Identification of nuclear dicing bodies containing proteins for microRNA biogenesis in living Arabidopsis plants

Abstract

MicroRNAs (miRNAs) are important for regulating gene expression in muticellular organisms. MiRNA processing is a two-step process. In animal cells, the first step is nuclear and the second step cytoplasmic, whereas in plant cells, both steps occur in the nucleus via the enzyme Dicer-like1 (DCL1) and other proteins including the zinc-finger-domain protein Serrate (SE) and a double-stranded RNA (dsRNA) binding-domain protein, Hyponastic Leaves1 (HYL1). Furthermore, plant miRNAs are methylated by Hua Enhancer (HEN1) at their 3' ends and loaded onto Argonaute1 (AGO1). However, little is known about the cellular basis of miRNA biogenesis. Using live-cell imaging, we show here that DCL1 and HYL1 colocalize in discrete nuclear bodies in addition to being present in a low-level diffuse nucleoplasmic distribution. These bodies, which we refer to as nuclear dicing bodies (D-bodies), differ from Cajal bodies. A mutated DCL1 with impaired function in miRNA processing fails to target to D-bodies, and an introduced primary (pri)-miRNA transcript is recruited to D-bodies. Furthermore, bimolecular fluorescence complementation (BiFC) shows that DCL1, HYL1, and SE interact in D-bodies. On the basis of these data, we propose that D-bodies are crucial for orchestrating pri-miRNA processing and/or storage/assembly of miRNA-processing complexes in the nuclei of plant cells.

Figure 1. Subnuclear localization and colocalization of DCL1, HYL1 and SE

(A) Introduced fusion proteins of DCL1-YFP, HYL1-YFP and YFP-SE complemented DCL1–9, HYL1–2 and SE-1 mutants, respectively. (B) DCL1 and HYL1 localize in discrete nuclear foci both in leaf and root cells, while SE localizes in nuclear speckles. (C) DCL1-YFP (green) fully colocalizes with HYL1-CFP (red), cells in root tip are shown. (D) YFP-SE (green) partially colocalizes with HYL1-CFP (red), cells in root tip are shown. (E) HcRed-SE and SR33-YFP were transiently co-expressed in tobacco leaf cells. Colocalization of HcRed-SE (red) with SR33-YFP (green) was observed in nuclear speckles. Scale bars, 5 μm.

Figure 2. DCL1 and HYL1 containing nuclear bodies do not correspond to Cajal bodies

(A) Restoration of Cajal bodies by introduction of an AtCoilin-CFP (red) fusion into non-Cajal bodies-1 mutant expressing U2B”-GFP (green). A pair of guard cells is shown in the image. (B) DCL1and HYL1-containing nuclear bodies were labeled with HYL1-CFP (red) and Cajal bodies were labeled with AtCoilin-YFP (green), HYL1 foci do not colocalize with Cajal bodies as shown in guard cells (upper panel) and a leaf epidermal cell (lower panel). (C) In non-Cajal bodies-1 mutant, U2B”-GFP (green) is diffusely distributed in the nucleoplasm, while HYL1-CFP (red) is localized to discrete nuclear bodies. Scale bar for all images, 5 μm.

Figure 3. D-bodies are involved in pri-miRNA processing

(A) DCL1-YFP localizes to D-bodies, while DCL1-9-YFP is diffusely distributed in the nucleoplasm. Representative nuclei of leaf epidermal cells are shown in upper panel. Lower panel shows anti-GFP immunoblot of extracts from plants expressing DCL1-YFP and DCL1–9-YFP (upper more intense bands), and wild type plants with no tagged protein. The lower band in lanes 1 and 3 may represent a commonly observed degradation product. (B) Genomic DNA flanking miR173 was fused to MS2 repeats, and coinfiltrated to tobacco leaves with MS2-YFP and DCL1-CFP. The miR173 transcript tracked by MS2-YFP (green) was observed to be recruited to DCL1-CFP foci (red, lower panel). In contrast, in nuclei expressing only MS2-YFP (upper panel) or MS2-YFP and MS2 repeats (middle panel), DCL1-CFP bodies (red) were distributed among a diffuse MS2-YFP signal (green). (C) Pair-wise BiFC experiments between SE, DCL1, HYL1, and DCL1–9. Protein partners was fused to an N-terminal fragment or C-terminal fragment of YFP, respectively, and co-infiltrated into tobacco leaves. Hoechst 33342 was used to label the nuclei (red). BiFC signals between SE, DCL1 and HYL1 were observed in D-bodies (green). No BiFC signals were observed between DCL1–9 and SE, DCL1 or HYL1. (D) Pair-wise BiFC experiments between SE, DCL1, HYL1, and HEN1, and AGO1. Interaction between HYL1 and AGO1 is observed in the D-body (arrow). Scale bars, 5 μm.