title: APC/C-mediated degradation of dsRNA-binding protein 4 (DRB4) involved in RNA silencing.
abstract: 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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  Our work identified a first plant substrate of the APC/C, which is not a regulator of the cell cycle. Though we cannot exclude that APC/C-dependent degradation of DRB4 has some regulatory roles under specific growth conditions, our work rather points to a housekeeping function of APC/C in maintaining precise cellular-protein concentrations and homeostasis of DRB4.
authors.affiliation: Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique, Unité Propre de Recherche 2357, Conventionné avec l'Université de Strasbourg, Strasbourg, France.
authors.forename: Katia
authors.initials: K
authors.lastname: Marrocco
authors.forename: Marie-Claire
authors.initials: MC
authors.lastname: Criqui
authors.forename: Jérôme
authors.initials: J
authors.lastname: Zervudacki
authors.forename: Gregory
authors.initials: G
authors.lastname: Schott
authors.forename: Herfried
authors.initials: H
authors.lastname: Eisler
authors.forename: Aude
authors.initials: A
authors.lastname: Parnet
authors.forename: Patrice
authors.initials: P
authors.lastname: Dunoyer
authors.forename: Pascal
authors.initials: P
authors.lastname: Genschik
mesh_terms: D064173:Anaphase-Promoting Complex-Cyclosome; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D018749:RNA, Plant / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000235:genetics / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; C511456:DRB4 protein, Arabidopsis; D018749:RNA, Plant; D016601:RNA-Binding Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome