RNA structure significantly. Interestingly, we found that the G quartet recognition is necessary but not sufficient for the FMRP RGG box binding to this RNA target, indicating that additional interactions of the peptide, possibly with the stem and/or stem-G quartet junction region, are required. Our results also indicate that the G quartet RNA recognition is not a general feature of the RGG box motif but rather carries some sequence, protein and/or RNA, specificity.

Amino Acid Sequence↗
PubMed2002

Deficient dimerization of human immunodeficiency virus type 1 RNA caused by mutations of the u5 RNA sequences.

The human immunodeficiency virus type 1 (HIV-1) virion contains two copies of genomic RNA that are noncovalently attached along a region at their 5' ends, in which two contact sites have been observed by electron microscopy. One of these sites is believed to be the stem-loop 1 (SL1) sequence which serves as the dimerization initiation site (DIS), and the other site, closer to the 5' end of the viral RNA, may involve the R or U5 RNA sequences. In this study, we present biochemical evidence showing that alteration of the U5 RNA sequence in the context of full-length viral RNA leads to diminished dimerization of virion RNA. In particular, two stretches of GU-rich sequences, which are located at nucleotides (nt) 99 to 108 and nt 112 to 123 within U5, were either deleted or substituted with exogenous sequences. The mutated viruses thus generated all exhibited deficient RNA dimerization. This dimerization deficit was not corrected by second-site mutations that preserved local RNA structures, such as the poly(A) hairpin, and was overcome to only a limited extent by compensatory mutations within Gag; these mutations were identified after long-term culture of the relevant mutant viruses in permissive cell lines and were able to restore viral infectiousness and RNA packaging to wild-type levels. Therefore, these GU sequences do not regulate RNA dimerization by the formation of local secondary structures nor by the maintenance of efficient viral RNA packaging; instead, they may mediate direct RNA-RNA interactions in the dimer structure. In contrast, mutation of palindrome 5'-AAGCUU-3', which resides within R and crowns the poly(A) hairpin, did not affect either RNA dimerization or RNA packaging.

5' Untranslated Regions↗
PubMed1994

Extremely stable transcripts may compensate for the elimination of the gene fert-1 from all Ascaris lumbricoides somatic cells.

The single-copy gene fert-1 becomes eliminated from all somatic cells during the process of chromatin diminution in Ascaris lumbricoides var. suum. By using Northern blot and in situ hybridization techniques, we have analyzed its rather unusual expression pattern. Different splicing and 3' end formation events generate in a developmentally regulated manner various poly(A)+ and poly(A)- fert-1 RNA species. The lack of any significant open reading frame in most of its RNA products indicates that fert-1 may function as structural RNA rather than encoding a protein. Fert-1 transcripts are produced in the precursors of the gametes, but degraded at the time of meiosis and not passed on to the zygote. Embryonic transcription of fert-1 sets in as soon as the female nucleus has completed its meiosis. Our data thus demonstrate that the Ascaris transcription apparatus is active prior to the general onset of zygotic transcription, which we think takes place in the four- to six-cell-stage embryos. Upon elimination of fert-1 gene from the somatic cells, most of its transcripts disappear. Two short fert-1 RNA products, however, are stably maintained throughout development until the second larval stage, which is more than 1 month after the elimination of their coding sequences. Possible functions of fert-1 are discussed.

Amino Acid Sequence↗
PubMed1997

Assignment methodology for larger RNA oligonucleotides: application to an ATP-binding RNA aptamer.

The use of uniform 13C, 15N labeling in the NMR spectroscopic study of RNA structures has greatly facilitated the assignment process in small RNA oligonucleotides. For ribose spin system assignments, exploitation of these labels has followed previously developed methods for the study of proteins. However, for sequential assignment of the exchangeable and nonexchangeable protons of the nucleotides, it has been necessary to develop a variety of new NMR experiments. Even these are of limited utility in the unambiguous assignment of larger RNAs due to the short carbon relaxation times and extensive spectral overlap for all nuclei. These problems can largely be overcome by the additional use of basetype selectively 13C, 15N-labeled RNA in combination with a judicious use of related RNAs with base substitutions. We report the application of this approach to a 36-nucleotide ATP-binding RNA aptamer in complex with AMP. Complete sequential 1H assignments, as well as the majority of 13C and 15N assignments, were obtained.

Adenosine Triphosphate↗
PubMed1986

Translational regulation of expression of the bacteriophage T4 lysozyme gene.

The bacteriophage T4 lysozyme gene is transcribed at early and late times after infection of E. coli, but the early mRNA is not translated. DNA sequence analysis and mapping of the 5' ends of the lysozyme transcripts produced at different times after T4 infection show that the early mRNA is initiated some distance upstream from the gene. The early mRNA is not translated because of a stable secondary structure which blocks the translational initiation site. The stable RNA structure has been demonstrated by nuclease protection in vivo. After DNA replication begins, two late promoters are activated; the late transcripts are initiated at sites such that the secondary structure can not form, and translation of the late messages occurs.

Amino Acid Sequence↗
PubMed1986

Translation initiation of bacteriophage lambda gene cII requires integration host factor.

Escherichia coli integration host factor (IHF), a DNA-binding protein, positively regulates expression of the lambda cII gene. Purified IHF stimulates cII protein synthesis in vitro, suggesting a direct role for host factor in cII expression. Further evidence for a direct role for IHF was obtained with operon and gene fusions between cII and lacZ or cII and galE. Analysis of these fusions in vivo demonstrated that IHF is essential for the initiation of cII translation. Replacement of the entire cII coding sequence with lacZ yielded a gene fusion which was still IHF dependent. However, a cII-galE fusion carrying a hybrid ribosome binding region expressed galE in IHF mutants. These results indicate that sequences which make cII translation IHF dependent lie between the ribosome binding region and the initiating codon of cII. Failure to translate cII activates a transcription terminator located within cII and results in polar effects on downstream transcription. This polarity is suppressed by the lambda N antitermination function. When cloned into another context, the terminator is active in both wild-type and IHF mutant strains. The amino terminus of cII is located near an IHF binding site in a region with considerable dyad symmetry. The role of IHF in cII translation may be to prevent formation of an RNA-RNA duplex that sequesters the ribosome binding site of cII. The binding of IHF might influence RNA structure by altering the rate of the dissociation of RNA from the DNA template.

Bacterial Proteins↗