Protein poly(ADP-ribosyl)ation and ubiquitination are two key post-translational modifications regulating many biological processes. Through crystallographic and biochemical analysis, we show that the RNF146 WWE domain recognizes poly(ADP-ribose) (PAR) by interacting with iso-ADP-ribose (iso-ADPR), the smallest internal PAR structural unit containing the characteristic ribose-ribose glycosidic bond formed during poly(ADP-ribosyl)ation. The key iso-ADPR-binding residues we identified are highly conserved among WWE domains. Binding assays further demonstrate that PAR binding is a common function for the WWE domain family. Since many WWE domain-containing proteins are known E3 ubiquitin ligases, our results suggest that protein poly(ADP-ribosyl)ation may be a general mechanism to target proteins for ubiquitination.Supplemental material is available for this article.Received November 2, 2011; revised version accepted December 19, 2011. Protein ubiquitination regulates diverse biological processes; however, the mechanism by which proteins are earmarked for ubiquitination remains incompletely understood. Other than phosphorylation, which is a general mechanism for many cases, hydroxylation of a substrate (i.e., HIF1-a) and the binding of small molecules (e.g., the plant hormone auxin) to E3 ligases have been shown to control protein ubiquitination in sporadic cases (Willems et al. 1999;Min et al. 2002;Bergink and Most recently, PARylation has been shown to control the polyubiquitination and degradation of axin, a key regulator of the Wnt signaling pathway (Huang et al. 2009;Callow et al. 2011;Kang et al. 2011;Zhang et al. 2011). In all of these reports, RNF146 (aka Iduna), which contains a WWE domain and a RING domain (Supplemental Fig. 1), is the only known E3 ubiquitin ligase to date that requires PARylation of the substrate for subsequent polyubiquitination (Callow et al. 2011;Kang et al. 2011;Zhang et al. 2011). The RNF146 WWE domain has been shown to bind PAR (Callow et al. 2011;Zhang et al. 2011), and it was reported that a short PAR-binding motif (PBM) within the domain retains this binding activity ). The PBM was originally found in histones and several other proteins (Gagne et al. 2008). However, the PBM identified in RNF146 is not conserved in other WWE domains, so it remains unclear whether the WWE domain represents a novel PAR-binding domain. Here we reveal the structural basis of the RNF146 WWE domain/iso-ADPR interaction and, for the first time, define the PAR/iso-ADPR binding as a bona fide function of the WWE domain family. Importantly, the structural coupling of WWE domains and E3 ligase domains in many WWE domaincontaining proteins suggests a functional coupling of protein PARylation and ubiquitination. Results and DiscussionThe RNF146 WWE domain recognizes iso-ADPR, but not ADPR We sought to clarify the requirement of the entire RNF146 WWE domain structure for PAR binding through structural analysis. PAR polymers display high chemical heterogeneity (in both lengths and branching patterns) and ar...
Protein poly(ADP-ribosyl)ation (PARylation) plays a role in diverse cellular processes such as DNA repair, transcription, Wnt signaling, and cell death1–6. Recent studies have shown that PARylation can serve as a signal for the polyubiquitination and degradation of several critical regulatory proteins, including Axin and 3BP2 (refs 7–9). The RING-type E3 ubiquitin ligase RNF146 (a.k.a. Iduna) is responsible for PARylation-dependent ubiquitination (PARdU)10–12. Here we provide a structural basis for RNF146 catalyzed PARdU and how PARdU specificity is achieved. First, we show that iso-ADPr, the smallest internal poly(ADP-ribose) (PAR) structural unit, binds between the WWE and RING domains of RNF146 and functions as an allosteric signal that switches the RING domain from a catalytically inactive state to an active one. In the absence of PAR, the RING domain is unable to efficiently bind and activate an E2. Binding of PAR/iso-ADPr induces a major conformational change that creates a functional RING structure. Thus RNF146 represents a new mechanistic class of RING E3 ligases whose activities are regulated by non-covalent ligand binding, which may provide a template for designing inducible protein-degradation systems. Second, we found that RNF146 directly interacts with the PAR polymerase tankyrase (TNKS). Disruption of the RNF146/TNKS interaction inhibits turnover of the substrate Axin in cells. Thus, both substrate PARylation and PARdU are catalyzed by enzymes within the same protein complex, and PARdU substrate specificity may be primarily determined by the substrate-TNKS interaction. We propose that maintenance of unliganded RNF146 in an inactive state may serve to maintain the stability of the RNF146-TNKS complex, which in turn regulates the homeostasis of PARdU activity in the cell.
Loss- and gain-of-function mutations of the X-linked gene MECP2 (methyl-CpG binding protein 2) lead to severe neurodevelopmental disorders in humans, such as Rett syndrome (RTT) and autism. MeCP2 is previously known as a transcriptional repressor by binding to methylated DNA and recruiting histone deacetylase complex (HDAC). Here, we report that MeCP2 regulates gene expression posttranscriptionally by suppressing nuclear microRNA processing. We found that MeCP2 binds directly to DiGeorge syndrome critical region 8 (DGCR8), a critical component of the nuclear microRNA-processing machinery, and interferes with the assembly of Drosha and DGCR8 complex. Protein targets of MeCP2-suppressed microRNAs include CREB, LIMK1, and Pumilio2, which play critical roles in neural development. Gain of function of MeCP2 strongly inhibits dendritic and spine growth, which depends on the interaction of MeCP2 and DGCR8. Thus, control of microRNA processing via direct interaction with DGCR8 represents a mechanism for MeCP2 regulation of gene expression and neural development.
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