Immunoprecipitated proteins were eluted by adding 1 Bolt LDS Sample Buffer (Thermo Fisher Scientific, Waltham, MA, USA) mixed with -mercaptoethanol and subsequent incubation at 95 C for 5 min. a proteasome- and CRL-dependent manner. Next, we generated knockout mice and exhibited that this DCAF12-mediated degradation of MOV10 is usually conserved in mice and humans. Detailed analysis of Dcaf12-deficient mice revealed that their testes produce fewer mature sperms, phenotype accompanied by elevated MOV10 and imbalance in meiotic markers SCP3 and -H2AX. Additionally, the percentages of splenic CD4+ T and natural killer T (NKT) cell populations were significantly altered. In vitro, activated Dcaf12-deficient T cells displayed inappropriately stabilized MOV10 and increased levels of activated caspases. In summary, we recognized MOV10 as a novel substrate of CRL4-DCAF12 and exhibited the biological relevance of the DCAF12-MOV10 pathway in spermatogenesis and T cell activation. [16]. Furthermore, it was implicated in the Hippo pathway regulation [17] and showed to be essential for normal synaptic function and plasticity [18]. In placental mammals, DCAF12 has two close paralogsDCAF12L1 and DCAF12L2 (protein sequence similarity ~70%) [19]. Although DCAF12L2 probably emerged by retrotransposition in the placental mammal ancestor, DCAF12L1 is present only in Euarchontoglires (a clade that includes rodents and primates) and seems to be a result of tandem duplication [20]. The expression pattern of DCAF12 paralogs differs from DCAF12, and it is unknown whether they assemble into functional CRL4. In human cells, DCAF12 regulates the stability of proteins ending in a twin-glutamic acid degron (C-terminal -EE degron) [21]. So far, only the regulation of melanoma antigen gene (MAGE) family members by DCAF12 has been studied [22]. Expression of MAGEs is normally Rabbit Polyclonal to ECM1 restricted to male germ cells, but the genes are aberrantly reactivated in various cancers and drive tumorigenesis. In malignancy cells, DCAF12 targets MAGE-A3 and MAGE-A6 for degradation in response to starvation [22]. However, the physiological function of DCAF12 in vertebrates remains unknown. Moloney leukemia computer virus 10 (MOV10) is usually a highly conserved RNA helicase belonging to the UPF1-like group of helicase superfamily 1 [23,24]. MOV10 homologs have been found in plants (SDE3 in [25]), nematodes (ERI-6/7 in [26,27]), and insects (Armi in [28,29]). The vertebrate genome also encodes MOV10 paralog MOV10L1, which arose by gene duplication [30,31]. MOV10 and its homologs have an evolutionary conserved but enigmatic role in post-transcriptional gene silencing (RNA interference) and silencing of transposons, viruses, and recently duplicated genes [25,27,28,29,30,31,32,33,34]. These MOV10 activities are a crucial part of the host defense system across diverse species. MOV10 binds retrotransposon RNAs and is a potent inhibitor of retrotransposition [35,36,37,38,39]. Post-transcriptional reduction of retrotransposon transcripts [38] and inhibition NSC 185058 of reverse transcription [37] were shown to be involved in the inhibition. However, the exact mechanism of retrotransposon restriction remains unclear [40,41]. Overexpression of NSC 185058 MOV10 also inhibits replication and reduces infectivity of a wide range of exogenous retroviruses, including human immunodeficiency computer virus type-1 (HIV-1) [35,42,43,44,45]. Furthermore, MOV10 is an interferon-stimulated gene [46,47], which exhibits broad antiviral activity [46,48,49,50,51,52,53,54,55]. Apart from retrotransposon restricting and antiviral activities, MOV10 has an essential role in post-transcriptional gene regulation, especially NSC 185058 in the microRNA (miRNA) pathway [56,57,58,59]. Human MOV10 predominantly binds to the 3 UTR of mRNAs, in close proximity to miRNA recognition elements, and usually facilitates miRNA-mediated translational suppression [59]. Additionally, mouse MOV10 was shown to regulate miRNA biogenesis and was implicated in the splicing control [60]. Furthermore, MOV10 was suggested to facilitate nonsense-mediated mRNA decay [61] and implicated in Polycomb-mediated transcriptional silencing [62]. Here, we discovered that DCAF12 directly recognizes the C-terminal glutamic acid-leucine (-EL) degron of MOV10 and mediates its proteasome-dependent degradation. Additionally, we established knockout (KO) mice and found that DCAF12 controls the protein level NSC 185058 of MOV10 during spermatogenesis and in T cells, especially after their activation. deficiency led to a decreased sperm count, dysregulation of immune cell populations, and increased splenocyte apoptosis after T cell activation. These observations spotlight the biological importance of the DCAF12-mediated MOV10 degradation in vivo. 2. Results 2.1. Proteomic Analysis of DCAF12-Interacting Proteins To reduce the presence of non-specific interactors of DCAF12, we adopted a tandem purification method to analyze the composition of cullin-based ubiquitin ligases. The procedure is based on sequential purification of a substrate receptor and a cullin scaffold protein. We validated this method using canonical ubiquitin ligase SKP1-CUL1-F-box protein (SCF)-TRCP and its interaction with numerous well-known substrates. These experiments showed significant enrichment in substrate isolation and, at the same time, reduction of non-specific binding (data not shown). Subsequently, we employed the same plan to analyze potential substrates of multisubunit ubiquitin.