Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions

Abstract

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 ...

Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba Dictyostelium discoideum

“…Since D. discoideum apparently lacks a bona fide Drosha, required for generation of pre-miRNA in animals, the miRNA biogenesis may be more similar to that in plants where one Dicer-like enzyme is responsible for producing both the pre- and mature miRNAs [19,29,30,48]. Hence, we hypothesized that the reduction of miRNAs observed in DrnB depleted cells would be reflected in stabilization and accumulation of longer pri-miRNA transcripts not processed to mature miRNAs, allowing for subsequent analysis of their 5´- and 3´-ends.…”

“…The stem-loop, harboring the miRNA sequence, is recognized by a complex of proteins where Dicer-like protein DCL1 first releases the pre-miRNA and subsequently also cleaves the pre-miRNA to generate miRNAs. These miRNAs are transferred to the cytoplasm by HASTY, a homologue of Exportin-5 [18,19]. However, a revised model has recently been put forward where miRNAs are loaded onto AGO1 whereafter the complex is transported out of the nucleus by a HASTY-independent pathway [20].…”

On the origin and functions of RNA-mediated silencing: from protists to man

“…In animals, miRNAs are initially transcribed into long precursor transcripts that are processed to mature miRNAs in a series of steps involving two RNaseIII-like enzymes, Drosha and Dicer (Bartel 2004;Wienholds and Plasterk 2005). Plants lack a Drosha homolog and the production of miRNAs from precursor RNAs appears to be carried out by a single Dicer-like protein Kurihara and Watanabe 2004;Millar and Waterhouse 2005). Plant miRNAs are also methylated on the ribose of the last nucleotide, a modification presumably involved in protecting miRNAs from 3′ end uridylation and degradation, whereas animal miRNAs do not appear to be modified Yu et al 2005).…”

Principles of micro-RNA production and maturation

“…miRNAs are initially transcribed as part of a much longer primary transcript (Lee et al, 2002;reviewed by Cullen, 2004;Kim, 2005). Several primary transcripts have been analysed in mammals, plants, worms and flies (Tam, 2001;Aukerman and Sakai, 2003;Bracht et al, 2004;Cai et al, 2004;Kurihara and Watanabe, 2004;Lee et al, 2004;Houbaviy et al, 2005;Sokol and Ambros, 2005). Full-length versions of the primary transcripts are shown to be generally quite long (>1 kb) and contain a 5 0 7-methyl guanosine cap and a 3 0 poly(A) tail.…”

“…Many of the proteins involved in animal miRNA processing have homologs in plants (Figure 2), with the exception of Drosha and DGCR8. In Arabidopsis, a Dicer-like enzyme, DCL1, appears to perform the analogous functions of both Drosha and Dicer of animal cells (Reinhart et al, 2002;Papp et al, 2003;Kurihara and Watanabe, 2004). Precursor miRNAs in plants are longer, more heterogeneous in size and more difficult to detect than those in animals (Ambros et al, 2003a).…”