APC/C-Mediated Degradation of dsRNA-Binding Protein 4 (DRB4) Involved in RNA Silencing

Background: Selective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all Eukaryotes. In particular, the APC/C (Anaphase Promoting Complex or Cyclosome) is a master ubiquitin protein ligase (E3) that targets regulatory proteins for degradation allowing sister chromatid separation and exit from mitosis. Interestingly, recent work also indicates that the APC/C remains active in differentiated animal and plant cells. However, its role in post-mitotic cells remains elusive and only a few substrates have been characterized. Methodology/Principal Findings: In order to identify novel APC/C substrates, we performed a yeast two-hybrid screen using as the bait Arabidopsis APC10/DOC1, one core subunit of the APC/C, which is required for substrate recruitment. This screen identified DRB4, a double-stranded RNA binding protein involved in the biogenesis of different classes of small RNA (sRNA). This protein interaction was further confirmed in vitro and in plant cells. Moreover, APC10 interacts with DRB4 through the second dsRNA binding motif (dsRBD2) of DRB4, which is also required for its homodimerization and binding to its Dicer partner DCL4. We further showed that DRB4 protein accumulates when the proteasome is inactivated and, most importantly, we found that DRB4 stability depends on APC/C activity. Hence, depletion of Arabidopsis APC/C activity by RNAi leads to a strong accumulation of endogenous DRB4, far beyond its normal level of accumulation. However, we could not detect any defects in sRNA production in lines where DRB4 was overexpressed

Selective protein degradation: a rheostat to modulate cell-cycle phase transitions.

International audiencePlant growth control has become a major focus due to economic reasons and results from a balance of cell proliferation in meristems and cell elongation that occurs during differentiation. Research on plant cell proliferation over the last two decades has revealed that the basic cell-cycle machinery is conserved between human and plants, although specificities exist. While many regulatory circuits control each step of the cell cycle, the ubiquitin proteasome system (UPS) appears in fungi and metazoans as a major player. In particular, the UPS promotes irreversible proteolysis of a set of regulatory proteins absolutely required for cell-cycle phase transitions. Not unexpectedly, work over the last decade has brought the UPS to the forefront of plant cell-cycle research. In this review, we will summarize our knowledge of the function of the UPS in the mitotic cycle and in endoreduplication, and also in meiosis in higher plants

The Control of Arabidopsis thaliana Growth by Cell Proliferation and Endoreplication Requires the F-Box Protein FBL17.

International audienceA key step of the cell cycle is the entry into the DNA replication phase that typically commits cells to divide. However, little is known about the molecular mechanisms regulating this transition in plants. Here, we investigated the function of FBL17 (F BOX-LIKE17), an Arabidopsis thaliana F-box protein previously shown to govern the progression through the second mitosis during pollen development. Our work reveals that FBL17 function is not restricted to gametogenesis. FBL17 transcripts accumulate in both proliferating and postmitotic cell types of Arabidopsis plants. Loss of FBL17 function drastically reduces plant growth by altering cell division activity in both shoot and root apical meristems. In fbl17 mutant plants, DNA replication is severely impaired and endoreplication is fully suppressed. At the molecular level, lack of FBL17 increases the stability of the CDK (CYCLIN-DEPENDENT KINASE) inhibitor KIP-RELATED PROTEIN2 known to switch off CDKA;1 kinase activity. Despite the strong inhibition of cell proliferation in fbl17, some cells are still able to enter S phase and eventually to divide, but they exhibit a strong DNA damage response and often missegregate chromosomes. Altogether, these data indicate that the F-box protein FBL17 acts as a master cell cycle regulator during the diploid sporophyte phase of the plant

Specific interaction between APC10 and DRB4 occurs <i>in vitro</i> and <i>in vivo</i>.

<p>(<b>A</b>) Yeast two-hybrid analyses were performed by mating on non-selective (-LW) and selective (-LWA) media. DRB4 was fused to the binding-domain (BD) whereas APC10, CDC20-1/-2/-3/-4 and CCS52A1/A2/B were fused to the activation domain (AD). Empty BD and AD vectors were used as negative controls. (<b>B</b>) [³⁵S]methionine-labelled DRB4 was incubated with recombinant GST-APC10, GST-CDC20-1/-2/-3 or GST alone. After several washes, proteins were affinity-purified on glutathione-Sepharose beads, and loaded on an acrylamide gel. The pulled-down proteins were analyzed by autoradiography. The same result was obtained in three independent experiments. (<b>C</b>) Bimolecular fluorescence complementation showed APC10 and DRB4 interaction <i>in planta</i>. Recombinant YN-APC10 and YC-DRB1/2 or 4 were co-bombarded together with a NLS-CFP construct into 4 day-old mustard seedlings. Fluorescence was observed using an E800 fluorescence microscope. YN and YC alone were used as negative controls. Reconstitution of functional YFP as detected by YFP fluorescence occurs only in the nucleus. A strong YFP signal was observed in the nucleus of 91% of examined cells (64/70). No fluorescence signal was obtained after bombardment with the following plasmid combinations YN-+YC-DRB4 and YN-APC10+YC- or with YN-APC10+YC-DRB1 and YN-APC10+YC-DRB2. (<b>D</b>) Yeast two-hybrid analyses were performed by mating on non-selective (-LW) and selective (-LWA) media. APC10 was fused to the BD and DRB1/2/3/4 and 5 were fused to the AD. Empty BD and AD vectors were used as negative controls.</p

Viral infection of <i>drb4</i> and DRB4 OE line.

<p>(<b>A</b>) Pictures from infected plants with the TRV-PDS virus, 14 days post-infection (dpi). Scale bar: 1 cm. (<b>B</b>) Northern blot analysis of TRV-PDS viral RNA or viral-derived siRNA accumulation in Col-0, <i>drb4</i> and DRB4 OE-27 line using a PDS specific probe. TAS1 tasiRNA accumulation was detected using a siRNA255 probe. miR159 accumulation was used here as a loading control. Values are normalized to miR159 and are expressed as a ratio relative to the wild-type Col-0, which was arbitrarily set to 1.</p

Mapping of interaction domains between APC10 and DRB4.

<p>(<b>A</b>) Schematic representation of DRB4 deletion constructs generated and fused to the activation domain (AD). Interaction with full-length APC10 or DRB4 fused to the binding-domain (BD) was then scored by yeast two-hybrid assays (see B). A summary of three independent assays is indicated on the right. +, interactions scored based on growth on selective media. −, no growth on selective media. (<b>B</b>) One of the three yeast two-hybrid assays between DRB4 or APC10 and DRB4 deleted versions. After mating, yeast was grown at 28°C for 3 days on non-selective (−LW) or strong selection (−LWA) media. Empty AD and BD vectors were included as negative controls.</p

Reduced APC/C activity slightly affect polIV/polV-dependent heterochromatic siRNA accumulation.

<p>Northern blot analysis of polIV-dependent (siRNA02, TR2258) or polIV/polV-dependent (siRNA1003, simpleHAT) siRNA accumulation in Col-0, <i>drb4</i>, <i>dcl4</i>, DRB4 OE27, RNAi APC10-38 and RNAi APC6-20 lines. miR173 accumulation is used here as a loading control. Values are normalized to miR173 and are expressed as a ratio relative to the wild-type Col-0, which was arbitrarily set to 1.</p