[Abstract unavailable

We made a series of improved Gateway binary vectors (pGWBs) for plant transformation. Fifteen different reporters and tags, sGFP, GUS, LUC, EYFP, ECFP, G3GFP, mRFP, 6ÂHis, FLAG, 3ÂHA, 4ÂMyc, 10ÂMyc, GST, T7, and TAP, were employed. Some vectors carry the 2Â35S-promoter for higher-level expression. The kanamycin-and hygromycin-resistant markers are independently available for each of the 43 types of vectors, thus an additional transformation of once-transformed plants can be carried out easily. Their small size and high-copy number in Escherichia coli make possible easier handling at plasmid preparation and sequencing. Improved pGWBs should be a powerful tool for transgenic research in plants.

Micro-RNAs (miRNAs) are small, noncoding RNAs of 18 -25 nt in length that negatively regulate their complementary mRNAs at the posttranscriptional level. Previous work has shown that some RNase III-like enzymes such as Drosha and Dicer are known to be involved in miRNA biogenesis in animals. However, the mechanism of plant miRNA biogenesis still remains poorly understood. In this article, the process of Arabidopsis miR163 biogenesis was examined. The results revealed that two types of miR163 primary transcripts (pri-miR163s) are transcribed from a single gene by RNA polymerase II and that miR163 biogenesis requires at least three cleavage steps by RNase III-like enzymes at 21-nt-long intervals. The first step is from pri-miR163 to long miR163 precursor (premiR163), the second step is from long pre-miR163 to short premiR163, and the last step is from short pre-miR163 to mature miR163 and the remnant. It is interesting that, during the process, four small RNAs including miR163 are released. By using dcl1 mutants, it was demonstrated that Arabidopsis Dicer homologue Dicer-like 1 (DCL1) catalyzes at least the first and second cleavage steps and that double-stranded RNA-binding domains of DCL1 are involved in positioning of the cleavage sites. Our result is direct evidence that DCL1 is involved in processing of pri-and pre-miRNA.M icro-RNAs (miRNAs) are endogenous, small, noncoding RNAs of 18-25 nt in length that negatively regulate their complementary mRNAs at the posttranscriptional level in many eukaryotic organisms (1). A large number of miRNAs have been discovered in both plants and animals (2-4).In animals, especially in the human, miRNA biogenesis and functional pathways have been examined and partly described. First, miRNA primary transcripts (pri-miRNAs) are trimmed in the nucleus into miRNA precursors (pre-miRNAs) by an RNase III-like enzyme called Drosha (5). After this initial processing, the pre-miRNAs are exported to the cytoplasm by the function of exportin-5 (6, 7) and are cleaved there to generate mature miRNAs by another RNase III-like enzyme called Dicer (8).These miRNAs are then incorporated into the RNA-induced silencing complex (RISC) endonuclease, which seems to negatively regulate the genes required for several developmental processes by promoting RISC-mediated degradation of target mRNAs, or the inhibition of target mRNA translation (9).In the genome of Arabidopsis thaliana, four Dicer-like (DCL) enzymes (DCL1-DCL4) are encoded (10). It has been shown that one of the enzymes, DCL1, is involved in miRNA accumulation (11). It is also known that DCL1 mRNA is subject to negative feedback regulation by miR162-guided mRNA degradation (12). On the other hand, DCL2 and DCL3 proteins are partly involved in viral short interfering RNA (siRNA) biogenesis and endogenous siRNA biogenesis such as retrotransposon siRNA, respectively (13).However, the mechanism of plant miRNA biogenesis, including the step where DCL1 acts, remains poorly understood, because it has been difficult to detect pri-and pre-miRNAs ...

It has been reported that some double-stranded RNA (dsRNA) binding proteins interact with small RNA biogenesis-related RNase III enzymes. However, their biological significance is poorly understood. Here we examine the relationship between the Arabidopsis microRNA-(miRNA) producing enzyme DCL1 and the dsRNA binding protein HYL1. In the hyl1-2 mutant, the processing steps of miR163 biogenesis were partially impaired; increased accumulation of pri-miR163 and reduced accumulation of short pre-miR163 and mature miR163 as well as misplaced cleavages in the stem structure of pri-miR163 were detected. These misplaced cleavages were similar to those previously observed in the dcl1-9 mutant, in which the second double-stranded RNA binding domain of the protein was disrupted. An immunoprecipitation assay using Agrobacterium-mediated transient expression in Nicotiana benthamiana showed that HYL1 was able to form a complex with wild-type DCL1 protein, but not with the dcl1-9 mutant protein. We also examined miR164b and miR166a biogenesis in hyl1-2 and dcl1-9. Increased accumulation of pri-miRNAs and reduced accumulation of pre-miRNAs and mature miRNAs were detected. Misplaced cleavage on pri-miR164b was observed only in dcl1-9 but not in hyl1-2, whereas not on pri-miR166a in either mutant. These results indicate that HYL1 has a function in assisting efficient and precise cleavage of pri-miRNA through interaction with DCL1.