DAWDLE Interacts with DICER-LIKE Proteins to Mediate Small RNA Biogenesis

Abstract

DAWDLE (DDL) is a conserved forkhead-associated (FHA) domain-containing protein with essential roles in plant development and immunity. It acts in the biogenesis of microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs), which regulate gene expression at the transcriptional and/or posttranscriptional levels. However, the functional mechanism of DDL and its impact on exogenous siRNAs remain elusive. Here, we report that DDL is required for the biogenesis of siRNAs derived from sense transgenes and inverted-repeat transgenes. Furthermore, we show that a mutation in the FHA domain of DDL disrupts the interaction of DDL with DICER-LIKE1 (DCL1), which is the enzyme that catalyzes miRNA maturation from primary miRNA transcripts (pri-miRNAs), resulting in impaired pri-miRNA processing. Moreover, we demonstrate that DDL interacts with DCL3, which is a DCL1 homolog responsible for siRNA production, and this interaction is crucial for optimal DCL3 activity. These results reveal that the interaction of DDL with DCLs is required for the biogenesis of miRNAs and siRNAs in Arabidopsis (Arabidopsis thaliana).

Figure 1.

DDL regulates the accumulation of miRNAs and siRNAs. A, Deep sequencing analysis of mature miRNA and siRNA accumulation in ddl-1. Libraries of small RNA were produced from inflorescences of ddl-1 and Ws. Each circle represents a small RNA calculated as reads per million, and a log₂-transformed ratio of ddl-1/Ws was plotted. The thick lines in the middle of circles indicate median values. Rep1 and Rep2 were two biological replicates of sequencing. B, miRNA abundance in inflorescences of ddl-1 and Ws. C, siRNA abundance in inflorescences of ddl-1 and Ws. Ws, wild-type control of ddl-1; U6, spliceosomal RNA U6, used as loading control. Small RNAs were detected by RNA blot. Radioactive signals were detected with a phosphor imager and quantified with ImageQuant (v5.2). The amount of miRNA or siRNA in ddl-1 was normalized to U6 RNA and compared with that in Ws (set as 1) to determine the relative abundance of small RNAs in ddl-1. The number below ddl-1 indicates the relative abundance of miRNAs or siRNAs, which is the average value of three replicates. P < 0.05. For miR159/319, upper band, miR159; lower band, miR319. The numbers represent the relative abundance quantified by three replicates (t test, P < 0.05).

Figure 2.

DDL is required for transgene induced siRNA accumulation. A, Histochemical staining of GUS in plants containing the L1 locus. Wild-type (DDL⁺) and ddl-1 containing the L1 locus were segregated from the sixth generation of a recombined inbred line through repeated five generations of self-crossing of DDL/ddl-1 harboring the L1 locus. Twenty plants containing GUS were analyzed for each genotype. B, GUS mRNA levels in DDL⁺ and ddl-1 detected by RNA blot. Equal total RNAs were loaded for RNA blot. C, The accumulation of GUS siRNA in DDL⁺ and ddl-1. D. The accumulation of AP1 siRNAs in DDL⁺, DDL⁺/AP1-IR, ddl-1, and ddl-1/AP1-IR plants detected by RNA blot. U6 RNA was used as loading control. E, AP1 expression levels in DDL⁺, DDL⁺/AP1-IR, ddl-1, and ddl-1/AP1-IR. DDL⁺, DDL⁺/AP1-IR, ddl-1, and ddl-1/AP1-IR plants were segregated from a recombined inbred line that was produced by five generations of repeated self-crossing of DDL/ddl-1 containing the AP1-IR locus. Equal total RNAs were loaded for the RNA blot.

Figure 3.

DDL interacts with DCL3. A, Schematic diagram of DCL3 domains and truncated DCL3 fragments used for protein interaction assay. B, The interaction between DDL and various DCL3 fragments detected by co-IP. Full-length and truncated DCL3 proteins fused with a MYC tag at their N terminus were expressed in N. benthamiana leaves. For DDL, protein extract from plants expressing 35S::DDL-GFP. The protein pairs in the protein extracts were indicated by the labels on the left side and above the picture. An anti-MYC antibody was used to detect MYC fusion proteins in immunoblots. Labels on left side of picture indicate proteins detected by immunoblot. Five percent input proteins were used for MYC-tagged proteins, while 20% inputs were used for DDL-GFP. C, Bimolecular fluorescence complementation analysis of the interaction DDL with DCL3. Paired nVenus and cCFP-fused proteins were coinfiltrated into N. benthamiana leaves. Yellow fluorescence (green in image) signals were examined at 48 h after infiltration by confocal microscopy. The red spots were autofluorescence from chlorophyll. One hundred nuclei were examined randomly for each pair of proteins and one image is shown; the percentage of cells with fluorescence is shown. Bar = 20 μm.

Figure 4.

ddl-1 reduces the accumulation of miR162 and siRNA in an in vitro processing assay. A, miRNA production from pri-miR162b in the protein extracts of ddl-1 and Ws. [³²P]-labeled MIR162b that contains the stem-loop of pri-miR162b flanked by 6-nucleotide arms at each end was generated through in vitro transcription. B, Quantification of miRNAs generated from pri-miR162b in ddl-1 compared to those in Ws at 40, 80, and 120 min. C, siRNA production from dsRNAs in the protein extracts isolated from inflorescences of ddl-1 and Ws. dsRNAs were synthesized through in vitro transcription of a DNA fragment (5′ portion of UBQ5 gene, ∼460 bp) under the presence of [α-³²P]UTP. D. Quantification of siRNAs produced from dsRNAs in ddl-1 compared to those in Ws at 40, 80, and 120 min. The amounts of miRNAs or siRNAs produced at 40 min from Ws were set as 1, and means ± sd were calculated from three biological replicates. E, The protein levels of DCL1 and DCL3 in protein extracts of Ws and ddl-1 detected by immunoblot. Hsc70 was used as loading control.

Figure 5.

The FHA domain of DDL is required for miRNA biogenesis. A, Schematic diagram of DDL protein domains and the G222R mutation of ddl-3. NLS, nuclear localization signal. B, miRNA and siRNA abundance in inflorescences of Ws, ddl-1, er105, and ddl-3 detected by RNA blotting. C, The phenotypes of ddl-1 harboring the DDL-GFP or the ddl-3-GFP transgenes. D, miRNA abundance in various genotypes detected by RNA blotting. The radioactive signals were detected with a phosphor imager and quantified with ImageQuant (v5.2). The amount of a small RNA was normalized to U6 RNA and compared with Ws. The numbers represent the relative abundance quantified in three replicates (t test, P < 0.05). Ws, background control of ddl-1; er-105, control of ddl-3.

Figure 6.

The G222R mutation disrupts the DDL-DCL1 and DDL-DCL3 interactions and the activity of DCLs. A and B, Interactions of ddl-3 with DCL1 (A) and DCL3 (B) examined by co-IP. GFP, ddl-3 fused with GFP at its C terminus (ddl-3-GFP) or DDL-GFP were coexpressed with N-terminal MYC-fused DCL1 (MYC-DCL1) or DCL3 (MYC-DCL3) in N. benthamiana. Anti-GFP antibodies were used to perform IP, and anti-MYC antibody was used to detect MYC fusion proteins in immunoblots. Five percent input proteins were used for immunoblots. C and D, MIR162b (C) and dsRNA (D) processing by protein extracts of ddl-3 and er-105, respectively. [³²P]-labeled MIR162b that contains the stem-loop of pri-miR162b flanked by 6-nucleotide arms at each end was generated through in vitro transcription. dsRNAs were synthesized through in vitro transcription of a DNA fragment (5′ portion of UBQ5 gene, ∼460 bp) under the presence of [α-³²P]UTP. Proteins were isolated from inflorescences of ddl-3 or er-105. The in vitro processing reactions were stopped at 80 min and RNAs were extracted for gel running. Radioactive signals were quantified with ImageQuant (v5.2).