Covalent circularization of exogenous RNA during incubation with a wheat embryo cell extract

Cell extracts from wheat embryos have been widely used for mRNA-directed protein production. Here, we found that a significant fraction of exogenous linear RNAs are circularized in a wheat embryo extract. The circularization was seen only in uncapped RNAs. The amount of the circular species reached around 1% of the initial RNA and increased along with an increase in the initial concentration more than proportionally. The circular RNAs were stable but unable to be translated in the extract. The circularization was competitively inhibited in the presence of a known substrate of a wheat embryo RNA ligase. Thus, we cloned the RNA ligase cDNAs. Three isoform sequences were homologous to the other plant RNA ligases. An addition of a cell-free synthesized wheat RNA ligase abolished the inhibition, which indicates a participation of its activity in the circularization. A possible role in RNA metabolism, RNA silencing in particular, is discussed.     

Use of domain enzymes from wheat RNA ligase for in vitro preparation of RNA molecules

Wheat RNA ligase can be dissected into three isolated domain enzymes that are responsible for its core ligase, 5′-kinase, and 2′,3′-cyclic phosphate 3′-phosphodiesterase activities, respectively. In the present study, we pursued a practical strategy using the domain enzymes for in vitro step-by-step ligation of RNA molecules. As a part of it, we demonstrated that a novel side reaction on 5′-tri/diphosphate RNAs is dependent on ATP, a 2′-phosphate-3′-hydroxyl end, and the ligase domain. Mass spectroscopy and RNA cleavage analyses strongly suggested that it is an adenylylation on the 5′ terminus. The ligase domain enzyme showed a high productivity for any of the possible 16 combinations of terminal bases and a high selectivity for the 5′-phosphate and 2′-phosphate-3′-hydroxyl ends. Two RNA molecules having 5′-hydroxyl and 2′,3′-cyclic monophosphate groups were ligated almost stoichiometrically after separate conversion of respective terminal phosphate states into reactive ones. As the product has the same terminal state as the starting material, the next rounds of ligation are also possible in principle. Thus, we propose a flexible method for in vitro RNA ligation.     

In vitro selection of RNA aptamers that selectively bind danofloxacin

Danofloxacin is a synthetic fluoroquinolone with broad spectrum antibacterial activity that is used for the treatment of respiratory diseases in animal husbandry. However, danofloxacin has many adverse reactions and is toxic to humans. Especially, it detrimentally affects muscle, central nerve system, peripheral nerve system, liver, and skin in those who ingest foods in which danofloxacin has accumulated. Prescreening and determination of the level of danofloxacin in foods or food products is necessary for human health. Aptamers are composing of oligonucleotides that specifically interact with target molecules. They are emerging as detection/diagnostic ligands. Here, we used the SELEX in vitro selection technology to identify specific and high-affinity RNA aptamers with 2′-fluoro-2′-deoxyribonucleotide modified pyrimidine nucleotides against danofloxacin. Selected RNA aptamers bound specifically to danofloxacin, but not to tetracycline. Truncation of RNA aptamer up to 36 mer did not comprise specificity and affinity. The truncated RNA aptamer specifically bound to target chemical, allowing the discrimination of danofloxacin from other fluoroquinolones. The isolated specific aptamer could be a potential agent used for the rapid and cost-effective detection and sensing of danofloxacin, replacing instrumental methods including the more expensive and time-consuming methods of high performance liquid chromatography and liquid chromatography/mass spectrometry.     

Different activities of the conserved lysine residues in the double-stranded RNA binding domains of RNA helicase A in vitro and in the cell

Background

RNA helicase A regulates a variety of RNA metabolism processes including HIV-1 replication and contains two double-stranded RNA binding domains (dsRBD1 and dsRBD2) at the N-terminus. Each dsRBD contains two invariant lysine residues critical for the binding of isolated dsRBDs to RNA. However, the role of these conserved lysine residues was not tested in the context of enzymatically active full-length RNA helicase A either in vitro or in the cells.

Methods

The conserved lysine residues in each or both of dsRBDs were substituted by alanine in the context of full-length RNA helicase A. The mutant RNA helicase A was purified from mammalian cells. The effects of these mutations were assessed either in vitro upon RNA binding and unwinding or in the cell during HIV-1 production upon RNA helicase A–RNA interaction and RNA helicase A-stimulated viral RNA processes.

Results

Unexpectedly, the substitution of the lysine residues by alanine in either or both of dsRBDs does not prevent purified full-length RNA helicase A from binding and unwinding duplex RNA in vitro. However, these mutations efficiently inhibit RNA helicase A-stimulated HIV-1 RNA metabolism including the accumulation of viral mRNA and tRNA^Lys3 annealing to viral RNA. Furthermore, these mutations do not prevent RNA helicase A from binding to HIV-1 RNA in vitro as well, but dramatically reduce RNA helicase A–HIV-1 RNA interaction in the cells.

Conclusions

The conserved lysine residues of dsRBDs play critical roles in the promotion of HIV-1 production by RNA helicase A.

General significance

The conserved lysine residues of dsRBDs are key to the interaction of RNA helicase A with substrate RNA in the cell, but not in vitro.     

Primary structure of a precursor to the aspartic proteinase from Rhizomucor miehei shows that the enzyme is synthesized as a zymogen

In order to characterize the zymogen of the milk-clotting enzyme from Rhizomucor miehei, we constructed a cDNA library on pBR327 in Escherichia coli. Aspartic proteinase-specific recombinants were isolated by colony hybridization to a specific oligonucleotide mixture, and the cDNA sequence corresponding to a precursor form of the enzyme was determined. The deduced amino acid sequence shows that this secreted fungal proteinase is synthesized as a precursor. The first 22 amino acid residues in this precursor constitute a typical signal peptide. The amino acid sequence of the following 47-amino-acid-long prosegment shows homology to the prosegments from both the extracellular and intracellular vertebrate aspartic proteinases, and to the prosegments from the yeast and Mucor pusillus aspartic proteinases as well. These observations suggest that all aspartic proteinases are synthesized with a prosegment and that this prosegment is essential for the correct folding of all the mature enzymes. The active Rhizomucor miehei enzyme consists of 361 amino acid residues with a total molecular weight of 38,701. Clusters of identities around the active site cleft support the assumption that these proteinases have a common folding of their peptide chains. The disulphide bridges were localized in the fungal enzyme, and 2 N-glycosylation sites were identified.     

RIP and RALyase cleave the sarcin/ricin domain, a critical domain for ribosome function, during senescence of wheat coleoptiles

Type-I ribosome-inactivating protein (RIP), which is found in many plants, catalyzes depurination of a specific adenine in the sarcin/ricin domain (SRD) of the large rRNA causing loss of ribosomal activity. Previously, we found a RNA apurinic site-specific lyase (RALyase) that catalytically cleaved the phosphodiester bond at the RIP-dependent depurination site by β-elimination reaction. Here we show that both the RIP activity and RIP-RALyase-mediated cleavage of SRD in the cytoplasmic ribosome were induced at the late stage of senescence of wheat coleoptiles. Following this process, tissue death was observed. Furthermore, transgenic tobacco plants expressing glucocorticoid-induced RIP developed senescence-like phenotype. Our results suggest that ribosome inactivation due to the cleavage of SRD by the inducible RIP and constitutively expressed RALyase may be a unique plant system that mediates programmed cell death at the late senescent stage.     

A general method of domain closure is applied to phosphoglycerate kinase and the result compared with the crystal structure of a closed conformation of the enzyme

The occurrence of large domain motions associated with the mechanism of action of many proteins is well established. We present a general method of predicting domain closure applicable to proteins containing domains separated by an apparent hinge. The method attempts to allow for natural directional bias within the closing protein by repeatedly applying a weak pulling force over a short distance between pairs of atoms chosen at random in the two domains in question. Appropriate parameters governing the pulling function were determined empirically. The method was applied to the bi-lobal protein PGK and a closed-form activated ternary complex generated for Bacillus stearothermophilus PGK. This model was compared with the recently determined crystal structure of closed-form Trypanosoma brucei PGK. The model predicts the correct hinge regions, although the magnitude of movement at one hinge point was overestimated, and provides a reasonable representation of the closed-form ternary complex. Proteins 30:372–380, 1998. © 1998 Wiley-Liss, Inc.     

Dynamic elements govern the catalytic activity of CapE,a capsular polysaccharide-synthesizing enzyme from Staphylococcus aureus

CapE is an essential enzyme for the synthesis of capsular polysaccharide (CP) of pathogenic strains of Staphylococcus aureus. Herein we demonstrate that CapE is a 5-inverting 4,6-dehydratase enzyme. However, in the absence of downstream enzymes, CapE catalyzes an additional reaction (5-back-epimerization) affording a by-product under thermodynamic control. Single-crystal X-ray crystallography was employed to identify the structure of the by-product. The structural analysis reveals a network of coordinated motions away from the active site governing the enzymatic activity of CapE. A second dynamic element (the latch) regulates the enzymatic chemoselectivity. The validity of these mechanisms was evaluated by site-directed mutagenesis.     

Modulation of the expression of ryanodine receptor mRNA from Plutella xylostella as a result of diamide insecticide application

Ryanodine receptors (RyRs), members of the largest family of calcium channel proteins, have been studied because of their key roles in calcium signalling within cells. With the development of diamide insecticides that exhibit a novel mode of action on the RyRs from Lepidoptera, research on insect RyRs has become more attractive in the field of plant protection. To enhance our understanding of the effects of diamides on RyRs, we cloned the Plutella xylostella RyR gene (Px-RyR), which is the most serious pest of Brassicaceae plants throughout the world. Furthermore, we investigated the modulation of the expression of Px-RyR as a result of the application of diamide insecticides. The full-length cDNAs of Px-RyR contain an open reading frame (ORF) of 15,372 bp with a predicted protein consisting of 5123 amino acids. Px-RyR possesses a high level of overall amino acid homology with other isoforms (77–92% identity with insect isoforms and 45–47% identity with vertebrate isoforms). The weight of Px. gradually decreased as the concentration of the diamides increased. However, the relative expression levels of the RyRs from larvae were dependent on the insecticide concentration and gradually increased with increasing insecticide concentrations.