Multisubunit UBIQUITIN LIGASES (E3s) that are assembled on a CULLIN scaffold were first reported seven years ago 1,2. The discovery of the archetypical cullin-RING ubiquitin ligase-SCF Cdc4-benefited from a strong foundation of genetic studies on cell division in Saccharomyces cerevisiae and Caenorhabditis elegans. The model established by SCF can now be extended to a superfamily of CULLIN-RING LIGASES (CRLS) that are found throughout eukaryotes. Together, these enzymes regulate a dazzling array of cellular and organismal processes-from glucose sensing and DNA replication to limb patterning and circadian rhythms. Although there is a great diversity of CRLs in terms of their composition and function, we propose that these enzymes can be characterized by a set of general principles that will apply to most members of the superfamily. In this review, we summarize what has been learned about SCF and other CRLs over the past seven years and, from this, we extract the key features that typify these enzymes. We also highlight the murky areas in which our understanding remains far from clear. Cullin-RING ligases are modular The cullin family. Human cells express seven different cullins (CUL1, 2, 3, 4A, 4B, 5 and 7) that each nucleate a multisubunit ubiquitin ligase (FIG. 1). In addition, at least two other proteins (the APC2 subunit of the ANAPHASE-PROMOTING COMPLEX/CYCLOSOME (APC/C) and the p53 cytoplasmic anchor protein PARC) contain a 'cullin-homology domain' 3-5. Although APC2 and PARC have ubiquitin-ligase activity, they are clearly distinct from the other cullins and are not considered here. The archetypal CRLs, which contain CUL1, are named SCF ubiquitin ligases, whereas the other CRLs have distinct subunit compositions and have been referred to by various names (TABLE 1). Cullin-RING ligases have an extended, rigid architecture. Much of what is known and inferred about the architecture of CRLs comes from protein-protein interaction studies and sequence comparisons that have been interpreted in light of three-dimensional (3D) X-ray crystal structures (for a summary of the solved structures for CRL complexes and related proteins, see online supplementary information S1 (table)). CUL1 and presumably all other cullins have a curved, yet rigid, N-terminal stalk that is comprised of three repeats of a five-helix bundle (cullin repeat (CR)1-3) and is linked to a C-terminal globular domain 6 (FIG. 2). The SKP1 adaptor binds to the N-terminal CR1 region, whereas the zinc-binding RING-H2-DOMAIN proteinwhich is known as either ROC1, RBX1 or HRT1 (REFS 7-10; and is referred to here as the 'RING' subunit)-binds 100 Å away from SKP1 and interdigitates itself with the C-terminal globular domain. SKP1 recruits substrate receptors and the RING subunit recruits the ubiquitin-conjugating enzyme (E2) to form the active ligase complex. The rigidity of the N-terminal stalk of CUL1 might juxtapose the E2 and the substrate to favour ubiquitin transfer, because a mutation that

Historically, N6-methyladenosine (m6A) has been identified as the most abundant internal modification of messenger RNA (mRNA) in eukaryotes 1. Its mammalian function remained unknown until recently, when it was reported that thousands of mammalian mRNAs and long noncoding RNAs (lncRNAs) show m6A modification 2,3 and that m6A demethylases are required for mammalian energy homeostasis and fertility 4,5. As yet, the identity of m6A methyltransferases (MTase) and the molecular mechanisms regulated by m6A remains unclear. Here, we show that two proteins, the putative m6A MTase, methyltransferase-like 3 (Mettl3) 6, and a related but uncharacterized protein Mettl14, function synergistically to control m6A formation in mammalian cells. Since m6A modification is involved in cell fate determination in yeast 7,8 and embryo development in plant 9,10, we knocked down Mettl3 and Mettl14, respectively, in mouse embryonic stem cells (mESCs). The resulting cells displayed equivalent phenotypes characterized by lack of m6A RNA methylation and lost self-renewal capability. We also observed that a large number of transcripts, including many encoding developmental regulators, showed m6A methylation inversely correlated with mRNA stability and gene expression. Further analysis suggested that some of these effects were mediated through Human antigen R (HuR) and microRNA pathways. Overall our work provides first experimental evidence of mammalian m6A MTases and reveals a previously unknown gene regulatory mechanism operating in mESCs through m6A methylation. This mechanism is required to keep mESCs at their ground state and may be relevant to thousands of mRNAs and lncRNAs in various cell types.

Ubiquitin chains linked via lysine 48 (K48) of ubiquitin mediate recognition of ubiquitinated proteins by the proteasome. However, the mechanisms underlying polymerization of this targeting signal on a substrate are unknown. Here we dissect this process using the cyclin-dependent kinase inhibitor Sic1 and its ubiquitination by the cullin-RING ubiquitin ligase SCF(Cdc4) and the ubiquitin-conjugating enzyme Cdc34. We show that Sic1 ubiquitination can be separated into two steps: attachment of the first ubiquitin, which is rate limiting, followed by rapid elongation of a K48-linked ubiquitin chain. Mutation of an acidic loop conserved among Cdc34 orthologs has no effect on attachment of the first ubiquitin onto Sic1 but compromises the processivity and linkage specificity of ubiquitin-chain synthesis. We propose that the acidic loop favorably positions K48 of a substrate-linked ubiquitin to attack SCF bound Cdc34 approximately ubiquitin thioester and thereby enables processive synthesis of K48-linked ubiquitin chains by SCF-Cdc34.

The cellular response to hypoxia involves several signalling pathways that mediate adaptation and survival. REDD1 (regulated in development and DNA damage responses 1), a hypoxiainducible factor-1 target gene, has a crucial role in inhibiting mammalian target of rapamycin complex 1 (mTORC1) signalling during hypoxic stress. However, little is known about the signalling pathways and post-translational modifications that regulate REDD1 function. Here, we show that REDD1 is subject to ubiquitin-mediated degradation mediated by the CUL4A-DDB1-ROC1-b-TRCP E3 ligase complex and through the activity of glycogen synthase kinase 3b. Furthermore, REDD1 degradation is crucially required for the restoration of mTOR signalling as cells recover from hypoxic stress. Our findings define a mechanism underlying REDD1 degradation and its importance for regulating mTOR signalling.