Highly stable and efficient mRNA templates for mRNA-protein fusions and C-terminally labeled proteins

For high-throughput in vitro protein selection using genotype (mRNA)-phenotype (protein) fusion formation and C-terminal protein labeling as a post-selection analysis, it is important to improve the stability and efficiency of mRNA templates for both technologies. Here we describe an efficient single-strand ligation (90% of the input mRNAs) using a fluorescein-conjugated polyethylene glycol puromycin (Fluor-PEG Puro) spacer. This ligation provides a stable c-jun mRNA with a flexible Fluor-PEG Puro spacer for efficient fusion formation (70% of the input mRNA with the PEG spacer) in a cell-free wheat germ translation system. When using a 5' untranslated region including SP6 promoter and Omega29 enhancer (a part of tobacco mosaic virus Omega), an A(8) sequence (eight consecutive adenylate residues) at the 3' end is suitable for fusion formation, while an XA(8) sequence (XhoI and the A(8) sequence) is suitable for C-terminal protein labeling. Further, we report that Fluor-PEG N-t-butyloxycarbonylpuromycin [Puro(Boc)] spacer enhances the stability and efficiency of c-jun mRNA template for C-terminal protein labeling. These mRNA templates should be useful for puromycin-based technologies (fusion formation and C-terminal protein labeling) to facilitate high-throughput in vitro protein selection for not only evolutionary protein engineering, but also proteome exploration.     

2'-O-[2-[(N,N-dimethylamino)oxy]ethyl]-modified oligonucleotides inhibit expression of mRNA in vitro and in vivo

Synthesis and antisense activity of oligonucleotides modified with 2′-O-[2-[(N,N-dimethylamino)oxy] ethyl] (2′-O-DMAOE) are described. The 2′-O-DMAOE-modified oligonucleotides showed superior metabolic stability in mice. The phosphorothioate oligonucleotide ‘gapmers’, with 2′-O-DMAOE- modified nucleoside residues at the ends and 2′-deoxy nucleosides residues in the central region, showed dose-dependent inhibition of mRNA expression in cell culture for two targets. ‘Gapmer’ oligonucleotides have one or two 2′-O-modified regions and a 2′-deoxyoligonucleotide phosphorothioate region that allows RNase H digestion of target mRNA. To determine the in vivo potency and efficacy, BalbC mice were treated with 2′-O-DMAOE gapmers and a dose-dependent reduction in the targeted C-raf mRNA expression was observed. Oligonucleotides with 2′-O-DMAOE modifications throughout the sequences reduced the intercellular adhesion molecule-1 (ICAM-1) protein expression very efficiently in HUVEC cells with an IC₅₀ of 1.8 nM. The inhibition of ICAM-1 protein expression by these uniformly modified 2′-O-DMAOE oligonucleotides may be due to selective interference with the formation of the translational initiation complex. These results demonstrate that 2′-O-DMAOE- modified oligonucleotides are useful for antisense-based therapeutics when either RNase H-dependent or RNase H-independent target reduction mechanisms are employed.     

Activation of cap-independent translation by variant eukaryotic initiation factor 4G in vivo

Protein synthesis is tightly controlled by assembly of an intricate ribonucleoprotein complex at the m⁷GTP-cap on eukaryotic mRNAs. Ensuing linear scanning of the 5′ untranslated region (UTR) is believed to transfer the preinitiation complex to the initiation codon. Eukaryotic mRNAs are characterized by significant 5′ UTR heterogeneity, raising the possibility of differential control of translation initiation rate at individual mRNAs. Curiously, many mRNAs with unconventional, highly structured 5′ UTRs encode proteins with central biological roles in growth control, metabolism, or stress response. The 5′ UTRs of such mRNAs may influence protein synthesis rate in multiple ways, but most significantly they have been implicated in mediating alternative means of translation initiation. Cap-independent initiation bypasses strict control over the formation of initiation intermediates at the m⁷GTP cap. However, the molecular mechanisms that favor alternative means of ribosome recruitment are not understood. Here we provide evidence that eukaryotic initiation factor (eIF) 4G controls cap-independent translation initiation at the c-myc and vascular endothelial growth factor (VEGF) 5′ UTRs in vivo. Cap-independent translation was investigated in tetracycline-inducible cell lines expressing either full-length eIF4G or a C-terminal fragment (Ct) lacking interaction with eIF4E and poly(A) binding protein. Expression of Ct, but not intact eIF4G, potently stimulated cap-independent initiation at the c-myc/VEGF 5′ UTRs. In vitro RNA-binding assays suggest that stimulation of cap-independent translation initiation by Ct is due to direct association with the c-myc/VEGF 5′ UTR, enabling 43S preinitiation complex recruitment. Our work demonstrates that variant translation initiation factors enable unconventional translation initiation at mRNA subsets with distinct structural features.     

Cloning, Expression in Escherichia coli, and Characterization of Arabidopsis thaliana UMP/CMP Kinase

A cDNA encoding the Arabidopsis thaliana uridine 5′-monophosphate (UMP)/cytidine 5′-monophosphate (CMP) kinase was isolated by complementation of a Saccharomyces cerevisiae ura6 mutant. The deduced amino acid sequence of the plant UMP/CMP kinase has 50% identity with other eukaryotic UMP/CMP kinase proteins. The cDNA was subcloned into pGEX-4T-3 and expressed as a glutathione S-transferase fusion protein in Escherichia coli. Following proteolytic digestion, the plant UMP/CMP kinase was purified and analyzed for its structural and kinetic properties. The mass, N-terminal sequence, and total amino acid composition agreed with the sequence and composition predicted from the cDNA sequence. Kinetic analysis revealed that the UMP/CMP kinase preferentially uses ATP (Michaelis constant [K_m] = 29 μm when UMP is the other substrate and K_m = 292 μm when CMP is the other substrate) as a phosphate donor. However, both UMP (K_m = 153 μm) and CMP (K_m = 266 μm) were equally acceptable as the phosphate acceptor. The optimal pH for the enzyme is 6.5. P¹, P⁵-di(adenosine-5′) pentaphosphate was found to be a competitive inhibitor of both ATP and UMP.     

A Novel Phosphorylation-Dependent RNase Activity of GAP-SH3 Binding Protein: a Potential Link between Signal Transduction and RNA Stability

A potential p120 GTPase-activating protein (RasGAP) effector, G3BP (RasGAP Src homology 3 [SH3] binding protein), was previously identified based on its ability to bind the SH3 domain of RasGAP. Here we show that G3BP colocalizes and physically interacts with RasGAP at the plasma membrane of serum-stimulated but not quiescent Chinese hamster lung fibroblasts. In quiescent cells, G3BP was hyperphosphorylated on serine residues, and this modification was essential for its activity. Indeed, G3BP harbors a phosphorylation-dependent RNase activity which specifically cleaves the 3′-untranslated region of human c-myc mRNA. The endoribonuclease activity of G3BP can initiate mRNA degradation and therefore represents a link between a RasGAP-mediated signaling pathway and RNA turnover.     

Characterization of Metabolites in the Biotransformation of 2,4,6-Trinitrotoluene with Anaerobic Sludge: Role of Triaminotoluene

The present study describes the biotransformation of 2,4,6-trinitrotoluene (TNT) (220 μM) by using anaerobic sludge (10%, vol/vol) supplemented with molasses (3.3 g/liter). Despite the disappearance of TNT in less than 15 h, roughly 0.1% of TNT was attributed to mineralization (¹⁴CO₂). A combination of solid-phase microextraction–gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry identified two distinctive cycles in the degradation of TNT. One cycle was responsible for the stepwise reduction of TNT to eventually produce triaminotoluene (TAT) in relatively high yield (160 μM). The other cycle involved TAT and was responsible for the production of azo derivatives, e.g., 2,2′,4,4′-tetraamino-6,6′-azotoluene (2,2′,4,4′-TA-6,6′-azoT) and 2,2′,6,6′-tetraamino-4,4′-azotoluene (2,2′,6,6′-TA-4,4′-azoT) at pH 7.2. These azo compounds were also detected when TAT was treated with the anaerobic sludge but not with an autoclaved sludge, suggesting the biotic nature of their formation. When the anaerobic conditions in the TAT-containing culture medium were removed by aeration and/or acidification (pH 3), the corresponding phenolic compounds, e.g., hydroxy-diaminotoluenes and dihydroxy-aminotoluenes, were observed at room temperature. Trihydroxytoluene was detected only after heating TAT in water at 100°C. When ¹³CH₃-labeled TNT was used as the N source in the above microcosms, we were unable to detect ¹³C-labeled p-cresol or [¹³CH₃]toluene, indicating the absence of denitration or deamination in the biodegradation process. The formation and disappearance of TAT were not accompanied by mineralization, suggesting that TAT acted as a dead-end metabolite.     

Psoralen photo-crosslinked mRNA–puromycin conjugates: a novel template for the rapid and facile preparation of mRNA–protein fusions

We describe the synthesis of a novel type of mRNA template and its use in the preparation of mRNA–protein fusions. A light-induced psoralen crosslinking reaction was used to attach a puromycin-containing oligonucleotide to the 3′-end of an mRNA template. The photo-crosslinked template was found to undergo efficient mRNA–protein fusion formation in rabbit reticulocyte lysate. Fusion formation was subsequently tested with templates carrying puromycin linkers of different length and chemical composition. Short linkers with multiple triethyleneglycol phosphate building blocks allowed the most efficient fusion formation under a wide range of salt conditions. The present method simplifies the preparation of mRNA–protein fusions and thus significantly accelerates the in vitro protein evolution procedure which involves repetitive cycles of fusion production and selection.     

Chemical synthesis and translesion replication of a cis-syn cyclobutane thymine-uracil dimer

The cytosine base in DNA undergoes hydrolytic deamination at a considerable rate when UV radiation induces formation of a cyclobutane pyrimidine dimer (CPD) with an adjacent pyrimidine base. We have synthesized a phosphoramidite building block of a cis–syn cyclobutane thymine–uracil dimer (T[]U), which is the deaminated form of the CPD at a TC site, and incorporated it into oligodeoxyribonucleotides. The previously reported method for synthesis of the thymine dimer (T[]T) was applied, using partially protected thymidylyl-(3′–5′)-2′-deoxyuridine as the starting material, and after triplet- sensitized irradiation, the configuration of the base moiety in the major product was determined by NMR spectroscopy. Presence of the cis–syn cyclobutane dimer in the obtained oligonucleotides was confirmed by UV photoreversal and reaction with T4 endonuclease V. Using a 30mer containing T[]U, translesion synthesis by human DNA polymerase η was analyzed. There was no difference in the results between the templates containing T[]T and T[]U and pol η bypassed both lesions with the same efficiency, incorporating two adenylates. This enzyme showed fidelity to base pair formation, but this replication causes a C→T transition because the original sequence is TC.     

Docosahexaenoic acid-containing phosphatidylethanolamine enhances HL-60 cell differentiation by regulation of c-jun and c-myc expression

18:1/docosahexaenoic acid (DHA)-containing phosphatidylethanolamine (PE) enhanced cell differentiation and growth inhibition of HL-60 induced by dibutyryl cAMP (dbcAMP) in a dose-dependent manner. The combined treatment of 200 μM dbcAMP and 50 μM 18:1/DHA-PE increased the NBT reducing activity, which is as an indicator of cell differentiation, to more than 75% from 40% of cells treated with 200 μM dbcAMP alone. In HL-60 cells treated with 50 μM 18:1/DHA-PE and 200 μM dbcAMP for 24 h, the expression level of c-jun mRNA and c-Jun protein were remarkably elevated compared to cells treated with dbcAMP alone. In contrast, there was no difference in the expression levels of c-fos mRNA and c-Fos protein between the combination of 18:1/DHA-PE + dbcAMP or dbcAMP alone. On the other hand, the combine treatment of 18:1/DHA-PE and dbcAMP markedly reduced the expression level of c-myc oncogene during 48 h incubation. The decreases of c-myc mRNA by 18:1/DHA-PE and/or dbcAMP was correlated with growth inhibition effect. Thus, 18:1/DHA-PE might enhance dbcAMP-induced HL-60 cell differentiation and growth inhibition by regulation of c-jun and c-myc mRNA and their products.     

Identification of Genes Encoding Conjugated Bile Salt Hydrolase and Transport in Lactobacillus johnsonii 100-100

Cytosolic extracts of Lactobacillus johnsonii 100-100 (previously reported as Lactobacillus sp. strain 100-100) contain four heterotrimeric isozymes composed of two peptides, α and β, with conjugated bile salt hydrolase (BSH) activity. We now report cloning, from the genome of strain 100-100, a 2,977-bp DNA segment that expresses BSH activity in Escherichia coli. The sequencing of this segment showed that it contained one complete and two partial open reading frames (ORFs). The 3′ partial ORF (927 nucleotides) was predicted by BLAST and confirmed with 5′ and 3′ deletions to be a BSH gene. Thermal asymmetric interlaced PCR was used to extend and complete the 948-nucleotide sequence of the BSH gene 3′ of the cloned segment. The predicted amino acid sequence of the 5′ partial ORF (651 nucleotides) was about 80% similar to the C-terminal half of the largest, complete ORF (1,353 nucleotides), and these two putative proteins were similar to several amine, multidrug resistance, and sugar transport proteins of the major facilitator superfamily. E. coli DH5α cells transformed with a construct containing these ORFs, in concert with an extracellular factor produced by strain 100-100, demonstrated levels of uptake of [¹⁴C]taurocholic acid that were increased as much as threefold over control levels. [¹⁴C]Cholic acid was taken up in similar amounts by strain DH5α pSportI (control) and DH5α p2000 (transport clones). These findings support a hypothesis that the ORFs are conjugated bile salt transport genes which may be arranged in an operon with BSH genes.     

Specific bonding of puromycin to full-length protein at the C-terminus

Puromycin, an analog of the 3′ end of aminoacyl-tRNA, causes premature termination of translation by being linked non-specifically to growing polypeptide chains. Here we report the interesting phenomenon that puromycin acting as a non-inhibitor at very low concentration (e.g. 0.04 µM) can bond only to full-length protein at the C-terminus. This was proved by using a carboxypeptidase digestion assay of the products obtained by Escherichia coli cell-free translation of human tau 4 repeat (tau4R) mRNA in the presence of low concentrations of puromycin or its derivatives. The tau4R mRNA was modified to code for three C-terminal methionines, which were radioactively labeled, followed by a stop codon. The translation products could not be digested by carboxy-peptidase if puromycin or a derivative was present at the C-terminus of full-length tau4R. Puromycin and its derivatives at 0.04–1.0 µM bonded to 7–21% of full-length tau4R, depending on the ability to act as acceptor substrates. Furthermore, the bonding efficiency of a puromycin derivative to tau4R was decreased by addition of release factors. These results suggest that puromycin and its derivatives at concentrations lower than those able to compete effectively with aminoacyl-tRNA can bond specifically to full-length protein at a stop codon. This specific bonding of puromycin to full-length protein should be useful for in vitro selection of proteins and for in vitro and in vivo C-terminal end protein labeling.     

Hepatocyte Growth Factor-Induced Expression of Ornithine Decarboxylase, c-met,and c-mycIs Differently Affected by Protein Kinase Inhibitors in Human Hepatoma Cells HepG2

Binding of hepatocyte growth factor (HGF) to its receptor Met induces autophosphorylation and activation of the tyrosine kinase activity. In HGF-treated HepG2 cells, we studied: (i) the expression patterns of early(c-myc,c-jun,and c-fos) and delayed-early (ornithinedecarboxylase and c-met) response genes and (ii) thepossible involvement of protein kinase transducersin the control of the expression of c-metand of other genes eventually induced downstream. c-metand c-mycmRNAs peaked 1–2 h after HGF, while c-junandc-fosmRNAs slightly increased at 1 h. Ornithinedecarboxylase activity was induced earlier (4 h) thanthe mRNA (8–10 h). The transducers involved in HGF-triggered gene inductions were investigated using different protein kinase inhibitors: genistein for the receptor tyrosine kinase, herbimycin A for the nonreceptor tyrosine kinase (pp60^c-src), wortmannin for phosphatidylinositol 3-kinase (PI3K) and H7 for protein kinase C (PKC). The similarity of responses to PKC inhibition led to suppose that c-mycand ornithinedecarboxylase mRNAs were induced sequentially along the same transduction pathway triggered by HGF. Ornithine decarboxylase activity seemed to be largely regulated by phosphorylation(s). The mRNA expression of c-junwas likely to undergo a negative regulation through a mechanism involving PI3K, while that ofc-metseemed to be almost independent from various protein kinases (PI3K, pp60^c-src, and PKC).     

Complementarity of the 16S rRNA penultimate stem with sequences downstream of the AUG destabilizes the plastid mRNAs

Escherichia coli mRNA translation is facilitated by sequences upstream and downstream of the initiation codon, called Shine–Dalgarno (SD) and downstream box (DB) sequences, respectively. In E.coli enhancing the complementarity between the DB sequences and the 16S rRNA penultimate stem resulted in increased protein accumulation without a significant affect on mRNA stability. The objective of this study was to test whether enhancing the complementarity of plastid mRNAs downstream of the AUG (downstream sequence or DS) with the 16S rRNA penultimate stem (anti-DS or ADS region) enhances protein accumulation. The test system was the tobacco plastid rRNA operon promoter fused with the E.coli phage T7 gene 10 (T7g10) 5′-untranslated region (5′-UTR) and DB region. Translation efficiency was tested by measuring neomycin phosphotransferase (NPTII) accumulation in tobacco chloroplasts. We report here that the phage T7g10 5′-UTR and DB region promotes accumulation of NPTII up to ~16% of total soluble leaf protein (TSP). Enhanced mRNA stability and an improved NPTII yield (~23% of TSP) was obtained from a construct in which the T7g10 5′-UTR was linked with the NPTII coding region via a NheI site. However, replacing the T7g10 DB region with the plastid DS sequence reduced NPTII and mRNA levels to 0.16 and 28%, respectively. Reduced NPTII accumulation is in part due to accelerated mRNA turnover.     

Osmotic Stress Induces Expression of Choline Monooxygenase in Sugar Beet and Amaranth

Choline monooxygenase (CMO) catalyzes the committing step in the synthesis of glycine betaine, an osmoprotectant accumulated by many plants in response to salinity and drought. To investigate how these stresses affect CMO expression, a spinach (Spinacia oleracea L., Chenopodiaceae) probe was used to isolate CMO cDNAs from sugar beet (Beta vulgaris L., Chenopodiaceae), a salt- and drought-tolerant crop. The deduced beet CMO amino acid sequence comprised a transit peptide and a 381-residue mature peptide that was 84% identical (97% similar) to that of spinach and that showed the same consensus motif for coordinating a Rieske-type [2Fe-2S] cluster. A mononuclear Fe-binding motif was also present. When water was withheld, leaf relative water content declined to 59% and the levels of CMO mRNA, protein, and enzyme activity rose 3- to 5-fold; rewatering reversed these changes. After gradual salinization (NaCl:CaCl₂ = 5.7:1, mol/mol), CMO mRNA, protein, and enzyme levels in leaves increased 3- to 7-fold at 400 mm salt, and returned to uninduced levels when salt was removed. Beet roots also expressed CMO, most strongly when salinized. Salt-inducible CMO mRNA, protein, and enzyme activity were readily detected in leaves of Amaranthus caudatus L. (Amaranthaceae). These data show that CMO most probably has a mononuclear Fe center, is inducibly expressed in roots as well as in leaves of Chenopodiaceae, and is not unique to this family.     

Comparison of Gas Chromatography and Mineralization Experiments for Measuring Loss of Selected Polychlorinated Biphenyl Congeners in Cultures of White Rot Fungi

Two methods were used to compare the biodegradation of six polychlorinated biphenyl (PCB) congeners by 12 white rot fungi. Four fungi were found to be more active than Phanerochaete chrysosporium ATCC 24725. Biodegradation of the following congeners was monitored by gas chromatography: 2,3-dichlorobiphenyl, 4,4′-dichlorobiphenyl, 2,4′,5-trichlorobiphenyl (2,4′,5-TCB), 2,2′,4,4′-tetrachlorobiphenyl, 2,2′,5,5′-tetrachlorobiphenyl, and 2,2′,4,4′,5,5′-hexachlorobiphenyl. The congener tested for mineralization was 2,4′,5-[U-¹⁴C]TCB. Culture supernatants were also assayed for lignin peroxidase and manganese peroxidase activities. Of the fungi tested, two strains of Bjerkandera adusta (UAMH 8258 and UAMH 7308), one strain of Pleurotus ostreatus (UAMH 7964), and Trametes versicolor UAMH 8272 gave the highest biodegradation and mineralization. P. chrysosporium ATCC 24725, a strain frequently used in studies of PCB degradation, gave the lowest mineralization and biodegradation activities of the 12 fungi reported here. Low but detectable levels of lignin peroxidase and manganese peroxidase activity were present in culture supernatants, but no correlation was observed among any combination of PCB congener biodegradation, mineralization, and lignin peroxidase or manganese peroxidase activity. With the exception of P. chrysosporium, congener loss ranged from 40 to 96%; however, these values varied due to nonspecific congener binding to fungal biomass and glassware. Mineralization was much lower, ≤11%, because it measures a complete oxidation of at least part of the congener molecule but the results were more consistent and therefore more reliable in assessment of PCB biodegradation.     

Syntheses and structural studies of calix[4]arene-nucleoside and calix[4]arene-oligonucleotide hybrids

We have synthesized three types of calix[4]arene– nucleoside hybrid efficiently by amide bond formation between the amine functional groups of 1,3-diaminocalix[4]arene and the carboxyl groups of thymidine nucleoside derivatives. X-ray crystallography of a homocoupled calix[4]arene–nucleoside hybrid revealed an interesting hydrogen bonding pattern between thymine bases and the amide linkages. We designed the calix[4]arene–oligonucleotide hybrids (5′-AAAAGATATCAAXTTGATATCTTTT-3′, 5′-T₁₂-X-T₁₂-3′, and 5′-A₁₂-X-T₁₂-3′) to be V-shaped oligodeoxyribonucleotides and synthesized them by using a calix[4]arene–nucleoside hybrid (X) as a key building block. Thermal denaturation experiments, monitored by UV spectroscopy at 260 and 284 nm, and circular dichroism spectra of the calix[4]arene–oligonucleotide hybrids suggest that the modified oligonucleotides indeed adopt V-shaped conformations, making them suitable for use as building blocks in the construction of programmed oligonucleotide nanostructures.     

AUUUA Sequences Direct mRNA Deadenylation Uncoupled from Decay during Xenopus Early Development

To study the regulation of AUUUA-mediated RNA deadenylation and destabilization during Xenopus early development, we microinjected chimeric mRNAs containing Xenopus or mammalian 3′ untranslated region (3′-UTR) sequences into Xenopus oocytes, mature eggs, or fertilized embryos. We found that the AU-rich elements (ARE) of Xenopus c-myc II and the human granulocyte-macrophage colony-stimulating factor gene (GMCSF) both direct deadenylation of chimeric mRNAs in an AUUUA-dependent manner. In the case of the Xenopus c-myc II ARE, mutation of a single AUUUA within an absolutely conserved 11-nucleotide region in c-myc 3′-UTRs prevents ARE-mediated deadenylation. AUUUA-specific deadenylation appears to be developmentally regulated: low deadenylation activity is observed in the oocyte, whereas rapid deadenylation occurs following egg activation or fertilization. Deadenylation results in the accumulation of stable deadenylated RNAs that become degraded only following mid-blastula transition. We conclude that ARE-mediated mRNA deadenylation can be uncoupled from ARE-mediated mRNA decay and that AUUUAs directly signal deadenylation during Xenopus early development.