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. 2012 Jun;159(2):748-58.
doi: 10.1104/pp.112.193508. Epub 2012 Apr 3.

The helicase and RNaseIIIa domains of Arabidopsis Dicer-Like1 modulate catalytic parameters during microRNA biogenesis

Chenggang Liu  1 , Michael J Axtell, Nina V Fedoroff
Affiliations

The helicase and RNaseIIIa domains of Arabidopsis Dicer-Like1 modulate catalytic parameters during microRNA biogenesis

Chenggang Liu et al. Plant Physiol. 2012 Jun.

Abstract

Dicer-Like1 (DCL1), an RNaseIII endonuclease, and Hyponastic Leaves1 (HYL1), a double-stranded RNA-binding protein, are core components of the plant microRNA (miRNA) biogenesis machinery. hyl1 null mutants accumulate low levels of miRNAs and display pleiotropic developmental phenotypes. We report the identification of five new hyl1 suppressor mutants, all of which are alleles of DCL1. These new alleles affect either the helicase or the RNaseIIIa domains of DCL1, highlighting the critical functions of these domains. Biochemical analysis of the DCL1 suppressor variants reveals that they process the primary transcript (pri-miRNA) more efficiently than wild-type DCL1, with both higher K(cat) and lower K(m) values. The DCL1 variants largely rescue wild-type miRNA accumulation levels in vivo, but do not rescue the MIRNA processing precision defects of the hyl1 null mutant. In vitro, the helicase domain confers ATP dependence on DCL1-catalyzed MIRNA processing, attenuates DCL1 cleavage activity, and is required for precise MIRNA processing of some substrates.

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Figures

Figure 1.
Figure 1.
Identification of dcl1 alleles that suppress the hyl1-2 phenotype. A, Schematic representation of the DCL1 protein. Labeled boxes indicate protein domains. The positions and amino acid substitutions identified in dcl1 suppressor alleles are indicated. The RNaseIIIa region is expanded for clarity. B, Photographs of plants showing the phenotypes of dcl1 mutants that suppress the hyl1-2 phenotype. Five-week-old plants grown under a 12L/12D light cycle. C, Complementation of hyl1-2 by dcl1 suppressor alleles. hyl1-2 plants were transformed with wild-type DCL1, dcl1-7, and the indicated dcl1 suppressor alleles under the control of the native DCL1 promoter. Numbers indicate the fraction of T1 plants showing the pictured phenotype. D, Quantitative real-time PCR analysis of intact DCL1 mRNA accumulation. Mean accumulation levels (relative to the mean Columbia-0 value) normalized to ACTIN2 accumulation, ±1 sd are plotted (n = 4; two biological replicates × two technical replicates). Samples were from 25-d-old rosette leaves from long-day-grown plants. Data from transgenics were from pooled, herbicide-resistant T2 individuals that complemented the hyl1-2 leaf phenotype. E, Quantitative real-time PCR analysis of mature miR162 and miR838 accumulation. As above except normalized to U6 accumulation levels.
Figure 2.
Figure 2.
miRNA accumulation in dcl1/hyl1-2 plants. A, RNA blots for the indicated small RNAs. WT, Wild type. B, SmallRNAseq summary showing the fraction of genome-mapped small RNAs that match MIRNA hairpins. C, Relative accumulation of individual mature miRNAs. The log2-transformed ratios of mutant/wild-type mature miRNA derived from scaled smallRNAseq data were computed for each mutant/wild-type combination and plotted. Thick bars indicate median values for each population. P values were derived from Kruskal-Wallis tests. Values corresponding to miR162 and miR838 accumulation are highlighted.
Figure 3.
Figure 3.
miRNA processing precision is not restored in hyl1-2/dcl1 suppressor plants. The log2-transformed ratio of mutant imprecision/wild-type imprecision at each MIRNA hairpin was calculated for each possible mutant/wild-type combination. Only highly expressed hairpins were considered (top quartile in wild type). Values above zero indicate greater imprecision in the mutant compared to the wild type. Thick lines indicate median values. P values were derived from Kruskal-Wallis tests. N.S., Not significant.
Figure 4.
Figure 4.
Enhanced catalytic activity of suppressor DCL1 proteins. A, Reaction kinetics of pri-miRNA156a processed by DCL1, DCL1-20, and DCL1-21. B, Kinetic parameters of DCL1, DCL1-20, and DCL1-21.
Figure 5.
Figure 5.
Cleavage of pri-miRNA156a and pri-miRNA166b by wild-type DCL1 and DCL1Δhelicase. A, Schematic diagram illustrating the DCL1ΔHelicase variant. B, Processing of pri-miRNA156a with or without ATP. C, Processing of pri-miRNA166b with or without ATP. D, The predicted secondary structure of pri-miRNA156a. Predicted fragments from precise DCL1-catalyzed processing are labeled as F1 and F2 (arm fragments), F3 (loop fragments), and m/m* (miRNA and miRNA* fragments). E, The predicted secondary structure of pri-miRNA 166b. Labeling conventions as in section D. F, In vivo MIRNA processing precision, estimated from smallRNAseq. The percentages of imprecise reads (i.e. those not within ±2 nucleotides of the annotated miRNA or miRNA*) are plotted for MIR156a and MIR166b.
Figure 6.
Figure 6.
Processivity of DCL1 processing of pri-miRNA to miRNA. A, The secondary structure of pri-miRNA 159a. The mature miR159 is highlighted in black. B, Cleavage assays used an internally labeled pri-miRNA159a substrate and wild-type DCL1. DCL1 was preincubated with internally labeled substrate on ice and in the absence of Mg2+ in lanes 2 to 4. Processed RNAs were separated by urea-PAGE. [See online article for color version of this figure.]
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