Chromomycin A3 binds to left-handed poly(dG-m5dC)

The interaction of chromomycin A3 (an antitumor antibiotic) with right-handed and left-handed polynucleotides has been studied by absorbance, fluorescence, circular dichroism, 31P-NMR and 1H-NMR techniques. Binding to either the B form of poly(dG-dC) or the Z form of poly(dG-m5dC) shifts the absorbance maximum to higher wavelength and enhances the fluorescence of the drug. Circular dichroic spectra of solutions containing various concentrations of chromomycin A3 and fixed concentrations of either B or Z polynucleotides show well defined isoelliptic points at similar wavelengths. At the isoelliptic point, the drug complex with B DNA exhibits positive ellipticity while with Z DNA it exhibits negative ellipticity. 31P-NMR spectra of the chromomycin A3 complex with the Z form of poly(dG-m5dC) demonstrate that the Z conformation is retained in the drug complex up to one molecule drug/four base pairs. At Mg2+ concentrations lower than that necessary to stabilize the left-handed conformation of poly(dG-m5dC) alone, 31P analysis shows that chromomycin A3 can bind simultaneously to both the B and Z conformations of poly(dG-m5dC), with no effect on the B-Z equilibrium. These data demonstrate that chromomycin A3 binds to left-handed poly(dG-m5dC) with retention of the left-handed conformation up to saturating drug concentrations.     

Lsm proteins bind and stabilize RNAs containing 5' poly(A) tracts

Many orthopoxvirus messenger RNAs have an unusual nontemplated poly(A) tract of 5 to 40 residues at the 5' end. The precise function of this feature is unknown. Here we show that 5' poly(A) tracts are able to repress RNA decay by inhibiting 3'-to-5' exonucleases as well as decapping of RNA substrates. UV cross-linking analysis demonstrated that the Lsm complex associates with the 5' poly(A) tract. Furthermore, recombinant Lsm1-7 complex specifically binds 5' poly(A) tracts 10 to 21 nucleotides in length, consistent with the length of 5' poly(A) required for stabilization. Knockdown of Lsm1 abrogates RNA stabilization by the 5' poly(A) tract. We propose that the Lsm complex simultaneously binds the 3' and 5' ends of these unusual messenger RNAs and thereby prevents 3'-to-5' decay. The implications of this phenomenon for cellular mRNA decay are discussed.     

Studies on the interaction between coxsackievirus A9 and HeLa cells. I. Plaque-forming ability of coxsackievirus A9 in HeLa cell cultures.

Most of the coxsackievirus A9 (CA 9 virus) including the prototype strain formed plaques in HeLa cell monolayers under agar overlay, although they showed little or no cytopathogenicity under fluid medium. These viruses were isolated or passaged in primary cynomolgus monkey kidney (MK) cell cultures, and the infectivity of any strain in terms of plaque-forming units was much higher in MK cells than in HeLa cells, even after plaque purification of the virus in HeLa cell cultures. CA 9 virus contained in the original throat swabs as well as some clones obtained by plaque purification in MK cells failed to form plaques in HeLa cells, but virus preparations obtained after several undiluted passages through MK cells included plaque-formers in HeLa cells, suggesting that such plaque (HeLa)-forming viruses may have developed at a certain rate during multiplication of the original non-plaque (HeLa)-forming virus population in MK cells. Out of four lines of HeLa cells examined, two, including a clonal line S3, failed to support plaque formation by CA 9 virus.     

Nucleophosmin deposition during mRNA 3' end processing influences poly(A) tail length

During polyadenylation, the multi-functional protein nucleophosmin (NPM1) is deposited onto all cellular mRNAs analysed to date. Premature termination of poly(A) tail synthesis in the presence of cordycepin abrogates deposition of the protein onto the mRNA, indicating natural termination of poly(A) addition is required for NPM1 binding. NPM1 appears to be a bona fide member of the complex involved in 3' end processing as it is associated with the AAUAAA-binding CPSF factor and can be co-immunoprecipitated with other polyadenylation factors. Furthermore, reduction in the levels of NPM1 results in hyperadenylation of mRNAs, consistent with alterations in poly(A) tail chain termination. Finally, knockdown of NPM1 results in retention of poly(A)(+) RNAs in the cell nucleus, indicating that NPM1 influences mRNA export. Collectively, these data suggest that NPM1 has an important role in poly(A) tail length determination and may help network 3' end processing with other aspects of nuclear mRNA maturation.     

Properties of a HeLa cell 3' exonuclease specific for degrading poly(A) tails of mammalian mRNA.

A HeLa cell 3'-exonuclease with properties of a mammalian mRNA poly(A) tail-removing enzyme has been characterized. The exonuclease shows high specificity for the poly(A) tail, and it is single strand-specific and requires a 3'-hydroxyl group for its activity. During degradation 5'-AMP is liberated as a product, and a 3'-OH group is left on the last adenosine residue of the remaining poly(A) tail. The activity is inhibited by 5'-AMP and can be competed by poly(A)-containing mRNA or poly(A). Based on these findings we propose a reaction pathway for poly(A) tail removal catalyzed by the HeLa cell poly(A) tail-specific 3' exonuclease.     

3′-Terminal addition to HeLa cell nuclear and cytoplasmic poly(A)

Examination of poly(A) labeling in the nucleus and cytoplasm of HeLa cells indicates: (1) in accord with previous conclusions (Jelinek et al., 1973) that the nucleus is the site of de novo synthesis of poly(A), (2) both a nuclear and cytoplasmic 3′-terminal addition to poly(A) is described confirming and extending reports by Brawerman (1976) and colleagues, and (3) apparently only molecules bearing poly(A) ~230 nucleotides or longer exit to the cytoplasm.     

Rrp6p controls mRNA poly(A) tail length and its decoration with poly(A) binding proteins

Poly(A) (pA) tail binding proteins (PABPs) control mRNA polyadenylation, stability, and translation. In a purified system, S. cerevisiae PABPs, Pab1p and Nab2p, are individually sufficient to provide normal pA tail length. However, it is unknown how this occurs in more complex environments. Here we find that the nuclear exosome subunit Rrp6p counteracts the in vitro and in vivo extension of mature pA tails by the noncanonical pA polymerase Trf4p. Moreover, PABP loading onto nascent pA tails is controlled by Rrp6p; while Pab1p is the major PABP, Nab2p only associates in the absence of Rrp6p. This is because Rrp6p can interact with Nab2p and displace it from pA tails, potentially leading to RNA turnover, as evidenced for certain pre-mRNAs. We suggest that a nuclear mRNP surveillance step involves targeting of Rrp6p by Nab2p-bound pA-tailed RNPs and that pre-mRNA abundance is regulated at this level.     

Design of LNA-modified siRNAs against the highly structured 5' UTR of coxsackievirus B3

This study describes a strategy to develop LNA-modified small interfering RNA (siRNAs) against the highly structured 5' UTR of coxsackievirus B3 (CVB-3), which is an attractive target site due to its high degree of conservation. Accessible sites were identified based on structural models and RNase H assays with DNA oligonucleotides. Subsequently, LNA gapmers, siRNAs, siLNAs and small internally segmented interfering RNA (sisiLNAs) were designed against sites, which were found to be accessible in the in vitro assays, and tested in reporter assays and experiments with the infectious virus. The best siLNA improved viability of infected cells by 92% and exerted good antiviral activity in plaque reduction assays.     

The 3'-amido and 5'-amido analogues of adenosine 3':5'-monophosphate; interaction with cAMP-specific proteins.

The sensitivity for recognition of adenosine 3:5'-monophosphate (cAMP) by its coordinate proteins towards chemical changes in the six-membered cyclic phosphate ring has been investigated. A comparison of the interaction parameters of the 3' and 5'-amido analogues (I, II) and of unsubstituted cAMP has been made using two different protein kinases and the phosphodiesterase from bovine heart. Binding affinity and the capacity of the amido analogues to stimulate the phosphotransferase activity of the kinases is greatly reeuced relative to cAMP, the 3'-position being more sensitive towards the modification than the 5'-position. The coordinate noncyclic derivatives, 3'-deoxy-3'-amino-5'-AMP (IV) and 5'-deoxy-5'-amino-3'-amp (iii), were also tested. Surprisingly activity towards protein kinases was found to be considerable for the 5'-deoxy-5'-amino-3'-AMP (III), while the 3'-deoxy-3'-amino-5'-AMP (IV) is practically inactive. A possible reason for this is that the noncylic 5'-analogue (III) may be able to assume a cyclic structure maintained by internal salt formation. The phosphodiesterase splits both cyclic amido analogues but with reduced rates compared to that of natural cAMP. Kinetic data obtained from different methods reveal a stronger affinity for the 5'-analogue (I) than the 3'-analogue (II) for the active site, although the reaction rate at saturated substrate concentration is significantly higher with II than with I. The properties of the amido and the noncyclic amino analogues are discussed with available data from chemotaxis of the cellular slime moulds. Furthermore data of the respective methylene cyclic derivatives are used for a more comprehensive comparison. The above is interpreted in terms of the electronic features of the substitutions and of the changes in bond distances or angles upon replacement of O by NH or CH2 in the cyclic phosphate ring (obtained from X-ray work).     

Stabilization of "A" particles of coxsackievirus B3 by a HeLa cell plasma membrane extract.

Previous studies in our laboratory showed that HeLa cell plasma membranes were recovered from sucrose gradients in two major bands and that the heavier band possessed a putative inhibitor of uncoating of coxsackievirus B3. It has now been found that the mechanism of inhibition is the stabilization of "A" particles against inactivation at 37 degrees C. [3H]uridine-labeled virions converted to A particles by band 4, the heavier band, were four times more stable at 37 degrees C than those produced by band 3. Partially purified A particles from both bands were equally unstable. It was found that the stabilizing factor was extractable by saline from band 4 and remained soluble after centrifugation (109,000 X g for 2 h). Addition to A particles of this soluble factor isolated from either band 4 or band 3 stabilized the A particles. The stabilizing factor could not be replaced by an extract from band 3 or by bovine serum albumin. Thus, the finding that the membrane factor inhibits virus uncoating by stabilizing A particles against spontaneous disruption at 37 degrees C focuses attention on an inherent problem associated with defining receptor-mediated virus uncoating.     

Genomic determinants of cardiovirulence in coxsackievirus B3 clinical isolates: localization to the 5' nontranslated region

Coxsackievirus B3 (CVB3) infections can cause myocarditis in humans and are implicated in the pathogenesis of dilated cardiomyopathy. The natural genetic determinants of cardiovirulence for CVB3 have not been identified, although using strains engineered in the laboratory, cardiovirulence determinants have been identified in the CVB3 5' nontranslated region (5'NTR) and capsid. The myocarditic phenotypes of two CVB3 clinical isolates were determined using an established murine model of inflammatory heart disease. The 5'NTRs and capsid proteins of the noncardiovirulent CVB3/CO strain and cardiovirulent CVB3/AS strain were examined to determine their influence on the cardiovirulence phenotype. Six intratypic chimeric viruses were constructed in which 5'NTR and capsid sequences of the infectious cDNA copy of the cardiovirulent CVB3/20 genome were replaced by homologous sequences from CVB3/CO or CVB3/AS. Chimeric strains were tested for cardiovirulence by inoculation of C3H/HeJ mice. Sections of hearts removed at 10 days postinoculation were examined for evidence of myocarditis by light microscopy and assayed for the presence of virus. Replacement of the CVB3/20 capsid coding region by that from the homologous region of CVB3/CO resulted in no change in the cardiovirulent CVB3/20 phenotype, with virus recoverable from the heart at 10 days postinoculation. However, recombinant virus containing the CVB3/CO 5'NTR alone or the 5'NTR and capsid sequences together were not myocarditic, and infectious virus was not recovered from the myocardium. Chimeric viruses containing the CVB3/AS 5'NTR alone, capsid sequence alone, or both together preserved the myocarditic phenotype. These data support the 5'NTR as the primary site in the determination of the natural cardiovirulence phenotype of CVB3.     

Specific interactions of HeLa cell proteins with proposed translation domains of the poliovirus 5'' noncoding region.

To determine which sequences or structures in the poliovirus 5' noncoding region (5'NCR) are involved in binding proteins used for internal ribosome binding and protein synthesis initiation, translation competition assays were performed in rabbit reticulocyte lysates in the presence and absence of HeLa cell extract. The results revealed two functional domains in the poliovirus 5'NCR. One, requiring nucleotides (nts) 457 to 626, binds proteins that are required for translation of all mRNAs and that are present in both reticulocyte lysates and HeLa cell extracts. Another, contained within nts 286 to 456, interacts with proteins that are specific for poliovirus translation and are present in HeLa cells but not in significant amounts in rabbit reticulocyte lysates. In order to detect HeLa cell proteins that interact stably with the 5'NCR of poliovirus, UV cross-linking was used. At least four major protein-RNA complexes were identified, three of which were shown by RNA competition analysis to bind specifically to defined domains within the 5'NCR. Protein A (54 kDa) cross-linked to RNA sequences and/or structures located between nts 457 and 626; proteins B (48 kDa) and C (38 kDa) bound to nts 286 to 456.     

Mechanism of poly(ethylene glycol) interaction with proteins

Poly(ethylene glycol) (PEG) is one of the most useful protein salting-out agents. In this study, it has been shown that the salting-out effectiveness of PEG can be explained by the large unfavorable free energy of its interaction with proteins. Preferential interaction measurements of beta-lactoglobulin with poly(ethylene glycols) with molecular weights between 200 and 1000 showed preferential hydration of the protein for those with Mr greater than or equal to 400, the degree of hydration increasing with the increase in poly(ethylene glycol) molecular weight. The preferential interaction parameter had a strong cosolvent concentration dependence, with poly(ethylene glycol) 1000 having the sharpest decrease with an increase in concentration. The preferential hydration extrapolated to zero cosolvent concentration increased almost linearly with increasing size of the additive, suggesting steric exclusion as the major factor responsible for the preferential hydration. The poly(ethylene glycol) concentration dependence of the preferential interactions could be explained in terms of the nonideality of poly(ethylene glycol) solutions. All the poly(ethylene glycols) studied, when used at levels of 10-30%, decreased the thermal stability of beta-lactoglobulin, suggesting that caution must be exercised in the use of this additive at extreme conditions such as high temperature.     

Translational control of dosage compensation in Drosophila by Sex-lethal: cooperative silencing via the 5' and 3' UTRs of msl-2 mRNA is independent of the poly(A) tail.

Translational repression of male-specific-lethal 2 (msl-2) mRNA by Sex-lethal (SXL) controls dosage compensation in Drosophila. In vivo regulation involves cooperativity between SXL-binding sites in the 5' and 3' untranslated regions (UTRs). To investigate the mechanism of msl-2 translational control, we have developed a novel cell-free translation system from Drosophila embryos that recapitulates the critical features of mRNA translation in eukaryotes: cap and poly(A) tail dependence. Importantly, tight regulation of msl-2 translation in this system requires cooperation between the SXL-binding sites in both the 5' and 3' UTRs, as seen in vivo. However, in contrast to numerous other developmentally regulated mRNAs, the regulation of msl-2 mRNA occurs by a poly(A) tail-independent mechanism. The approach described here allows mechanistic analysis of translational control in early Drosophila development and has revealed insights into the regulation of dosage compensation by SXL.     

Presence of 2',5'-oligo(A) and of enzymes that synthesize, bind, and degrade 2',5'-oligo(A) in HeLa cell nuclei

Nuclei prepared from HeLa cells by lysis with nonionic detergents or by a nonaqueous fractionation procedure were assayed for enzymatic activities which synthesize, bind, and degrade 2',5'-oligo(A). Isolated nuclei synthesized micromolar concentrations of 2',5'-oligo(A) when incubated with poly(inosinic) . poly(cytidylic) acid. The products of nuclear synthesis were identified with authentic 2',5'-oligo(A) by several criteria. The nuclei synthesized nanomolar amounts of 2',5'-oligo(A) even when incubated without added double-stranded RNA. These oligonucleotides were identified by their pattern of degradation with different nucleases and by a specific competition-binding assay. This assay revealed the presence in nuclei of an activity which binds 2',5'-oligo(A) with an affinity constant similar to that of the cytoplasmic binding activity previously identified with the 2',5'-oligo(A)-dependent endoribonuclease (Nilsen, T. W., Wood, D. L., and Baglioni, C. (1981) J. Biol. Chem. 256, 10751-10754). The nuclei had also an activity which degraded 2',5'-oligo(A). Finally, unincubated nuclei isolated by the nonaqueous fractionation procedure contained detectable concentrations of 2',5'-oligo(A). These results show that an activator of the enzyme which synthesize 2',5'-oligo(A) is present in nuclei and that these oligonucleotides are normally formed in HeLa cells, and suggest a possible role for the 2',5'-oligo(A)-activated endoribonuclease in nuclear RNA metabolism.