The BRCA1 tumor suppressor forms a heterodimer with the BARD1 protein, and the resulting complex functions as an E3 ubiquitin ligase that catalyzes the synthesis of polyubiquitin chains. In theory, polyubiquitination can occur by isopeptide bond formation at any of the seven lysine residues of ubiquitin. The isopeptide linkage of a polyubiquitin chain is a particularly important determinant of its cellular function, such that K48-linked chains commonly target proteins for proteasomal degradation, while K63 chains serve non-proteolytic roles in various signaling pathways. To determine the isopeptide linkage formed by BRCA1/BARD1-dependent polyubiquitination, we purified a full-length heterodimeric complex and compared its linkage specificity with that of E6-AP, an E3 ligase known to induce proteolysis of its cellular substrates. Using a comprehensive mutation analysis, we found that E6-AP catalyzes the synthesis of K48-linked polyubiquitin chains. In contrast, however, the BRCA1/BARD1 heterodimer directs polymerization of ubiquitin primarily through an unconventional linkage involving lysine residue K6. Although heterologous substrates of BRCA1/BARD1 are not known, BRCA1 autoubiquitination occurs principally by conjugation with K6-linked polymers. The ability of BRCA1/BARD1 to form K6-linked polyubiquitin chains suggests that it may impart unique cellular properties to its natural enzymatic substrates.The BRCA1 tumor suppressor gene has been implicated in various cellular processes that include DNA repair, transcriptional regulation, and cell cycle checkpoint control (1). Its protein product contains an NH 2 -terminal RING domain and two COOH-terminal BRCT repeats. In vivo, BRCA1 exists as a heterodimer with BARD1, a related protein that displays a similar configuration of RING and BRCT motifs (2). The BRCA1/BARD1 heterodimer is stabilized by a 4-helix bundle formed by ␣-helices that immediately flank the RING domains of both polypeptides (3). Since most cellular BRCA1 polypeptides are found in association with BARD1 (4, 5), the BRCA1/ BARD1 heterodimer is likely to be an essential mediator of BRCA1 function (6). This conclusion is strongly supported by analysis of Bard1-null mice, which display a characteristic phenotype that is essentially indistinguishable from that of Brca1-null animals (7).Recent studies have uncovered an enzymatic role for the BRCA1/BARD1 heterodimer in protein ubiquitination (8 -15). Ubiquitination occurs through a sequential process involving ubiquitin-activating enzyme (E1), 1 a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3) (16,17). In the final step of this process, a ubiquitin monomer is covalently attached to a lysine residue on the ultimate substrate. The monoubiquitinated substrate can also serve as a nucleus for assembly of a polyubiquitin chain that, in many cases, will target the substrate for proteasome-mediated degradation. A common feature of many E3 ligases is the presence of a RING motif, and recent studies have established that sequences encompassing the RI...

Post-transcriptional gene silencing mediated by microRNAs (miRNAs) modulates numerous developmental and stress response pathways. For the last two decades, HASTY (HST), the ortholog of human EX-PORTIN 5, was considered to be a candidate protein that exports plant miRNAs from the nucleus to the cytoplasm. Here, we report that HST functions in the miRNA pathway independent of its cargo-exporting activity in Arabidopsis. We found that Arabidopsis mutants with impaired HST shuttling exhibit normal subcellular distribution of miRNAs. Interestingly, protein-protein interaction and microscopy assays showed that HST directly interacts with the microprocessor core component DCL1 through its N-terminal domain. Moreover, mass spectrometry analysis revealed that HST also interacts independently of its N-terminal domain with the mediator complex subunit MED37. Further experiments revealed that HST could act as a scaffold to facilitate the recruitment of DCL1 to genomic MIRNA loci by stabilizing the DCL1-MED37 complex, which in turn promotes the transcription and proper processing of primary miRNA transcripts (pri-miRNAs). Taken together, these results suggest that HST is likely associated with the formation of the miRNA biogenesis complex at MIRNA genes, promoting the transcription and processing of pri-miRNAs rather than the direct export of processed miRNAs from the nucleus.

DNA methylation patterns in plants are dynamically regulated by DNA methylation and active DNA demethylation in response to both environmental changes and development of plant. Beginning with the removal of methylated cytosine by ROS1/DME family of 5-methylcytosine DNA glycosylases, active DNA demethylation in plants occurs through base excision repair. So far, many components involved in active DNA demethylation remain undiscovered. Through a forward genetic screening of Arabidopsis mutants showing DNA hypermethylation at the EPF2 promoter region, we identified the conserved iron-sulfur cluster assembly protein MET18. MET18 dysfunction caused DNA hypermethylation at more than 1000 loci as well as the silencing of reporter genes and some endogenous genes. MET18 can directly interact with ROS1 in vitro and in vivo. ROS1 activity was reduced in the met18 mutant plants and point mutation in the conserved Fe-S cluster binding motif of ROS1 disrupted its biological function. Interestingly, a large number of DNA hypomethylated loci, especially in the CHH context, were identified from the met18 mutants and most of the hypo-DMRs were from TE regions. Our results suggest that MET18 can regulate both active DNA demethylation and DNA methylation pathways in Arabidopsis.