Inclusion of a Furin Cleavage Site Enhances Antitumor Efficacy against Colorectal Cancer Cells of Ribotoxin α-Sarcin- or RNase T1-Based Immunotoxins.

Immunotoxins are chimeric molecules that combine the specificity of an antibody to recognize and bind tumor antigens with the potency of the enzymatic activity of a toxin, thus, promoting the death of target cells. Among them, RNases-based immunotoxins have arisen as promising antitumor therapeutic agents. In this work, we describe the production and purification of two new immunoconjugates, based on RNase T1 and the fungal ribotoxin α-sarcin, with optimized properties for tumor treatment due to the inclusion of a furin cleavage site. Circular dichroism spectroscopy, ribonucleolytic activity studies, flow cytometry, fluorescence microscopy, and cell viability assays were carried out for structural and in vitro functional characterization. Our results confirm the enhanced antitumor efficiency showed by these furin-immunotoxin variants as a result of an improved release of their toxic domain to the cytosol, favoring the accessibility of both ribonucleases to their substrates. Overall, these results represent a step forward in the design of immunotoxins with optimized properties for potential therapeutic application in vivo.

X-ray crystallographic structure of RNase Po1 that exhibits anti-tumor activity.

RNase Po1 is a guanylic acid-specific ribonuclease member of the RNase T1 family from Pleurotus ostreatus. We previously reported that RNase Po1 inhibits the proliferation of human tumor cells, yet RNase T1 and other T1 family RNases are non-toxic. We determined the three-dimensional X-ray structure of RNase Po1 and compared it with that of RNase T1. The catalytic sites are conserved. However, there are three disulfide bonds, one more than in RNase T1. One of the additional disulfide bond is in the catalytic and binding site of RNase Po1, and makes RNase Po1 more stable than RNase T1. A comparison of the electrostatic potential of the molecular surfaces of these two proteins shows that RNase T1 is anionic whereas RNase Po1 is cationic, so RNase Po1 might bind to the plasma membrane electrostatically. We suggest that the structural stability and cationic character of RNase Po1 are critical to the anti-cancer properties of the protein.

The inhibition of human tumor cell proliferation by RNase Pol, a member of the RNase T1 family, from Pleurotus ostreatus.

RNase Po1 is a guanylic acid-specific ribonuclease (a RNase T1 family RNase) from Pleurotus ostreatus. We determined the cDNA sequence encoding RNase Po1 and expressed RNase Po1 in Escherichia coli. A comparison of the enzymatic properties of RNase Po1 and RNase T1 indicated that the optimum temperature for RNase Po1 activity was 20 °C higher than that for RNase T1. An MTT assay indicated that RNase Po1 inhibits the proliferation of human neuroblastoma cells (IMR-32 and SK-N-SH) and human leukemia cells (Jurkat and HL-60). Furthermore, Hoechst 33342 staining showed morphological changes in HL-60 cells due to RNase Po1, and flow cytometry indicated the appearance of a sub-G1 cell population. The extent of these changes was dependent on the concentration of RNase Pol. We suggest that RNase Po1 induces apoptosis in tumor cells.

Mechanism of T7 RNAP pausing and termination at the T7 concatemer junction: a local change in transcription bubble structure drives a large change in transcription complex architecture.

The T7RNA polymerase (RNAP) elongation complex (EC) pauses and is destabilized at a unique 8 nucleotide (nt) sequence found at the junction of the head-to-tail concatemers of T7 genomic DNA generated during T7 DNA replication. The paused EC may recruit the T7 DNA processing machinery, which cleaves the concatemerized DNA within this 8 nt concatemer junction (CJ). Pausing of the EC at the CJ involves structural changes in both the RNAP and transcription bubble. However, these structural changes have not been fully defined, nor is it understood how the CJ sequence itself causes the EC to change its structure, to pause, and to become less stable. Here we use solution and RNAP-tethered chemical nucleases to probe the CJ transcript and changes in the EC structure as the polymerase pauses and terminates at the CJ. Together with extensive mutational scanning of regions of the polymerase that are likely to be involved in recognition of the CJ, we are able to develop a description of the events that occur as the EC transcribes through the CJ and subsequently pauses. In this process, a local change in the structure of the transcription bubble drives a large change in the architecture of the EC. This altered EC structure may then serve as the signal that recruits the processing machinery to the CJ.

[Inhibition of mutant-type p53 by a chimeric U6 maxizyme in hepatocellular carcinoma cell lines].

OBJECTIVE: To study the inhibition of maxizyme (Mz) directed against the mutant-type p53 gene (mtp53) at codon 249 in exon 7 (AGG --> AGT) both in cell-free system and in MHCC97 cell lines. METHODS: Maxizyme and control mutant maxizyme (G5 --> A5) were designed by computer and cloned into the eukaryotic expression vector pBSKneoU6 (pU6Mz, pU6asMz). Mz was driven by T7 RNA polymerase promoter in vitro. In the cell lines, U6 promoter was driven by RNA PolIII. The mutant type p53 gene fragment was cloned into the pGEM-T vector under the T7 promoter control. The 32P-labeled mtp53 transcript was the target RNA. Cold maxizyme transcripts were incubated with 32P-labeled target RNA in vitro. pU6Mz was introduced into MHCC97 cells by Lipofectamine2000 and mtp53 expression was analyzed by RT-PCR and Western blot. RESULTS: In vitro cleavage showed that pU6Mz was very active with cleavage efficiency of 42% while pU6asMz was not. The wild type p53 was not cleaved. Partial down-regulation of mtp53 mRNA and mtp53 protein were observed in MHCC97 cells transfected with pU6Mz but not those with pU6asMz. The proliferation of MHCC cells was inhibited by MTT analysis. CONCLUSION: Our findings suggest that the chimeric U6 maxizyme against the mtp53 is a new promising gene therapeutic agent in treating hepatocellular carcinoma.

Association of ribosomal proteins with nascent transcripts in S. cerevisiae.

Although it is generally accepted that transcription and translation are spatially separated in eukaryotes, a number of recent observations have called this belief into question. In particular, several studies have shown that parts of the translation machinery, including ribosomal proteins, can be found associated with sites of active transcription in metazoans. Here we describe results of chromatin immunoprecipitation (ChIP) experiments designed to determine whether ribosomal proteins associate with nascent transcripts in Saccharomyces cerevisiae and whether this association reflects a functional engagement of the translation machinery. We find that HAT-tagged ribosomal proteins can be detected in association with nascent RNAs in budding yeast. However, our data clearly indicate that this binding is independent of transcript translatability, so is therefore not indicative of nuclear translation.

Regulated expression of functional external guide sequences in mammalian cells using a U6 RNA polymerase III promoter.

A regulatable promoter has been stably integrated into a human embryonic kidney cell line. The promoter is a pol III mouse promoter and is under the control of ponasterone A, an ecdysone inducer. The promoter controls transcription of an external guide sequence (EGS) targeted against Rpp38, a protein subunit of human RNase P, or of lamin A/C, a gene product located in the nucleus. The amounts of protein of both gene products are severely reduced when the EGSs are made. Several other, but not all, of the protein subunits of RNase P are also inhibited in both mRNA and protein levels when Rpp38 mRNA is targeted.

Chemical and enzymatic probing of effector-mediated changes in the conformation of a maxizyme.

The protein encoded by chimeric BCR-ABL mRNA causes chronic myelogenous leukemia (CML). We showed previously that a novel allosterically controllable ribozyme, of the type known as a maxizyme, can cleave this mRNA, with high specificity and high-level activity in vivo. We designed the maxizyme in such a way that it was able to form an active core with which to capture the catalytically indispensable Mg2+ ions only in the presence of the BCR-ABL mRNA junction. In order to probe the putative conformational changes, we used a weakly alkaline solution (pH 9.2) in the presence of 25 mM Mg2+ ions to hydrolyze differentially phosphodiester bonds that were located in different environments. Phosphodiester bonds in single-stranded regions were clearly more susceptible to attack by alkali than those within a double-stranded helix. As indicated by earlier data obtained in vivo, our results demonstrated that the active conformation was achieved only in the presence of the junction within the chimeric BCR-ABL mRNA. Moreover, we demonstrated that the use of mild alkaline solutions to probe RNA structures is very informative.

Peptidyl prolyl cis-trans-isomerase activity associated with the lumen of the endoplasmic reticulum.

Peptidyl prolyl cis-trans-isomerase (PPI) activity was detected in microsomal fractions from bovine and rat liver. Extensive washing, proteinase and sonication treatments indicated that although some of this activity was due to adsorbed cytosolic enzymes, there was also an active but latent microsomal PPI activity. Density-gradient subfractionation indicated that activity was associated with vesicles derived from both the rough and the smooth endoplasmic reticulum (ER), suggesting that the activity was located within the ER lumen. The luminal PPI activity was inhibited by cyclosporin A and was active towards an unfolded protein substrate as well as towards the standard peptide substrate.

Protein-S-S-glutathione mixed disulfides as models of unfolded proteins.

Mixed disulfides between glutathione and the reduced forms of disulfide-bonded proteins were generated and characterized to explore their suitability as models of the unfolded state of newly-synthesized secretory proteins. RNase T1 and alpha-lactalbumin were reduced and converted to mixed disulfide derivatives, named GS-RNase T1 and GS-alpha-lactalbumin, in good yield; the molecular masses of the derivatives were confirmed by electrospray mass spectrometry. The intrinsic fluorescence of the derivatives and the binding of the hydrophobic fluorescent dye ANS were characteristic of fully unfolded proteins. Fluorescence studies and enzyme activity data indicated that GS-RNase T1 could be refolded to a nativelike state at NaCl concentrations greater than 1.5 M, as was previously demonstrated for the reduced, carboxymethylated derivative of this protein. The [NaCl]-dependent folding/unfolding equilibrium for GS-RNase T1 was reversible and could be influenced by urea. Fluorescence studies indicated that GS-alpha-lactalbumin showed a [NaCl]-dependent partial shift toward a more nativelike state, which was enhanced by the presence of Ca2+ ions. Both of the GS derivatives stimulated the ATPase activity of BiP, with apparent affinities in the range 0.1-1.0 mM. The results indicate that these GS-S-protein mixed disulfide derivatives are ideal model unfolded proteins that can be used as substrates for detailed studies on secretory protein folding in vitro and on the interactions between unfolded proteins and facilitators of protein folding.

The role of posttranscriptional modification in stabilization of transfer RNA from hyperthermophiles.

The influence of posttranscriptional modification on structural stabilization of tRNA from hyperthermophilic archaea was studied, using Pyrococcus furiosus (growth optimum 100 degrees C) as a primary model. Optical melting temperatures (Tm) of unfractionated tRNA in 20 mM Mg2+ are 97 degrees C for P. furiosus and 101.5 degrees C for Pyrodictium occultum (growth optimum, 105 degrees C). These values are approximately 20 degrees C higher than predicted solely from G-C content and are attributed primarily to posttranscriptional modification. Twenty-three modified nucleosides were determined in total digests of P. furiosus tRNA by combined HPLC-mass spectrometry. From cells cultured at 70, 85, and 100 degrees C, progressively increased levels of modification were observed within three families of nucleosides, the most highly modified forms of which were N4-acetyl-2'-O-methylcytidine (ac4Cm), N2,N2,2'-O-trimethylguanosine (m2(2)Gm), and 5-methyl-2-thiouridine (m5s2U). Nucleosides ac4Cm and m2(2)Gm, which are unique to the archaeal hyperthermophiles, were shown in earlier NMR studies to exhibit unusually high conformational stabilities that favor the C3'-endo form [Kawai, G., et al. (1991) Nucleic Acids Symp. Ser. 21, 49-50; (1992) Nucleosides Nucleotides 11, 759-771]. The sequence location of m5s2U was determined by mass spectrometry to be primarily at tRNA position 54, a site of known thermal stabilization in the bacterial thermophile Thermus thermophilus [Horie, N., et al. (1985) Biochemistry 24, 5711-5715]. It is concluded that selected posttranscriptional modifications in archaeal thermophiles play major stabilizing roles beyond the effects of Mg2+ binding and G-C content, and are proportionally more important and have evolved with greater structural diversity at the nucleoside level in the bacterial thermophiles.

5S rRNA modification in the hyperthermophilic archaea Sulfolobus solfataricus and Pyrodictium occultum.

The 5S rRNAs from Sulfolobus solfataricus and Pyrodictium occultum were digested to nucleosides and analyzed using directly-combined HPLC/mass spectrometry. P. occultum 5S rRNA contains two modified nucleoside species, N4-acetylcytidine (ac4C) and N4-acetyl-2'-O-methylcytidine (ac4Cm). Oligonucleotides were generated from P. occultum 5S rRNA by RNase T1 hydrolysis, and their molecular weights were determined using electrospray mass spectrometry and compared with those predicted from the P. occultum 5S RNA gene sequence. Deviation in mass between expected and observed molecular weights permitted ac4Cm to be located at position 35, in the nonanucleotide CAA-CACC[ac4Cm]G, and the ac4C in one or both of two (C,U)G trinucleotides. 2'-O-Methylcytidine is unambiguously characterized in S. solfataricus 5S rRNA, confirming earlier tentative assignments at the analogous sequence position (Stahl, D.A., Luehrsen, K.R., Woese, C.R., and Pace, N.R. (1981) Nucleic Acids Res., Vol. 9, pp. 6129-6137; Dams, E., Londei, P., Cammarano, P., Vandenberghe, A., and De Wachter, R. (1983) Nucleic Acids Res. Vol. 11, pp. 4667-4676). Potential effects of the presence of ac4C and ac4Cm on thermal stabilization of 5S rRNA in thermophiles are discussed.

Direct evidence for interaction of the conserved GTPase domain within 28 S RNA with mammalian ribosomal acidic phosphoproteins and L12.

A complex consisting of the acidic phosphoproteins P0, P1, and P2 (P proteins), L12, and RNA fragments was isolated from rat liver ribosomes after treatment with RNase T1 in the presence of EDTA. The complex was reactive with the anti-28 S RNA antibody specific for the highly conserved "GTPase domain" within 28 S rRNA. This suggests an association of these proteins with the RNA domain. To characterize this complex, the P proteins and L12 were isolated and tested for their binding specificity to the RNA by RNase T1 protection and gel retardation assays. Protein L12 and the P protein complex (P complex) both bound to rat 28 S rRNA and protected sequences comprising residues 1859-1921 and 1838-1936, respectively. The sequences overlap each other and lie in the GTPase domain. An in vitro transcript covering residues 1841-1936 of the 28 S rRNA as well as the protected RNA fragments also showed an ability to bind to the P complex and L12, and the binding was cooperative. RNA sequence elements within residues 1841-1936 required for protein binding were defined using site-directed mutagenesis. A unique internal loop including residues 1858 and 1859 and a distinct subregion comprising residues 1867-1914 in this domain were necessary for the binding of the P complex and L12, respectively. These results indicate that P proteins and L12 bind to restricted sites in the GTPase domain and that the complex constitutes the GTPase-related functional site in mammalian ribosomes.

Endonucleolysis in the turnover of insulin-like growth factor II mRNA.

The overlapping transcription units constituting the rat insulin-like growth factor II (IGF-II) locus generate multiple mRNAs by using different promoters. Three promoters have been identified, giving rise to 4.6-, 3.8-, and 3.6-kilobase mRNAs. The latter, originating from promoter P3, is the most abundant IGF-II mRNA in the rat liver cell-line BRL-3A. Moreover, a non-polyadenylated 1.2-kilobase (kb) transcript and a 1.8-kb tail fragment are prominent transcripts at steady-state. In this study, we show that the 1.8-kb tail fragment is uncapped and sediments as a 30 S ribonucleoprotein particle, and is thus not actively engaged in protein synthesis. In contrast, both the 3.6-kb mRNA and the 1.2-kb transcript cosediment with polysomes. In the presence of cytoplasmic extracts, the full-length 3.6-kb mRNA is cleaved into the 1.8-kb tail fragment and a similar-sized upstream fragment. The cleavage occurs between a putative hairpin and a phylogenetically conserved guanosine-rich region which forms a stable higher order RNA structure in the presence of K+. We suggest that endonucleolysis is the initial step in IGF-II mRNA decay and that this event may participate in the post-transcriptional regulation of IGF-II production.

RNA hydrolysis via an oxyphosphorane intermediate.

From calculations of a model reaction scheme for base-catalyzed RNA hydrolysis, a pentacoodinate dianionic intermediate 2a (Storer, et al., J. Am. Chem. Soc., 1991, 113, 5216-5219) as well as two transition states, TS1 and TS2, to the intermediate have been located by ab initio calculations at the 3-21G* level. Although the intermediate, which has the well depth on the order of kBT, is unlikely to be kinetically significant, the overall rate-limiting transition state structure TS2 obtained at 3-21G* level is very close to the corresponding structure at the STO-3G level; it has an extended P-O(5') bond breaking character. These gas-phase calculation results are used to qualitatively interpret mutagenesis results of Barnase and RNase T1 where water molecules are absent from the active site.

Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNA(Lys).

Human immunodeficiency virus (HIV) reverse transcriptase (RT) uses host tRNA(Lys) partially annealed to the primer binding site (PBS) as primer for the initiation of cDNA synthesis. When assaying cDNA synthesis with a template-primer complex formed by an RNA fragment carrying the PBS site and bovine tRNA(Lys) we noticed that an excess of primer tRNA inhibited strongly the DNA polymerase activity of a recombinant HIV RT (p66-p51 heterodimeric form) produced in transformed yeast cells. The same inhibitory effect was observed with animal DNA polymerase alpha, while avian retrovirus RT was neither affected by tRNA(Lys) nor by its specific primer tRNA(Trp). Although the strongest inhibition was observed with tRNA(Lys), other tRNas like tRNA(Phe) and tRNA(Trp) inhibited also the HIV RT, whereas tRNAs specific for valine, proline and glycine had no effect on enzyme activity. Digestion of tRNA(Lys) with pancreatic RNase abolished the inhibition; on the other hand T1 RNase digestion had no effect on the inhibition suggesting a role of the anticodon region in this effect. The 12- and 14-mers corresponding to the anticodon regions of the three bovine tRNA(Lys) isoacceptors inhibited RT activity, indicating that at least an important part of the inhibitory effect could be ascribed to this tRNA region. A strong stimulation of DNA polymerase activity was observed when the effect of tRNA(Lys) was assayed on a recombinant HIV reverse transcriptase produced in a protease deficient yeast strain, which leads to the production of an active p66 enzyme. The same tRNAs that inhibited strongly the heterodimeric form stimulated the p66 form of HIV reverse transcriptase. The results suggest that although both enzymatic forms are able to interact with tRNA(Lys) the topography, as well as the functional implications of the interaction between the precursor and the mature form of HIV reverse transcriptase with the tRNA(Lys) primer, are different.

A cytosolic protein binds to structural elements within the iron regulatory region of the transferrin receptor mRNA.

The level of mRNA encoding the transferrin receptor (TfR) is regulated by iron, and this regulation is mediated by a portion of the 3' untranslated region (UTR) of the TfR transcript. This portion of 3' UTR of the human TfR mRNA contains five RNA elements that have structural similarity to the iron-responsive element (IRE) found as a single copy in the 5' UTR of the mRNA for ferritin, whose translation is regulated by iron. Moreover, five very similar elements are also contained in the 3' UTR of the chicken TfR mRNA. Cytosolic extracts of human cell lines are shown by a gel shift assay involving RNase T1 protection to contain an IRE-binding protein capable of specific interaction with the human TfR 3' UTR. When the protecting protein is removed, the protected RNA can be digested with RNase T1 to yield oligoribonucleotide fragments characteristic of two of the IREs contained in the TfR 3' UTR. As judged by cross-competition experiments, the same IRE-binding protein interacts with the ferritin IRE. The apparent affinity of RNA sequence elements for the IRE-binding protein is shown to depend upon the sequence of the RNA. A comprehensive secondary structure for the regulatory region of the TfR mRNA is proposed based on the experimentally demonstrated presence of at least two IRE-like structural elements.

Temperature dependent chemical and enzymatic probing of the tRNA-like structure of TYMV RNA.

In this paper we report on the thermal unfolding of the tRNA-like structure present at the 3' end of turnip yellow mosaic virus (TYMV) RNA. Diethyl pyrocarbonate (DEP), sodium bisulphite, nuclease S1 and ribonuclease T1 were used as structure probes at a broad range of temperatures. In this way most of the nucleotides present in the tRNA-like moiety were analysed. The melting behaviour of both secondary and tertiary interactions could be followed on the basis of the temperature dependent accessibility of the individual nucleotides or bases towards the various probes. The three-dimensional model of the tRNA-like domain (Dumas et al., J. Biomol. Struct. and Dyn. 4, 707 (1987] was supported by the results to a large extent. The interactions occurring between the T- and D-loop appear to be more complex than proposed in the latter model. Additional evidence for the presence of the RNA pseudoknot (Rietveld et al., Nucleic Acids Res. 10, 1929 (1982] was derived from the fact that the three coaxially stacked helical segments in the aminoacylacceptor arm displayed different melting transitions under certain experimental conditions. Aspects of melting behaviour and thermal stability of double helical regions within the tRNA-like structure are discussed, as well as the applicability of nucleases and modifying reagents at various temperatures in the analysis of RNA structure.