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.

Fig. 1.

Localization and sequence of the miR163 gene. (A) The miR163 gene is located on chromosome 1 between the At1g66720 and At1g66730 genes. The red box indicates the miR163 gene, and the black boxes indicate two adjacent genes. (B) Schematic representation of miR163 gene organization. Bold black lines and red lines indicate the sequences corresponding to the stem-loop-forming region of primary transcript (pri-miR163) and miR163, respectively. The positions of the polyadenylation signal (AATAAA) are pointed to by arrows. It was thought that type 2 pri-miR163 could emerge by splicing through exclusion of the polyadenylation signal for type 1 pri-miR163.

Fig. 2.

miR163 biogenesis requires at least three cleavage steps. (A) Positions of the four probes used in this study. Bold black lines and red lines indicate the sequences corresponding to the stem-loop-forming region of pri-miR163 and miR163, respectively. (B) Northern blot analysis for detection of pri-miR163 and/or pre-miR163s using the four kinds of probes shown in A. Size markers (Ambion, Austin, TX) are indicated to the left of each panel. Bands a-e correspond to those shown in the model (D). (C) Northern blot analysis for detection of miR163. Positions of the 26-nt DNA oligonucleotide and 100-nt RNA are indicated to the left. The image of 5S rRNA and tRNA was used as a loading control for both B and C. Samples were isolated from uninfected (mock) and TMV-Cg-infected (Cg) plants 10 days after inoculation. (D) Model of miR163 biogenesis. The pri-miR163 (a) is processed into long pre-miR163 (b) at the root of the stem-loop structure, releasing 5′ remnant (e). At the next step, the long pre-miR163 is cleaved into short pre-miR163 (c) at the edge of the miR163 sequence. Last, the short pre-miR163 is cleaved at the opposite edge of the miR163 sequence, producing mature miR163 and remnant (d). The predicted molecular length of each RNA from the result of Fig. 3C is shown in this model.

Fig. 3.

Determination of positions and forms of RNase III-cleavage sites. (A) Illustration of the method used in this study. RNAs were self-ligated, followed by RT-PCR using miR163 gene-specific primers. (B) Image of agarose gel electrophoresis of RT-PCR products. Three bands detected between 200- and 400-nt molecular markers were cloned and sequenced. Bands at a higher molecular weight position were from two rounds of RT-PCR products. (C) Sequence of the stem region of pri-miR163. The three cleavage sites are indicated by arrows b-d, which correspond to the roots of structures b-d shown in Fig. 2D. The number of sequenced clones corresponding to each site is indicated above each cleavage site. The sequence of miR163 is underlined. The gray box indicates the 21-nt-long miR163^*, the opposite strand from miR163. Three kinds of small RNAs produced by the three cleavage steps were designated as upper left (UL), lower left (LL), and miR163^*, respectively. (D) Northern blot analysis for detection of four kinds of small RNAs shown in C. The positions of the 21- and 26-nt oligonucleotides are indicated in Left and Center. The image of 5S rRNA and tRNA was used as a loading control.

Fig. 4.

miR163 biogenesis in dcl mutants. (A) Northern blot analysis for detection of pri-miR163 and pre-miR163 in dcl1-7 mutants using probe 1 (shown in Fig. 2A). (B) Northern blot analysis for detection of pri-miR163 and pre-miR163 in dcl1-9 mutants using probe 1′, which covers the stem-loop-forming region of DCL1-9 pri-miR163. (C) Northern blot analysis for detection of pri-miR163 and 5′ remnant in dcl1-7 mutants using probe 2 (shown in Fig. 2A). (D) Northern blot analysis for detection of pri-miR163 and pre-miR163 in dcl2-2 and dcl3-1 plants. miR163 was detected by using probe 3 (shown in Fig. 2A). The image of 5S rRNA and tRNA was used as a loading control.

Fig. 5.

Aberrant positioning of cleavage sites in the dcl1-9 mutant. (A) Image of agarose gel electrophoresis of RT-PCR products. (B) The two cleavage sites in the dcl1-9/dcl1-9 mutant. The gray boxes indicate the regions in which two cleavage sites extend. Zones f and g correspond to the roots of bands f and g of dcl1-9/dcl1-9 mutants, respectively, shown in Fig. 4B. The number of sequenced clones corresponding to each site is indicated above each gray box. The gray arrows indicate the three cleavage sites in the wild type.