Distinct Dynamic Modes Enable the Engagement of Dissimilar Ligands in a Promiscuous Atypical RNA Recognition Motif.

Conformational dynamics play a critical role in ligand binding, often conferring divergent activities and specificities even in species with highly similar ground-state structures. Here, we employ time-resolved electrospray ionization hydrogen-deuterium exchange (TRESI-HDX) to characterize the changes in dynamics that accompany oligonucleotide binding in the atypical RNA recognition motif (RRM2) in the C-terminal domain (CTD) of human La protein. Using this approach, which is uniquely capable of probing changes in the structure and dynamics of weakly ordered regions of proteins, we reveal that binding of RRM2 to a model 23-mer single-stranded RNA and binding of RRM2 to structured IRES domain IV of the hepatitis C viral (HCV) RNA are driven by fundamentally different dynamic processes. In particular, binding of the single-stranded RNA induces helical "unwinding" in a region of the CTD previously hypothesized to play an important role in La and La-related protein-associated RNA remodeling, while the same region becomes less dynamic upon engagement with the double-stranded HCV RNA. Binding of double-stranded RNA also involves less penetration into the RRM2 binding pocket and more engagement with the unstructured C-terminus of the La CTD. The complementarity between TRESI-HDX and Δδ nuclear magnetic resonance measurements for ligand binding analysis is also explored.

Enzyme assays for synthesis and degradation of 2-5As and other 2'-5' oligonucleotides.

BACKGROUND: The 5'-triphosphorylated, 2'-5'-linked oligoadenylate polyribonucleotides (2-5As) are central to the interferon-induced antiviral 2-5A system. The 2-5As bind and activate the RNase L, an endoRNase degrading viral and cellular RNA leading to inhibition of viral replication. The 2-5A system is tightly controlled by synthesis and degradation of 2-5As. Whereas synthesis is mediated by the 2'-5' oligoadenylate synthetase family of enzymes, degradation seems to be orchestrated by multiple enzyme nucleases including phosphodiesterase 12, the ectonucleotide pyrophosphatase/phosphodiesterase 1 and the A-kinase anchoring protein 7. RESULTS: Here we present assay tools for identification and characterization of the enzymes regulating cellular 2-5A levels. A procedure is described for the production of 2'-5' oligoadenylates, which are then used as substrates for development and demonstration of enzyme assays measuring synthetase and nuclease activities, respectively. The synthetase assays produce only a single reaction product allowing for very precise kinetic assessment of the enzymes. We present an assay using dATP and the A(pA)3 tetramer core as substrates, which requires prior isolation of A(pA)3. A synthetase assay using either of the dNTPs individually together with NAD(+) as substrates is also presented. The nuclease reactions make use of the isolated 2'-5' oligoadenylates in producing a mixture of shorter reaction products, which are resolved by ion-exchange chromatography to determine the enzyme activities. A purified human 2'-5' oligoadenylate synthetase and a purified human phosphodiesterase 12 along with crude extracts expressing those proteins, are used to demonstrate the assays. CONCLUSIONS: This paper comprises an assay toolbox for identification and characterization of the synthetases and nucleases regulating cellular 2-5A levels. Assays are presented for both enzyme families. The assays can also be used to address a broader cellular role of the OAS enzymes, based on the multiple substrate specificity intrinsic to these proteins.

Binding of the plant alkaloid aristololactam-ß-d-glucoside and antitumor antibiotic daunomycin to single stranded polyribonucleotides.

BACKGROUND: Interaction of the plant alkaloid aristololactam-ß-d-glucoside and the antitumor drug daunomycin with single stranded RNAs poly(G), poly(I), poly(C) and poly(U) has been investigated. METHODS: Biophysical techniques of absorption, fluorescence, competition dialysis, circular dichroism, and microcalorimetry have been used. RESULTS: Absorption and fluorescence studies have revealed noncooperative binding of ADG and DAN to the single stranded RNAs. The binding affinity of ADG varied as poly(G) > poly(I) > > poly(C) > poly(U). The affinity of DAN was one order higher than that of ADG and varied as poly(G) > poly(I) > poly(U) > poly(C). This binding preference was further confirmed by competition dialysis assay. The thermodynamics of the binding was characterised to be favourable entropy and enthalpic terms but their contributions were different for different systems. The major non-polyelectrolytic contribution to the binding revealed from salt dependent data appears to be arising mostly from stacking of DAN and ADG molecules with the bases leading to partial intercalation to single stranded RNA structures. Small negative heat capacity values have been observed in all the four cases. CONCLUSIONS: This study presents the comparative structural and thermodynamic profiles of the binding of aristololactam-ß-d-glucoside and daunomycin to single stranded polyribonucleotides. GENERAL SIGNIFICANCE: These results suggest strong, specific but differential binding of these drug molecules to the single stranded RNAs and highlight the role of their structural differences in the interaction profile.

Binding properties of Ru(II) polypyridyl complexes with poly(U)·poly(A)*poly(U) triplex: the ancillary ligand effect on third-strand stabilization.

The binding properties of [RuL(2)(mip)](2+) {where L is 1,10-phenanthroline (phen) or 4,7-dimethyl-1,10-phenanthrollne (4,7-dmp) and mip is 2'-(3",4"-methylenedioxyphenyl)imidazo[4',5'-f][1,10]phenanthroline} with regard to the triplex RNA poly(U)·poly(A)*poly(U) were investigated using various biophysical techniques and quantum chemistry calculations. In comparison with [Ru(4,7-dmp)(2)(mip)](2+), remarkably higher binding affinity of [Ru(phen)(2)(mip)](2+) for the triplex RNA poly(U)·poly(A)*poly(U) was achieved by changing the ancillary ligands. The stabilization of the Hoogsteen-base-paired third strand was improved by about 10.9 °C by [Ru(phen)(2)(mip)](2+) against 6.6 °C by [Ru(4,7-dmp)(2)(mip)](2+). To the best of our knowledge, [Ru(phen)(2)(mip)](2+) is the first metal complex able to raise the third-strand stabilization of poly(U)·poly(A)*poly(U) from 37.5 to 48.4 °C. The results reveal that the ancillary ligands have an important effect on third-strand stabilization of the triplex RNA poly(U)·poly(A)*poly(U) when metal complexes contain the same intercalative ligands.

Crystallization of oligonucleotides containing A-rich repeats suggests a structural contribution to the autoregulation mechanism of PABP translation.

Eukaryotic poly(A)-binding protein (PABP) commonly binds to the 3'-UTR poly(A) tail of every mRNA, but it also binds to the 5'-UTR of PABP mRNA for autoregulation of its expression. In the sequence of the latter binding site, the contiguous A residues are segmented discretely by the insertion of short pyrimidine oligonucleotides as linkers, so that (A)(6-8) segments are repeated six times. This differs from the poly(A)-tail sequence, which has a higher binding affinity for PABP. In order to examine whether the A-rich repeats have a functional structure, several RNA/DNA analogues were subjected to crystallization. It was found that some of them could be crystallized. Single crystals thus obtained diffracted to 4.1 Å resolution. The fact that the repeated sequences can be crystallized suggests the possibility that the autoregulatory sequence in PABP mRNA has a specific structure which impedes the binding of PABP. When PABP is excessively produced, it could bind to this sequence by releasing the structure in order to interfere with initiation-complex formation for suppression of PABP translation. Otherwise, PABP at low concentration preferentially binds to the poly(A) tail of PABP mRNA.

Small molecule-RNA interaction: spectroscopic and calorimetric studies on the binding by the cytotoxic protoberberine alkaloid coralyne to single stranded polyribonucleotides.

BACKGROUND: RNA has attracted recent attention for its key role in gene expression and hence targeting by small molecules for therapeutic intervention. This study is aimed to elucidate the specificity of the alkaloid coralyne to poly(G), poly(C), poly(I) and poly(U) in the light of its ability in inducing self-structure in poly(A). METHODS: Multifaceted experimental techniques like competition dialysis, absorption, fluorescence, circular dichroism and calorimetry were employed. Salt dependence and temperature dependence of the binding was also elucidated. RESULTS: Results of competition dialysis, absorption and fluorescence studies revealed that coralyne binds strongly to the polypurines, poly(G) and poly(I) compared to the polypyrimidines, poly(U) and poly(C). Partial intercalative binding due to the stacking of the molecules between the bases was envisaged. The binding was predominantly enthalpy driven with favourable entropy term with a large favourable non-electrostatic contribution revealed from salt dependent data and the dissection of the free energy. The heat capacity change of -125 and -119 cal/mol K(-1) respectively for poly(G) and poly(I) and the partial enthalpy-entropy compensation phenomenon observed confirmed the involvement of multiple weak noncovalent interactions. Circular dichroism studies provided evidence for significant perturbation of the conformation of the RNAs, but no self-structure induction was evident in any of the polymers under the condition of the study. CONCLUSIONS: This study presents a complete structural and thermodynamic profile of coralyne interaction to four single stranded RNA polymers. GENERAL SIGNIFICANCE: The study for the first time elucidates the base specificity of coralyne-RNA complexation at the single stranded level.

A strong exonic splicing enhancer in dystrophin exon 19 achieve proper splicing without an upstream polypyrimidine tract.

Proper splicing is known to proceed under the control of conserved cis-elements located at exon-intron boundaries. Recently, it was shown that additional elements, such as exonic splicing enhancers (ESEs), are essential for the proper splicing of certain exons, in addition to the splice donor and acceptor site sequences; however, the relationship between these cis-elements is still unclear. In this report, we utilize dystrophin exon 19 to analyse the relationship between the ESE and its upstream acceptor site sequences. Dystrophin exon 19, which maintains adequate splicing donor and acceptor consensus sequences, encodes exonic splicing enhancer (dys-ESE19) sequences. Splice pattern analysis, using a minigene reporter expressed in HeLa cells, showed that either a strong polypyrimidine tract (PPT) or a fully active dys-ESE19 is sufficient for proper splicing. Each of these two cis-elements has enough activity for proper exon 19 splicing suggesting that the PPT, which is believed to be an essential cis-element for splicing, is dispensable when the downstream exon contains a strong ESE. This compensation was only seen in living cells but not in 'in vitro splicing'. This suggests the possibility that the previous splicing experiments using an in vitro splicing system could underestimate the activity of ESEs.

Purification and characterization of an extracellular, uracil specific ribonuclease from a Bizionia species isolated from the marine environment of the Sundarbans.

The first ribonuclease (RNase) from the Cytophaga-Flavobacterium-Bacteroides phylum, dominant in the marine environment, and also from the first Bizionia species isolated from the tropics was purified and characterized. Extracellular RNase production occurred when the culture medium contained 5-7% (w/v) NaCl. The 53.0 kDa enzyme was purified 29 folds with a recovery of 4% and specific activity of 630unit/mg protein. The pH and temperature optima are 6.5 and 35 degrees C, respectively and the enzyme retains more than half of its activity (relative to optimal assay conditions) after 1h pre-incubation separately with 5% (w/v) NaCl or from pH 5.0 to 8.5 or at 50 degrees C. Dithiothreitol and beta-mercaptoethanol do not inhibit whereas human placental RNase inhibitor protein halves the RNase activity. While Mg(2+), Ba(2+) and Ca(2+) enhanced the enzyme activity, Fe(2+), Cu(2+) and Hg(2+) inactivated it. This RNase degrades uracil containing nucleic acids only. Our isolate could be a novel renewable source of deoxyribonuclease (DNase)--free RNase enzyme.

Binding properties of replication protein A from human and yeast cells.

Replication protein A (RP-A; also known as replication factor A and human SSB), is a single-stranded DNA-binding protein that is required for simian virus 40 DNA replication in vitro. RP-A isolated from both human and yeast cells is a very stable complex composed of 3 subunits (70, 32, and 14 kDa). We have analyzed the DNA-binding properties of both human and yeast RP-A in order to gain a better understanding of their role(s) in DNA replication. Human RP-A has high affinity for single-stranded DNA and low affinity for RNA and double-stranded DNA. The apparent affinity constant of RP-A for single-stranded DNA is in the range of 10(9) M-1. RP-A has a binding site size of approximately 30 nucleotides and does not bind cooperatively. The binding of RP-A to single-stranded DNA is partially sequence dependent. The affinity of human RP-A for pyrimidines is approximately 50-fold higher than its affinity for purines. The binding properties of yeast RP-A are similar to those of the human protein. Both yeast and human RP-A bind preferentially to the pyrimidine-rich strand of a homologous origin of replication: the ARS307 or the simian virus 40 origin of replication, respectively. This asymmetric binding suggests that RP-A could play a direct role in the process of initiation of DNA replication.

Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities.

Several proteins of heterogeneous nuclear ribonucleoprotein (hnRNP) particles display very high binding affinities for different ribonucleotide homopolymers. The specificity of some of these proteins at high salt concentrations and in the presence of heparin allows for their rapid one-step purification from HeLa nucleoplasm. We show that the hnRNP C proteins are poly(U)-binding proteins and compare their specificity to that of the previously described cytoplasmic poly(A)-binding protein. These findings provide a useful tool for the classification and purification of hnRNP proteins from various tissues and organisms and indicate that different hnRNP proteins have different RNA-binding specificities.

Endothelial cytosolic proteins bind to the 3' untranslated region of endothelial nitric oxide synthase mRNA: regulation by tumor necrosis factor alpha.

Changes in endothelial nitric oxide synthase (eNOS) expression may be involved in the endothelium-dependent vasorelaxation dysfunction associated with several vascular diseases. In the present work, we demonstrate that eNOS mRNA contains a previously undescribed cis element in the 3' untranslated region (3' UTR). A U+C-rich segment in the 3' UTR is critical in complex formation with bovine aortic endothelial cell cytosolic proteins. Tumor necrosis factor alpha (TNF-alpha), which destabilizes eNOS mRNA, increased the binding activity of the cytosolic proteins in a time-dependent manner. These data suggest that endothelial cytosolic proteins bind to the 3' UTR of eNOS mRNA. These proteins may play a role in TNF-alpha-induced eNOS mRNA destabilization.

iPABP, an inducible poly(A)-binding protein detected in activated human T cells.

The poly(A)-binding protein (PABP) binds to the poly(A) tail present at the 3' ends of most eukaryotic mRNAs. PABP is thought to play a role in both translation and mRNA stability. Here we describe the molecular cloning and characterization of an inducible PABP, iPABP, from a cDNA library prepared from activated T cells. iPABP shows 79% sequence identity to PABP at the amino acid level. The RNA binding domains of iPABP and PABP are nearly identical, while their C termini are more divergent. Like PABP, iPABP is primarily localized to the cytoplasm. iPABP is expressed at low levels in resting normal human T cells; following T-cell activation, however, iPABP mRNA levels are rapidly up-regulated. In contrast, PABP is constitutively expressed in both resting and activated T cells. iPABP mRNA was also expressed at much higher levels than PABP mRNA in heart and skeletal muscle tissue. These data suggest that the regulation of cytoplasmic poly(A)-binding activity is more complex than previously believed. In most tissues, poly(A)-binding activity is likely to be the result of the combined effects of constitutively expressed PABP and iPABP, whose expression is subject to more complex regulation.

Molecular mimicry of the NF-kappaB DNA target site by a selected RNA aptamer.

During the past two decades, structural and biophysical studies of DNA-protein and RNA-protein complexes have enhanced our understanding of the physico-chemical basis of nucleic acid recognition by proteins. However, it remains unclear what protein surface features are most important for nucleic acid binding and whether the same protein surface could bind specifically to both DNA and RNA. The recently described X-ray crystal structure of the transcription factor NF-kappaB p50 homodimer bound to a high-affinity RNA aptamer allows the direct comparison of NF-kappaB-RNA and NF-kappaB-DNA binding modes. The RNA aptamer, which bears no sequence homology to natural NF-kappaB DNA targets, adopts a structure with similar physico-chemical properties to kappaB DNA and contacts a common nucleic-acid-binding 'consensus surface' on the p50 homodimer.

Small molecule-RNA recognition: Binding of the benzophenanthridine alkaloids sanguinarine and chelerythrine to single stranded polyribonucleotides.

Single stranded RNAs are biologically potent as they participate in various key cellular processes. The binding efficacy of two potent anticancer alkaloids, sanguinarine (here after SANG) and chelerythrine (here after CHEL), with single-stranded ribonucleic acids poly(rI), poly(rG), and poly(rC) were studied using spectroscopic and thermodynamic tools. Results reveal that both SANG and CHEL binds well with single stranded RNAs with affinity in the order poly(rI)>poly(rG)>poly(rC). CHEL showed slightly higher affinity compared to SANG with all the single stranded RNAs. Both SANG and CHEL showed association affinity of the lower 106 order with poly(rI), higher 105 order binding with poly(rG) and lower 105 order with poly(rC). The binding mode was partial intercalation due to the staking interaction between the bases and the alkaloids. The complexation of both the SANG and CHEL to the RNAs were mainly enthalpy driven and also favoured by entropy changes. Perturbation was observed in the RNA conformation due to binding of the alkaloids. In this present study we have deciphered the fundamental structural and calorimetric aspects of the interaction of the natural benzophenanthridine alkaloids with single stranded RNAs and these results may help to develop new generation alkaloid based therapeutics targeting single stranded RNAs.

Improved hybridisation potential of oligonucleotides comprising O-methylated anhydrohexitol nucleoside congeners.

The hybridising potential of anhydrohexitol nucleoside analogues (HNAs) is well documented, but tedious synthesis of the monomers hampers their development. In a search for better analogues, the synthesis of two new methylated anhydrohexitol congeners 1 and 2 was accomplished and the physico-chemical properties of their respective oligomers were evaluated. Generally, oligonucleotides (ONs) containing the 3'-O-methyl derivative 1 showed a small increase in thermal stability towards complementary sequences as compared to HNA. Compared to the altritol modification, 3'-O-methylation seems to cause a small decrease in thermal stability of duplexes, especially when targeting RNA. These results suggest the possibility of derivatisation of the 3'-hydroxyl group of altritol-containing congeners without significantly affecting the thermal stability of the duplexes. The methyl glycosidic analogues 2 likewise increased the affinity for RNA in comparison with well-known HNA, while at the same time being economically more favorable monomers. However, homopolymers of 2 displayed self-pairing, but not so homopolymers of 1. Upon incorporation of the hexitols within RNA sequences in an effort to induce a beneficial pre-organised structure, the positive effect of the 3'-O-methyl derivative 1 proved larger than that of 2.

RNA-binding characteristics of the chloroplast S1-like ribosomal protein CS1.

The chloroplast ribosomal protein CS1, the homolog of the bacterial ribosomal protein S1, is believed to be involved in the process of ribosome binding to mRNA during translation. Since translation control is an important step in chloroplast gene expression, and in order to study initiation complex formation, we studied the RNA-binding properties of CS1 protein. We found that most of the CS1 protein in spinach chloroplast co-purified with the 30S ribosomal subunit. The relative binding affinity of RNA to CS1 was determined using the UV-crosslinking competition assay. CS1 protein binds the ribohomopolymer poly(U) with a relatively high binding affinity. Very low binding affinities were obtained for the other ribohomopolymers, poly(G), poly(A) and poly(C). In addition, no specific binding of CS1, either in the 30S complex or as a recombinant purified protein, was obtained to the 5'-untranslated region of the mRNA in comparison to the other parts. RNA-binding experiments, in which the N- and C-termini of the protein were analyzed, revealed that the RNA-binding site is located in the C-terminus half of the protein. These results suggest that CS1 does not direct the 30S complex to the initiation codon of the translation site by specific binding to the 5'-untranslated region. In bacteria, specific binding is derived by base pairing between 16S rRNA and the Shine-Dalagarno sequences. In the chloroplast, nuclear encoded and gene-specific translation factors may be involved in the determination of specific binding of the 30S subunit to the initiator codon.

Coordination of interstrand cross-links between polydeoxyguanylic acid and polydeoxycytidylic acid by cis-diamminedichloroplatinum (II).

Cisplatin has been shown to create interstrand DNA crosslinks by several methods. These experiments were performed to determine if cisplatin coordinates cross-links between deoxyguanylic and deoxycytidylic acids. After incubation at pH 7.2 with cisplatin at various platinum/base nucleotide ratios, ri, for 3 days at 20 degrees, polydeoxyguanylic acid . polydeoxycytidylic acid [poly(dG) . poly(dC)] was centrifuged in alkaline cesium sulfate for 3 days at 35,000 rpm. Untreated samples resulted in a low-density deoxycytidylic acid peak and a high-density deoxyguanylic acid peak, as identified by the ultraviolet absorption spectrum. The density gradient profiles of cisplatin-treated poly(dG). poly(dC) had an additional hybrid-density peak the area of which increased with the dose (ri) as the areas under the polydeoxycytidylic acid [poly(dC)] and polydeoxyguanylic acid [poly(dG)] peaks diminished. With increasing ri, the densities of both the poly(dG) and hybrid peaks increased while that of the poly(dC) peak remained unchanged. Similarly, the amount of bound platinum in the poly(dG) and hybrid-density peaks increased with ri, while no platinum could be detected in the poly(dC) peak. Therefore, cisplatin appears to react first with the deoxyguanylic acid strand, followed by a second reaction with the deoxycytidylic acid strand to form the interstrand cross-links that are necessary for the formation of the hybrid-density bands. Based upon the intercept of the percent cross-linked poly(dG) . poly(dC) versus ri curve with the abscissa, the minimum amount of cisplatin or the threshold amount of cisplatin required to observe cross-links was ri = 0.017. When multiplied by the molecular weight of the poly(dG) . poly(dC) used, this was equivalent to 1 cross-link per 15 molecules of bound platinum. However, based upon the amount of bound drug, the frequency of cross-links per platinum reaction was calculated to range from one of 50 to 67 reactions. Due to limitations in the sensitivity of the platinum assay and to a progressively lower recovery of the cross-linked material at lower ri values, it was not possible to calculate the cross-linking efficiency based upon the amount of bound drug for ri values equal to or less than 0.025. The monofunctional analogue, [diethylenetriaminechloroplatinum] chloride, did not cause a hybrid peak to form. These studies demonstrate that cisplatin coordinates interstrand cross-links between poly(dG) and poly(dC).

Possible sites of origin of human plasma ribonucleases as evidenced by isolation and partial characterization of ribonucleases from several human tissues.

The ribonucleases (RNases) present in a number of human tissues, including heart, brain, lung, and kidney, were purified, partially characterized, and compared in their properties to the previously described RNases from human liver, spleen, pancreas, and serum. The enzymes appeared to fall into two major classes: liver-spleen type RNase and plasma-type RNase. These two types of enzymes were present in varying proportions in all tissues examined. The extent to which the tissues studied possibly contribute to serum RNase levels is discussed.

Detection in human ovary and prostate tumors of DNA polymerase activity that copies poly(2'-O-methylcytidylate) . oligodeoxyguanylate.

Particulate DNA polymerase activity that copied poly(2'-O-methylcytidylate) . oligodeoxyguanylate and banded at a density of 1.15 to 1.20 g/ml in sucrose gradients was detected in 8 of 16 human ovary tumors and in 11 of 16 malignant prostate tissues. None of the 10 nonmalignant ovary and prostate tissues examined contained detectable particulate DNA polymerase activity that copied poly(2'-O-methylcytidylate) . oligodeoxyguanylate. Since poly(2'-O-methylcytidylate) . oligodeoxyguanylate is effectively copied by oncornavirus RNA-directed DNA polymerase (reverse transcriptase) although not by the known species of human cell DNA polymerase, these results are interpreted as supporting the concept that some malignant human tissues contain particle-associated reverse transcriptase activity.

Trapping of DNA-reactive metabolites of therapeutic or carcinogenic agents by carbon-14-labeled synthetic polynucleotides.

Many substances which do not react with DNA directly are metabolized into important DNA-modifying intermediates. We have devised a method for trapping these intermediates with 14C-labeled nucleosides contained in a synthetic polynucleotide. The polynucleotide structure protects the labeled nucleoside from metabolism; thus, it is unaltered when the polymer is incubated with a drug-metabolizing system. However, when the polymer is incubated with this system and a compound which can be metabolized into a reactive species, these intermediates are trapped by the 14C-labeled nucleoside and subsequently are detected as new peaks of radioactivity in a digest of the labeled polynucleotide. This system has been used to detect reactive intermediates of cyclophosphamide generated by a liver homogenate.