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.

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The nonsense-mediated mRNA decay (NMD) pathway is a wellknown eukaryotic surveillance mechanism that eliminates aberrant mRNAs that contain a premature termination codon (PTC). The UP-Frameshift (UPF) proteins, UPF1, UPF2, and UPF3, are essential for normal NMD function. Several NMD substrates have been identified, but detailed information on NMD substrates is lacking. Here, we noticed that, in Arabidopsis, most of the mRNA-like nonprotein-coding RNAs (ncRNAs) have the features of an NMD substrate. We examined the expression profiles of 2 Arabidopsis mutants, upf1-1 and upf3-1, using a whole-genome tiling array. The results showed that expression of not only protein-coding transcripts but also many mRNA-like ncRNAs (mlncRNAs), including natural antisense transcript RNAs (nat-RNAs) transcribed from the opposite strands of the coding strands, were up-regulated in both mutants. The percentage of the up-regulated mlncRNAs to all expressed mlncRNAs was much higher than that of the upregulated protein-coding transcripts to all expressed proteincoding transcripts. This finding demonstrates that one of the most important roles of NMD is the genome-wide suppression of the aberrant mlncRNAs including nat-RNAs.ncRNA ͉ tiling array ͉ UPF1 ͉ UPF3 In Arabidopsis thaliana, hundreds of nonprotein-coding RNA (ncRNA) transcripts have been discovered based on the cloning of full-length cDNAs, whole-genome tiling array, and deep-sequencing analysis (1-8). They include microRNA (miRNA) precursors, transacting siRNA (ta-siRNA) precursors, and mRNA-like ncRNAs (mlncRNA). The mlncRNAs were divided into 2 types-natural antisense transcript RNAs (natRNAs) that arise from the strands opposite the coding strands and other mlncRNAs.Nonsense-mediated mRNA decay (NMD) is a eukaryotic mRNA quality-control mechanism that eliminates aberrant mRNAs containing a premature termination codon (PTC) from cells to avoid the production of truncated proteins (9-13). Such transcripts can arise by genomic frameshifts, nonsense mutations, inefficiently spliced premRNAs, and so on. Several proteins involved in NMD have already been discovered (10). Among them, the UP-Frameshift (UPF) proteins, UPF1, UPF2, and UPF3, are core components of mRNA surveillance complexes and are essential for normal NMD function. In yeasts and mammals, aberrant transcripts with nonsense mutations are recognized by the UPF complex and then degraded from the 5Ј and 3Ј ends by recruiting-decapping and 5Ј33Ј exonuclease activities and deadenylating and 3Ј35Ј exonuclease activities (14-16).Plants also have a sophisticated NMD system (10). The Arabidopsis genome encodes homologues of 3 UPF genes (UPF1, UPF2, and UPF3). Some UPF1 and UPF3 mutants were obtained in Arabidopsis and analyzed to investigate the NMD system in plants and to identify several mRNA substrates for NMD by using microarrays (17-20). For example, the aberrant mRNAs containing PTC were overaccumulated in the upf1 and upf3 mutants (17)(18)(19).In plants, aberrant mRNAs with termination codons located distant (м300 nt) f...

MicroRNAs (miRNAs) have important roles in gene regulation during plant development. Previous studies revealed that some miRNAs are highly shared by most land plants. Recently, the liverwort, Marchantia polymorpha, has been studied by molecular genetic approaches, and sequencing of its genome is currently underway. The expression pattern and the detailed functions of miRNAs during Marchantia development are unknown. Here, we profiled the small RNAs expressed in thalli, antheridiophores and archegoniophores of M. polymorpha using high-throughput RNA sequencing. We revealed that a limited number of miRNAs are shared between M. polymorpha and the moss, Physcomitrella patens, and that a number of miRNAs are M. polymorpha specific. Like other land plants, cognate target genes corresponding to conserved miRNAs could be found in the genome database and were experimentally confirmed to guide cleavage of target mRNAs. The results suggested that two genes in the SPL (SQUAMOSA PROMOTER BINDING-LIKE) transcription factor family, which are regulated by miR156 in most land plants, were instead targeted by two distinct miRNAs in M. polymorpha. In order to demonstrate the physiological roles of miRNAs in M. polymorpha, we constructed an miRNA ectopic expression system to establish overexpression transformants for conserved miRNAs, miR166 and miR319. Ectopic expression of these miRNAs induced abnormal development of the thallus and gemma cups, suggesting that balanced expression of miRNA/target mRNAs has a crucial role in developmental regulation in M. polymorpha. Profiling data on miRNA together with the ectopic expression system would provide new information on the liverwort small RNA world and evolutionary divergence/conservation of small RNA function among land plants.