Regio-selective preparation of vinyl adenosine ester catalyzed by alkaline protease

The transesterification of divinyladipate with adenosine in DMF containing 20% (v/v) DMSO was catalyzed by Streptomyces sp. alkaline protease and esterification occurred exclusively at the 3-position of hydroxyl group of ribofuranose in adenosine to give 3-O-vinyladipoyl adenosine without other products.     

Specific interactions between Dicer-like proteins and HYL1/DRB- family dsRNA-binding proteins in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Proteins that specifically bind double-stranded RNA (dsRNA) are involved in the regulation of cellular signaling events and gene expression, and are characterized by a conserved dsRNA-binding motif (dsRBM). Here we report the biochemical properties of nine such gene products, each containing one or two dsRBMs: four ArabidopsisDicer-like proteins (DCL1-4), ArabidopsisHYL1 and four of its homologs (DRB2, DRB4, DRB5 and OsDRB1). DCL1, DCL3, HYL1 and the four HYL1 homologs exhibit significant dsRNA-binding activity, indicating that these proteins are involved in RNA metabolism. The dsRBMs from dsRBM-containing proteins (dsRBPs) also function as a protein–protein interaction domain and homo- and heterodimerization are essential for biological functioning of these proteins. We show that DRB4 interacts specifically with DCL4, and HYL1 most strongly interacts with DCL1. These results indicate that each HYL1/DRB family protein interacts with one specific partner among the four Dicer-like proteins. Localization studies using GFP fusion proteins demonstrate that DCL1, DCL4, HYL1 and DRB4 localize in the nucleus, while DRB2 is present in the cytoplasm. Subcellular localizations of HYL1, DRB4, DCL1 and DCL4 further strengthen the notion that HYL1 and DCL1, and DRB4 and DCL4, exist as complexes. The presented data suggest that each member of the HYL1/DRB protein family may individually modulate Dicer function through heterodimerization with a Dicer-like protein in vivo.     

Specific requirement of DRB4, a dsRNA-binding protein, for the in vitro dsRNA-cleaving activity of Arabidopsis Dicer-like 4

Arabidopsis thaliana Dicer-like 4 (DCL4) produces 21-nt small interfering RNAs from both endogenous and exogenous double-stranded RNAs (dsRNAs), and it interacts with DRB4, a dsRNA-binding protein, in vivo and in vitro. However, the role of DRB4 in DCL4 activity remains unclear because the dsRNA-cleaving activity of DCL4 has not been characterized biochemically. In this study, we biochemically characterize DCL4's Dicer activity and establish that DRB4 is required for this activity in vitro. Crude extracts from Arabidopsis seedlings cleave long dsRNAs into 21-nt small RNAs in a DCL4/DRB4-dependent manner. Immunoaffinity-purified DCL4 complexes produce 21-nt small RNAs from long dsRNA, and these complexes have biochemical properties similar to those of known Dicer family proteins. The DCL4 complexes purified from drb4-1 do not cleave dsRNA, and the addition of recombinant DRB4 to drb4-1 complexes specifically recovers the 21-nt small RNA generation. These results reveal that DCL4 requires DRB4 to cleave long dsRNA into 21-nt small RNAs in vitro. Amino acid substitutions in conserved dsRNA-binding domains (dsRBDs) of DRB4 impair three activities: binding to dsRNA, interacting with DCL4, and facilitating DCL4 activity. These observations indicate that the dsRBDs are critical for DRB4 function. Our biochemical approach and observations clearly show that DRB4 is specifically required for DCL4 activity in vitro.     

Distinct substrate specificities of Arabidopsis DCL3 and DCL4

In Arabidopsis thaliana, Dicer-like 3 (DCL3) and Dicer-like 4 (DCL4) cleave long, perfect double-stranded RNAs (dsRNAs) into 24 and 21 nucleotides (nt) small interfering RNAs, respectively, which in turn function in RNA-directed DNA methylation and RNA interference, respectively. To reveal how DCL3 and DCL4 individually recognize long perfect dsRNAs as substrates, we biochemically characterized DCL3 and DCL4 and compared their enzymatic properties. DCL3 preferentially cleaves short dsRNAs with 5′ phosphorylated adenosine or uridine and a 1 nt 3′ overhang, whereas DCL4 cleaves long dsRNAs with blunt ends or with a 1 or 2 nt 3′ overhang with similar efficiency. DCL3 produces 24 nt RNA duplexes with 2 nt 3′ overhangs by the 5′ counting rule. Inorganic phosphate, NaCl and KCl enhance DCL3 activity but inhibit DCL4 activity. These results indicate that plants use DCLs with distinct catalytic profiles to ensure each dsRNA substrate generates only a specific length of siRNAs that trigger a unique siRNA-mediated response.     

An Arabidopsis RNase III-like protein,AtRTL2, cleaves double-stranded RNA in vitro

Class 1 ribonuclease III (RNase III), found in bacteria and yeast, is involved in processing functional RNA molecules such as ribosomal RNAs (rRNAs). However, in Arabidopsis thaliana, the lack of an obvious phenotype or quantitative change in mature rRNAs in class 1 RNase III (AtRTL2) mutants and overexpressing plants suggests that AtRTL2 is not involved in rRNA maturation. We characterized the in vitro activity of AtRTL2 to consider its in vivo function. AtRTL2 cleaved double-stranded RNA (dsRNA) specifically in vitro, yielding products of approximately 25 nt or longer in length, in contrast to 10–20 nt long products in bacteria and yeasts. Although dsRNA-binding activity was not detected, the dsRNA-binding domains in AtRTL2 were essential for its dsRNA-cleaving activity. Accumulation of small RNAs derived from transgene dsRNAs was increased when AtRTL2 was transiently expressed in Nicotiana benthamiana leaves by agroinfiltration. These results raise the possibility that AtRTL2 has functions distinct from those of other class 1 RNase IIIs in vivo.     

Selective enzymatic preparation of vinyl sugar esters using DMSO as a denaturing co-solvent

The protease-catalyzed transesterifications between hexoses and divinyladipate were examined. In dimethylformamide hexoses such as d-glucose, d-mannose, d-galactose and -methyl d-galactoside were esterified with divinyladipate by alkaline protease from Streptomyces sp. to give corresponding 6-O-vinyl adipoyl sugars. When the denaturing cosolvent, DMSO, was added to the solvent, galactose was selectively esterified at only the C-2 position.     

Relationship between symptoms and gene expression induced by the infection of three strains of Rice dwarf virus

Background

Rice dwarf virus (RDV) is the causal agent of rice dwarf disease, which often results in severe yield losses of rice in East Asian countries. The disease symptoms are stunted growth, chlorotic specks on leaves, and delayed and incomplete panicle exsertion. Three RDV strains, O, D84, and S, were reported. RDV-S causes the most severe symptoms, whereas RDV-O causes the mildest. Twenty amino acid substitutions were found in 10 of 12 virus proteins among three RDV strains.

Methodology/Principal Findings

We analyzed the gene expression of rice in response to infection with the three RDV strains using a 60-mer oligonucleotide microarray to examine the relationship between symptom severity and gene responses. The number of differentially expressed genes (DEGs) upon the infection of RDV-O, -D84, and -S was 1985, 3782, and 6726, respectively, showing a correlation between the number of DEGs and symptom severity. Many DEGs were related to defense, stress response, and development and morphogenesis processes. For defense and stress response processes, gene silencing-related genes were activated by RDV infection and the degree of activation was similar among plants infected with the three RDV strains. Genes for hormone-regulated defense systems were also activated by RDV infection, and the degree of activation seemed to be correlated with the concentration of RDV in plants. Some development and morphogenesis processes were suppressed by RDV infection, but the degree of suppression was not correlated well with the RDV concentration.

Conclusions/Significance

Gene responses to RDV infection were regulated differently depending on the gene groups regulated and the strains infecting. It seems that symptom severity is associated with the degree of gene response in defense-related and development- and morphogenesis-related processes. The titer levels of RDV in plants and the amino acid substitutions in RDV proteins could be involved in regulating such gene responses.     

The dsRNA-binding protein DRB4 interacts with the Dicer-like protein DCL4 in vivo and functions in the <Emphasis Type="Italic">trans</Emphasis>-acting siRNA pathway

Arabidopsis thaliana encodes four Dicer-like (DCL) proteins and five dsRNA-binding (DRB) proteins. We have previously demonstrated that DCL4 specifically interacts with DRB4 in vitro. Here we describe the interaction between DCL4 and DRB4 in vivo. The phenotype of a mutant with a defect in DCL4 (dcl4-2) was similar to that of a mutant with a defect in DRB4 (drb4-1): both mutant plants had elongated and downwardly curled rosette leaves and over-accumulated anthocyanin. In immunoprecipitation experiments with either anti-DCL4 or anti-DRB4 antibody and crude extracts of wild-type Arabidopsis plants, co-immunoprecipitation of DCL4 and DRB4 was detected, indicating that DCL4 interacts with DRB4 in vivo. This interaction was confirmed by immunoprecipitation experiments using extracts from dcl4-2, drb4-1, or transgenic plants expressing the hemagglutinin-tagged version of DCL4 or DRB4. The results of immunoprecipitation experiments also suggest that most DCL4 is associated with DRB4, but that some DRB4 is free or associated with other proteins. Reduced accumulation of the TAS1 and TAS3 trans-acting siRNA (ta-siRNA) and over accumulation of their target mRNAs (At5g18040 and auxin response factors ARF3 and ARF4) were detected in both drb4-1 and dcl4-2 mutants. These results indicate that DRB4, together with DCL4, functions in the ta-siRNA biogenesis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Yukihiro Nakazawa and Akihiro Hiraguri contributed equally to this work.     

Synthesis of vinyl arabinose ester catalyzed by protease from Streptomyces sp.

The transesterification of 0.5 M divinyladipate with 0.25 M arabinose in dimethylformamide for 7 days was catalyzed by Streptomyces sp. alkaline protease to give 5-O-vinyladipoyl-d-arabinofuranose at ca. 50% yield. Only enzymatic transesterification of primary hydroxyl group of arabinofuranose proceeded without esterification of arabinopyranose.