DICER-LIKE 4 functions in trans-acting small interfering RNA biogenesis and vegetative phase change in Arabidopsis thaliana

Abstract

Arabidopsis thaliana contains four DICER-LIKE (DCL) genes with specialized functions in small RNA biogenesis for RNA interference-related processes. A mutant with defects in DCL4 was identified and analyzed for microRNA- and endogenous, small interfering RNA (siRNA)-related functions. The dcl4-2 mutant contained normal or near-normal levels of microRNAs (21 nt) and heterochromatin-associated siRNAs (24 nt). In contrast, this mutant lacked each of three families of 21-nt trans-acting siRNAs (ta-siRNAs) and possessed elevated levels of ta-siRNA target transcripts. The dcl4-2 mutant resembled an rna-dependent RNA polymerase 6 mutant in that both mutants lacked ta-siRNAs and displayed heterochronic defects in which vegetative phase change was accelerated. Double mutant analyses with dcl2-1, dcl3-1, and dcl4-2 alleles revealed hierarchical redundancy among DCL activities, leading to alternative processing of ta-siRNA precursors in the absence of DCL4. These data support the concept that plants have specialized and compartmentalized DCL functions for biogenesis of distinct small RNA classes.

Fig. 1.

Characterization of Arabidopsis dcl4-2 mutant. (A) Organization of DCL4 genomic DNA (Upper) and mRNA (Lower). In the genomic diagram, exons and introns are illustrated as black bars and lines, respectively, and 5′ and 3′ UTRs are shown as gray bars. The T-DNA insertion site in dcl4-2 is indicated. Regions encoding conserved domains are indicated by colors in the mRNA diagram. The arrowhead indicates the dcl4-2 insertion point. Locations of cDNA-derived probe sequences are indicated by thin lines. dsRBM, dsRNA binding motif; DUF283, domain of unknown function 283. (B) RNA blot analyses of dcl4-2 mRNA. Blot assays with duplicate samples from inflorescence tissue of wild-type (lanes 1 and 2) and mutant (lanes 3 and 4) plants were done by using three DCL4-specific or tyrosine aminotransferase (TyrAT) probes. (C) Phenotypes of early rosette leaves of dcl4-2, rdr6-15, and wild-type plants. Arrowheads indicate rosette leaves 5 and 6. (D) Leaf growth (length/width) in rosette leaves of dcl4-2, rdr6-15, and wild-type plants. Mean and SD are shown for each data point (n = 40).

Fig. 2.

Expression profiling of miRNA and ta-siRNA target genes in dcl1-7, dcl4-2, and rdr6-15 mutant lines. Mean fold-change values were calculated for miRNA (A) and ta-siRNA (B) target transcripts in Col-0 (1.0 by definition) and mutant plants. The primary principal components analysis eigenvectors that accounted for >60% or >90% of the variation with miRNA and ta-siRNA targets, respectively, are shown by the red lines. Targets for ta-siRNAs from TAS1 (purple), TAS2 (green), and TAS3 (blue) are color-coded. Note that only seven of nine predicted or validated ta-siRNA targets are represented on the array (Table 1).

Fig. 3.

RNA blot assays for small RNAs in dcl4-2 plants. Triplicate RNA samples from leaf and inflorescence (Inflor.) tissues of dcl4-2 mutant and wild-type plants were analyzed. A representative blot image and mean (±SD) small RNA levels (fmol/mg total RNA based on quantitative standards) are presented in columns in each panel. Small RNAs tested were AtSN1 siRNAs (A), miR171 (B), miR159 (C), miR163 (D), miR173 (E), and miR390 (F) and ta-siRNAs siR255 (G), siR1511(H), and TAS3.5′D7(+) (I). In blot assays for ta-siRNAs, signals from the 21- plus 22-nt zone and the 24-nt zone were measured and presented in separate graphs. Note that it was not possible to accurately measure 22-nt ta-siRNA forms in the presence of high levels of 21-nt forms.

Fig. 4.

RNA blot assays for ta-siRNAs in single and double mutant plants. Duplicate total RNA samples from inflorescence tissue of mutant and wild-type plants were analyzed for siR255 (A) and siR1511 (B) RNAs. In each panel, a blot image and a bar graph showing signal intensity from the 21- plus 22-nt (open bars) and 24-nt (filled bars) zones are presented.

Fig. 5.

Model for trans-acting siRNA biogenesis in Arabidopsis. RISC, RNA-induced silencing complex.