**2. RNF183**

The E3 ubiquitin ligase RNF183 has been identified as a new biomarker of endometrial carcinoma (EC) via gene expression screening and protein level experiments on carcinoma samples. Furthermore, the di fferential expression of RNF183 in primary endometrial tumors has been shown to be correlated with its expression level in corresponding uterine fluid samples and it exhibits an analogous value in the initial stage of EC [22]. EC is the most common invasive tumor of the female genital tract, which is usually detected in its initial stages. However, 20% of patients are at an advanced stage at the time of detection. Because molecular markers for the diagnosis of EC have ye<sup>t</sup> to be validated, new methods for the medical prognostication and classification of EC are needed to combat this deadly disease. RNF183 could be helpful as a precise molecular tool to diagnose EC and reduce unnecessary biopsies.

Another study has indicated that RNF183 interacts with fetal and adult testis-expressed 1 (FATE1) in tumors and negatively regulates the apoptosis e ffector Bcl-2-interacting killer (BIK), leading to increased viability of tumor cells [15]. FATE1 is one of the cancer/testis antigens whose expression is biased to the testes but is also activated in cancer [49,50]. Depletion of FATE1 reduces the viability of cancer cells. Large-scale proteomic studies have revealed that BIK is a FATE1-interacting partner. At the same time, RNF183 has also been revealed to be a FATE1-interacting partner. BIK associates with both FATE1 and RNF183, and both RNF183 depletion and the mutant RNF183, which exhibits the loss of enzyme activity, increase BIK protein accumulation. Thus, FATE1 and RNF183 collaborate to suppress BIK protein levels and escape from BIK-related apoptotic signaling [15]. However, in Ewing sarcoma cells, no appreciable levels of BIK protein are detectable even in the presence of the proteasome inhibitor MG132, and FATE1 depletion does not induce BIK accumulation.

There is another context-selective mechanism in Ewing sarcoma. FATE1 is most robustly induced by the Ewing sarcoma breakpoint region 1-Friend Leukemia Integration 1 (EWSR1-FLI1) chimeric transcription factor caused by a pathognomonic chromosomal translocation of Ewing sarcoma and interacts with Bcl-2/adenovirus E1B 19 kDa protein-interacting protein 3-like (BNIP3L). Then, BNIP3L is degraded in the presence of RNF183 [16]. Because BNIP3L is a tumor suppressor [51], its depletion increases tumorigenesis in vivo [16].

Moreover, *RNF183* has been identified as a gene conferring resistance to trametinib. Trametinib is one of the anticancer drugs inhibiting MEK1/2 [52]. RNF183 expression is increased after trametinib treatment, which in turn activates the NF-κB pathway. Then, the activated NF-κB increases the expression of the pro-inflammatory cytokine interleukin-8 (IL-8), which is a downstream target of NF-κB [20]. IL-8 signaling increases the proliferation and survival of cancer cells and potentiates their migration [53]. Thus, RNF183 confers resistance to trametinib on colorectal cancer (CRC) cells and promotes their proliferation and metastasis [20].

Under physiological conditions, RNF183 is not expressed in the large intestine, but is specifically expressed in the kidney [10]. The abnormal expression of RNF183 is thought to be involved in several diseases, not only tumorigenesis but also inflammatory conditions such as inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), which is a chronic, idiopathic, inflammatory, gastrointestinal disease, the molecular mechanism underlying the development and pathophysiology of which have not been fully elucidated. [15–17,20–22]. However, FATE1 is not expressed in the intestine. Therefore, there is a FATE1-independent inflammatory mechanism involving RNF183 in the large intestine. In fact, some studies have shown that RNF183 is upregulated in colon samples of the intestinal tissues of IBD patients [17] and the colons of mice with colitis treated with trinitrobenzene sulfonic acid (TNBS) or dextran sulfate sodium (DSS) [17,18].

It has been reported that RNF183 is largely involved in executing apoptosis in response to prolonged ER stress. It is considered that the mechanism of apoptosis involving RNF183 features the ubiquitylation and degradation of B-cell lymphoma extra-large (Bcl-xL), which functions as an inhibitor of apoptosis by preventing cytochrome c release [11]. Bcl-xL is usually localized to the mitochondria [54], whereas RNF183 is predominantly localized to the ER, Golgi, and lysosome [12]. Some Bcl-xL may be targeted to the ER [55], where it is in the vicinity of RNF183. Then, since

their cytosolic domains can interact with each other, they interact directly and RNF183 ubiquitylates Bcl-xL [11]. The detailed mechanism behind this involves inositol requiring 1 α (IRE1 α) being activated by prolonged ER stress and readily decreasing microRNA-7 (miR-7) and microRNA-96 (miR-96), presumably by the digestion of miR precursors through the IRE1-dependent decay of mRNA [56,57]. Since miR-7 and miR-96 negatively regulate RNF183 by directly interacting with its 3--UTR [17], their decrease eventually stabilizes the RNF183 mRNA and leads to increased protein levels. This increase in RNF183 in turn promotes its binding to Bcl-xL, polyubiquitylation, and subsequent degradation. The gradual decrease in Bcl-xL levels eventually triggers the intrinsic apoptotic pathway [11]. It has also been reported that increased RNF183 due to decreased miR-7 may contribute to the pathogenesis of IBD by recognizing NF-κB inhibitor α (IκBα), not Bcl-xL, as a substrate and degrading ubiquitylated IκB α [17]. Because IκB α is a suppressor of NF-κB, the reduction of IκB α by ubiquitylation and degradation induces NF-κB activation.

Recently, another mechanism of RNF183-related IBD pathogenesis has also been reported. Specifically, RNF183 recognizes DR5 as a substrate protein and K63-ubiquitylated DR5 is transported to lysosomes for degradation. In addition, RNF183 promotes TRAIL-induced caspase activation and apoptosis, providing new insights into the potential roles of RNF183 in DR5-mediated caspase activation in the pathogenesis of IBD [18]. RNF183-mediated ubiquitylation of substrates, Bcl-xL, IκBα, and DR5, and the negative regulation of RNF183 by miR-7 may be important novel epigenetic mechanisms in the pathogenesis of IBD.

In human and mouse tissues, RNF183 is specifically expressed in the kidney [10]. In particular, high Rnf183 expression in the renal medullary collecting duct has been reported from a tissue analysis using GFP-knock-in mice [13]. The kidney is the only tissue that is continuously under hypertonic conditions, and this hypertonicity gradually increases from the outer medulla down to the inner medulla. Nuclear factor of activated T cells 5 (NFAT5)/tonicity-responsive enhancer-binding protein is a transcription factor essential for the adaptation to hypertonic conditions, under which it stimulates the transcription of some genes [58]. The Rnf183 gene is also downstream of NFAT5 [14]. Indeed, the expression of Rnf183 in the renal medulla is dramatically decreased upon treatment with the loop diuretic furosemide, which can downregulate NFAT5 levels by inhibiting the Na-K-Cl cotransporter type 2 (NKCC2) and inducing hypotonicity in the medulla [13]. This is consistent with the decrease in NFAT5 protein and the mRNA expression of its target gene. Additionally, Rnf183 expression increases markedly in mouse inner-medullary collecting duct (mIMCD-3) cells treated with hypertonic NaCl. Rnf183, as well as several NFAT5 downstream genes, protects renal medullary cells from hypertonicity-induced apoptosis. mIMCD-3 cells transfected with siRNA targeting Rnf183 exhibit significant increases in cleaved caspase-3 protein levels. Therefore, Rnf183 expression is involved in the osmotic tolerance of mIMCD-3 cells [14].

In terms of its subcellular localization, RNF183 is predominantly localized to the endoplasmic reticulum (ER), Golgi apparatus and lysosome. Its stability depends on its interaction with Sec16A, which is involved in the formation of coat protein complex II (COPII) vesicles. However, Sec16A is not ubiquitylated by RNF183 [12]. Recently, it has been identified that the Na,K-ATPase β1 subunit, which forms a complex with Na,K-ATPase α1 subunit on the plasma membrane, is one of the substrates for RNF183. Na,K-ATPase contributes to the regulation of cell volume and solute absorption by the active transport of Na<sup>+</sup> and K<sup>+</sup> across the plasma membrane [59], and the expression of both α1 and β1 subunits is increased for adapting hypertonic condition in human renal cells [60]. RNF183 ubiquitylates only the β1 subunit, not the α1 subunit. Then, a complex with α1 and β1 subunits translocates from the plasma membrane to the lysosome, where it is degraded [19]. Therefore, RNF183 may play an important role in the kidney for the adaptation to hyperosmotic stress by regulating the level of Na,K-ATPase.

As described above, the kidney-specific ubiquitin ligase RNF183 protects cells from apoptosis induced by hypertonic stress in the kidney, whereas its aberrant expression such as in the colon induces inflammatory and tumorigenesis. Understanding the function of RNF183 could lead to new therapeutic strategies for patients with IBD and various types of cancer.
