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

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

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

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

Over-accumulation of DRB4 does not affect <i>trans</i>-acting siRNA biogenesis and their targets accumulation.

<p>(<b>A</b>) Northern blot analysis of various sRNA accumulation in Col-0, <i>drb4</i>, <i>dcl4</i>, DRB4 OE27, RNAi APC10-38 and RNAi APC6-20 lines. <i>Trans</i>-acting siRNA and miRNA accumulation were detected using a <i>TAS1</i> tasiRNA255, a miR159 specific probe and a miR173 specific probe, respectively. APC10 and APC6 probes were used to score the accumulation of siRNA targeted against APC10 and APC6 genes in their respective RNAi lines. U6 was used for the loading control. (<b>B</b>) Quantitative real-time PCR reactions were performed on total RNA from the same background depicted in (A). Specific primers against <i>APC10</i>, <i>APC6</i>, <i>DRB4</i> and three target genes of the tasiRNA pathway (<i>TAS1</i> target, <i>ARF3</i> and <i>ARF4</i>) were used. RNA levels were normalized to that of Actin2 (At3g18780) and then to the value of the wild-type plants, which was arbitrarily set to 1. Error bars represent standard deviation from 2 independent experiments involving triplicate PCR reactions each.</p

DRB4 over-accumulates after MG132 treatment and in APC/C RNAi lines.

<p>(<b>A</b>) Characterization of DRB4 overexpressing lines. Northern blot analysis (upper panels) of <i>DRB4</i> mRNA accumulation in flower extracts from Col-0 and two independent transgenic lines expressing DRB4 under the control of the strong 35S promoter (referred hereafter as lines DRB4 OE-4 and -27). Accumulation of EF1α mRNA is used as a loading control. Western blot analysis (lower panel) performed using an antibody directed against DRB4. The asterisk indicates a non-specific cross-reacting band that can also be used as a loading control. Pictures of 5 week-old Col-0 and DRB4 OE-27 plants. (<b>B</b>) Three week-old DRB4 OE-27 seedlings were incubated in liquid medium supplemented or not with 100 µM MG132. After 4 h and 6 h of incubation, seedlings were collected and total protein extracted. Western blot was performed using antibodies against DRB4 or TSN as a loading control. (<b>C</b>) Western blot analysis of DRB4 accumulation in flower extracts from Col-0, <i>drb4</i>, <i>dcl4</i>, DRB4 OE-27 line, RNAi APC10-38 and RNAi APC6-20 lines. Coomassie staining was used as a loading control (LC).</p

Demandes Territoriales

National audiencePourquoi a-t-on (encore) besoin de territoires ? Comment s’expriment les demandes pour que soient créés, recréés, renouvelés des territoires ? Quels types de territoires sont attendus, souhaités ou réclamés ? Cet ouvrage propose de poursuivre la réflexion sur les enjeux contemporains du rapport des sociétés à l’espace, au pouvoir et à l’action en partant du constat que la demande territoriale est la fois sociale, culturelle, économique, politique, environnementale, matérielle et symbolique. Elle met au centre des préoccupations la dynamique de transformation sociale tendant vers une infinité de territoires bien au-delà des seuls territoires offerts par les États. Les textes, issus du 3e colloque du Collège international des sciences territoriales, sont organisés en 4 parties : politiques publiques environnementales, d’économie sociale et solidaire ou d’accès à la santé ; implantation universitaire dans la diversité des demandes exprimées et latentes ; demande de données territoriales pour mieux connaître les inégalités, la métropolisation ou la patrimonialisation ; diversité des refus et réticences à l’institutionnalisation ou tactiques utilisées par les territoires existants pour se relégitimer en prenant en compte de nouvelles demandes.Cet ouvrage constate qu’à chaque demande ne correspond pas une offre et réfute l’existence d’un marché territorial régulant les attentes sociales. Bien au contraire, la quête territoriale incomplète, imparfaite et infinie de nombreux collectifs tend à montrer une réinvention continuelle des cadres dans lesquels les sociétés contemporaines organisent les interactions entre les humains, les intérêts, les enjeux. La dimension territoriale de ces actions apparaît alors déterminante pour comprendre ce qui se joue avec la territorialisation des demandes sociale