RING Finger Protein Research Articles

E3 ligase RNF2 inhibits porcine circovirus type 3 replication by targeting its capsid protein for ubiquitination-dependent degradation.

Porcine circovirus type 3 (PCV3) is closely associated with various diseases, such as the porcine dermatitis, nephropathy syndrome, and multisystemic clinicopathological diseases. PCV3-associated diseases are increasingly recognized as severe diseases in the global swine industry. Ring finger protein 2 (RNF2), an E3 ubiquitin ligase exclusively located in the nucleus, contributes to various biological processes. This ligase interacts with the PCV3 Cap. However, its role in PCV3 replication remains unclear. This study confirmed that the nuclear localization signal domain of the Cap and the RNF2 N-terminal RING domain facilitate the interaction between the Cap and RNF2. Furthermore, RNF2 promoted the binding of K48-linked polyubiquitination chains to lysine at positions 139 and 140 (K139 and K140) of the PCV3 Cap, thereby degrading the Cap. RNF2 knockdown and overexpression increased or decreased PCV3 replication, respectively. Moreover, the RING domain-deleted RNF2 mutant eliminated the RNF2-induced degradation of the PCV3 Cap and RNF2-mediated inhibition of viral replication. This indicates that both processes were associated with its E3 ligase activity. Our findings demonstrate that RNF2 can interact with and degrade the PCV3 Cap via its N-terminal RING domain in a ubiquitination-dependent manner, thereby inhibiting PCV3 replication.IMPORTANCEPorcine circovirus type 3 is a recently described pathogen that is prevalent worldwide, causing substantial economic losses to the swine industry. However, the mechanisms through which host proteins regulate its replication remain unclear. Here, we demonstrate that ring finger protein 2 inhibits porcine circovirus type 3 replication by interacting with and degrading the Cap of this pathogen in a ubiquitination-dependent manner, requiring its N-terminal RING domain. Ring finger protein 2-mediated degradation of the Cap relies on its E3 ligase activity and the simultaneous existence of K139 and K140 within the Cap. These findings reveal the mechanism by which this protein interacts with and degrades the Cap to inhibit porcine circovirus type 3 replication. This consequently provides novel insights into porcine circovirus type 3 pathogenesis and facilitates the development of preventative measures against this pathogen.

SMARCA2 and SMARCA4 Participate in DNA Damage Repair.

The switching/sucrose non-fermentable (SWI/SNF) Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A (SMARCA) member 2 and member 4 (SMARCA2/4) are paralogs and act as the key enzymatic subunits in the SWI/SNF complex for chromatin remodeling. However, the role of SMARCA2/4 in DNA damage response remains unclear. Laser microirradiation assays were performed to examine the key domains of SMARCA2/4 for the relocation of the SWI/SNF complex to DNA lesions. To examine the key factors that mediate the recruitment of SMARCA2/4, the relocation of SMARCA2/4 to DNA lesions was examined in HeLa cells treated with inhibitors of Ataxia-telangiectasia-mutated (ATM), Ataxia telangiectasia and Rad3-related protein (ATR), CREB-binding protein (CBP) and its homologue p300 (p300/CBP), or Poly (ADP-ribose) polymerase (PARP) 1/2 as well as in H2AX-deficient HeLa cells. Moreover, by concomitantly suppressing SMARCA2/4 with the small molecule inhibitor FHD286 or Compound 14, the function of SMARCA2/4 in Radiation sensitive 51 (RAD51) foci formation and homologous recombination repair was examined. Finally, using a colony formation assay, the synergistic effect of PARP inhibitors and SMARCA2/4 inhibitors on the suppression of tumor cell growth was examined. We show that SMARCA2/4 relocate to DNA lesions in response to DNA damage, which requires their ATPase activities. Moreover, these ATPase activities are also required for the relocation of other subunits in the SWI/SNF complex to DNA lesions. Interestingly, the relocation of SMARCA2/4 is independent of γH2AX, ATM, ATR, p300/CBP, or PARP1/2, indicating that it may directly recognize DNA lesions as a DNA damage sensor. Lacking SMARCA2/4 prolongs the retention of γH2AX, Ring Finger Protein 8 (RNF8) and Breast cancer susceptibility gene 1 (BRCA1) at DNA lesions and impairs RAD51-dependent homologous recombination repair. Furthermore, the treatment of an SMARCA2/4 inhibitor sensitizes tumor cells to PARP inhibitor treatment. This study reveals SMARCA2/4 as a DNA damage repair factor for double-strand break repair.

RNF213 promotes Treg cell differentiation by facilitating K63-linked ubiquitination and nuclear translocation of FOXO1

Autoreactive CD4+ T helper cells are critical players that orchestrate the immune response both in multiple sclerosis (MS) and in other neuroinflammatory autoimmune diseases. Ubiquitination is a posttranslational protein modification involved in regulating a variety of cellular processes, including CD4+ T cell differentiation and function. However, only a limited number of E3 ubiquitin ligases have been characterized in terms of their biological functions, particularly in CD4+ T cell differentiation and function. In this study, we found that the RING finger protein 213 (RNF213) specifically promoted regulatory T (Treg) cell differentiation in CD4+ T cells and attenuated autoimmune disease development in an FOXO1-dependent manner. Mechanistically, RNF213 interacts with Forkhead Box Protein O1 (FOXO1) and promotes nuclear translocation of FOXO1 by K63-linked ubiquitination. Notably, RNF213 expression in CD4+ T cells was induced by IFN-β and exerts a crucial role in the therapeutic efficacy of IFN-β for MS. Together, our study findings collectively emphasize the pivotal role of RNF213 in modulating adaptive immune responses. RNF213 holds potential as a promising therapeutic target for addressing disorders associated with Treg cells.

M6A methylation of RNF43 inhibits the progression of endometriosis through regulating oxidative phosphorylation via NDUFS1.

Oxidative phosphorylation is becoming increasingly important in the induction and development of endometriosis. Recently, it has been reported that ring finger protein 43 (RNF43) is involved in the process of oxidative phosphorylation, but the mechanism remains unclear. Our investigation is to delve into the roles of RNF43 in endometriosis and elucidate the related mechanisms. We found RNF43 was downregulated in ectopic endometrial tissue and primary ectopic endometrial stromal cells (ECESCs). Knockdown of RNF43 enhanced cell viability and migration by activating oxidative phosphorylation in eutopic endometrial stromal cells (EUESCs), while overexpression of RNF43 led to the opposite results. Moreover, RNF43 reinforced the ubiquitination and degradation of NADH dehydrogenase Fe-S protein 1 (NDUFS1) by interacting with it. Likewise to RNF43 overexpression, NDUFS1 silencing inhibited cell viability, migration, and oxidative phosphorylation in ECESCs. NDUFS1 was a downstream target of RNF43, mediating its biological role in endometriosis. Interestingly, the expression and stability of RNF43 mRNA were regulated by the Methyltransferase-like 3 (METTL3)/IGF2BP2 m6A modification axis. The results of rat experiments showed decreased RNF43 expression and increased NDUFS1 expression in endometriosis rats, which was enhanced by METTL3 inhibition. Those observations indicated that m6A methylation-mediated RNF43 negatively affects viability and migration of endometrial stromal cells through regulating oxidative phosphorylation via NDUFS1. The discovery of METTL3/RNF43/NDUFS1 axis suggested promising therapeutic targets for endometriosis.

RNF31-mediated IKKα ubiquitination aggravates inflammation and intestinal injury through regulating NF-κB activation in human and mouse neonates

AimsNeonatal necrotizing enterocolitis (NEC) is a leading cause of intestine inflammatory disease, and macrophage is significantly activated during NEC development. Posttranslational modifications (PTMs) of proteins, particularly ubiquitination, play critical roles in immune response. This study aimed to investigate the effects of ubiquitin-modified proteins on macrophage activation and NEC, and discover novel NEC-related inflammatory proteins. Materials and methodsProteomic and ubiquitin proteomic analyses of intestinal macrophages in NEC/healthy mouse pups were carried out. In vitro macrophage inflammation model and in vivo NEC mouse model, as well as clinical human samples were used for further verification the inhibitor of nuclear factor-κB kinase α (IKKα) ubiquitination on NEC development through Western blot, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry. Key findingsWe report here that IKKα was a new ubiquitin-modified protein during NEC through ubiquitin proteomics, and RING finger protein 31 (RNF31) acted as an E3 ligase to be involved in IKKα degradation. Inhibition of IKKα ubiquitination and degradation with siRNF31 or proteasome inhibitor decreased nuclear factor-κB (NF-κB) activation, thereby decreasing the expression of pro-inflammatory factors and M1 macrophage polarization, resulting in reliving the severity of NEC. SignificanceOur study suggests the activation of RNF31-IKKα-NF-κB axis triggering NEC development and suppressing RNF31-mediated IKKα degradation may be therapeutic strategies to be developed for NEC treatment.

Ubiquitination of p21 by E3 ligase RNF135 promotes the proliferation of human glioblastoma cells.

Glioblastoma multiforme (GBM) is a highly heterogeneous tumor of the central nervous system with a high mortality rate. The upregulation of RING finger protein 135 (RNF135), an E3 ligase, has been observed in GBM, but the associated mechanisms have not been fully elucidated. The aim of the present study was to identify the substrate of RNF135 and study its functions in GBM. Bioinformatics analyses were performed. In addition, RNF135 was overexpressed or knocked down in human U87 and U251 GBM cells, and the effect on cell proliferation was analyzed using Cell Counting Kit-8 and colony formation assays. Furthermore, the interaction of RNF135 with its potential substrate was analyzed using glutathione S-transferase, yeast two-hybrid, immunoprecipitation (IP), co-IP and immunoblotting assays. Bioinformatics analysis indicated that RNF135 serves as a marker of poor prognosis in GBM. The overexpression of RNF135 was demonstrated to promote the proliferation of GBM cells, while the knockdown of RNF135 inhibited GBM cell growth. In addition, the results of the interaction experiments indicate that RNF135 interacts with p21 and mediates the degradation of p21 by ubiquitination. The major site of RNF135-mediated p21 ubiquitination was identified as K163. Further experiments demonstrated that RNF135 promotes the proliferation of GBM cells mainly via p21. In summary, these findings suggest that RNF135 has potential as a therapeutic target for the treatment of GBM.

LIMA1 links the E3 ubiquitin ligase RNF40 to lipid metabolism

LIMA1 is a LIM domain and Actin binding 1 protein that acts as a skeleton protein to promote cholesterol absorption, which makes it an ideal target for interfering with lipid metabolism. However, the detailed regulation of LIMA1 remains unclear. Here, we identified that ring finger protein 40 (RNF40), an E3 ubiquitin ligase previously known as an epigenetic modifier to increase H2B ubiquitination, mediated the ubiquitination of LIMA1 and thereby promoted its degradation in a proteasome-dependent manner. Fraction studies revealed that the 1–166aa fragment of LIMA1 was indispensable for the interaction with RNF40, and at least two domains of RNF40 might mediate the association of RNF40 with LIMA1. Notably, treatment with simvastatin dramatically decreased the levels of CHO and TG in control cells rather than cells with overexpressed LIMA1. Moreover, RNF40 significantly decreased lipid content, which could be reversed by LIMA1 overexpression. These findings suggest that E3 ubiquitin ligase RNF40 could directly target LIMA1 and promote its protein degradation in cytoplasm, leading to the suppression of lipid accumulation mediated by LIMA1. Collectively, this study unveils that RNF40 is a novel E3 ubiquitin ligase of LIMA1, which underpins its high therapeutic value to combat dysregulation of lipid metabolism.

Membralin is required for maize development and defines a branch of the endoplasmic reticulum–associated degradation pathway in plants

Endoplasmic reticulum (ER)-associated degradation (ERAD) plays key roles in controlling protein levels and quality in eukaryotes. The Ring Finger Protein 185 (RNF185)/membralin ubiquitin ligase complex was recently identified as a branch in mammals and is essential for neuronal function, but its function in plant development is unknown. Here, we report the map-based cloning and characterization of Narrow Leaf and Dwarfism 1 (NLD1), which encodes the ER membrane-localized protein membralin and specifically interacts with maize homologs of RNF185 and related components. The nld1 mutant shows defective leaf and root development due to reduced cell number. The defects of nld1 were largely restored by expressing membralin genes from Arabidopsis thaliana and mice, highlighting the conserved roles of membralin proteins in animals and plants. The excessive accumulation of β-hydroxy β-methylglutaryl-CoA reductase in nld1 indicates that the enzyme is a membralin-mediated ERAD target. The activation of bZIP60 mRNA splicing-related unfolded protein response signaling and marker gene expression in nld1, as well as DNA fragment and cell viability assays, indicate that membralin deficiency induces ER stress and cell death in maize, thereby affecting organogenesis. Our findings uncover the conserved, indispensable role of the membralin-mediated branch of the ERAD pathway in plants. In addition, ZmNLD1 contributes to plant architecture in a dose-dependent manner, which can serve as a potential target for genetic engineering to shape ideal plant architecture, thereby enhancing high-density maize yields.

Microglial Sp1 induced LRRK2 upregulation in response to manganese exposure, and 17β-estradiol afforded protection against this manganese toxicity

Chronic exposure to elevated levels of manganese (Mn) causes a neurological disorder referred to as manganism, presenting symptoms similar to those of Parkinson’s disease (PD), yet the mechanisms by which Mn induces its neurotoxicity are not completely understood. 17β-estradiol (E2) affords neuroprotection against Mn toxicity in various neural cell types including microglia. Our previous studies have shown that leucine-rich repeat kinase 2 (LRRK2) mediates Mn-induced inflammatory toxicity in microglia. The LRRK2 promoter sequences contain three putative binding sites of the transcription factor (TF), specificity protein 1 (Sp1), which increases LRRK2 promoter activity. In the present study, we tested if the Sp1-LRRK2 pathway plays a role in both Mn toxicity and the protection afforded by E2 against Mn toxicity in BV2 microglial cells. The results showed that Mn induced cytotoxicity, oxidative stress, and tumor necrosis factor-α production, which were attenuated by an LRRK2 inhibitor, GSK2578215A. The overexpression of Sp1 increased LRRK2 promoter activity, mRNA and protein levels, while inhibition of Sp1 with its pharmacological inhibitor, mithramycin A, attenuated the Mn-induced increases in LRRK2 expression. Furthermore, E2 attenuated the Mn-induced Sp1 expression by decreasing the expression of Sp1 via the promotion of the ubiquitin-dependent degradation pathway, which was accompanied by increased protein levels of RING finger protein 4, the E3-ligase of Sp1, Sp1 ubiquitination, and SUMOylation. Taken together, our novel findings suggest that Sp1 serves as a critical TF in Mn-induced LRRK2 expression as well as in the protection afforded by E2 against Mn toxicity through reduction of LRRK2 expression in microglia.

Genome-Wide Identification of CHYR Gene Family in Sophora alopecuroides and Functional Analysis of SaCHYR4 in Response to Abiotic Stress.

Sophora alopecuroides has important uses in medicine, wind breaking, and sand fixation. The CHY-zinc-finger and RING-finger (CHYR) proteins are crucial for plant growth, development, and environmental adaptation; however, genetic data regarding the CHYR family remain scarce. We aimed to investigate the CHYR gene family in S. alopecuroides and its response to abiotic stress, and identified 18 new SaCHYR genes from S. alopecuroides whole-genome data, categorized into 3 subclasses through a phylogenetic analysis. Gene structure, protein domains, and conserved motifs analyses revealed an exon-intron structure and conserved domain similarities. A chromosome localization analysis showed distribution across 12 chromosomes. A promoter analysis revealed abiotic stress-, light-, and hormone-responsive elements. An RNA-sequencing expression pattern analysis revealed positive responses of SaCHYR genes to salt, alkali, and drought stress. SaCHYR4 overexpression considerably enhanced alkali and drought tolerance in Arabidopsis thaliana. These findings shed light on SaCHYR's function and the resistance mechanisms of S. alopecuroides, presenting new genetic resources for crop resistance breeding.

RNF216 Inhibits the Replication of H5N1 Avian Influenza Virus and Regulates the RIG-I Signaling Pathway in Ducks.

The RING finger (RNF) family, a group of E3 ubiquitin ligases, plays multiple essential roles in the regulation of innate immunity and resistance to viral infection in mammals. However, it is still unclear whether RNF proteins affect the production of IFN-I and the replication of avian influenza virus (AIV) in ducks. In this article, we found that duck RNF216 (duRNF216) inhibited the duRIG-I signaling pathway. Conversely, duRNF216 deficiency enhanced innate immune responses in duck embryonic fibroblasts. duRNF216 did not interacted with duRIG-I, duMDA5, duMAVS, duSTING, duTBK1, or duIRF7 in the duck RIG-I pathway. However, duRNF216 targeted duTRAF3 and inhibited duMAVS in the recruitment of duTRAF3 in a dose-dependent manner. duRNF216 catalyzed K48-linked polyubiquitination of duck TRAF3, which was degraded by the proteasome pathway. Additionally, AIV PB1 protein competed with duTRAF3 for binding to duRNF216 to reduce degradation of TRAF3 by proteasomes in the cytoplasm, thereby slightly weakening duRNF216-mediated downregulation of IFN-I. Moreover, although duRNF216 downregulated the IFN-β expression during virus infection, the expression level of IFN-β in AIV-infected duck embryonic fibroblasts overexpressing duRNF216 was still higher than that in uninfected cells, which would hinder the viral replication. During AIV infection, duRNF216 protein targeted the core protein PB1 of viral polymerase to hinder viral polymerase activity and viral RNA synthesis in the nucleus, ultimately strongly restricting viral replication. Thus, our study reveals a new mechanism by which duRNF216 downregulates innate immunity and inhibits AIV replication in ducks. These findings broaden our understanding of the mechanisms by which the duRNF216 protein affects AIV replication in ducks.

Interaction between RNF4 and SART3 is associated with the risk of schizophrenia

The pathogenesis of schizophrenia (SCZ) is heavily influenced by genetic factors. Ring finger protein 4 (RNF4) and squamous cell carcinoma antigen recognized by T cells 3 (SART3) are thought to be involved in nervous system growth and development via oxidative stress pathways. Moreover, they have previously been linked to SCZ. Yet the role of RNF4 and SART3 in SCZ remains unclear. Here, we investigated how these two genes are involved in SCZ by studying their variants observed in patients. We first observed significantly elevated mRNA levels of RNF4 and SART3 in the peripheral blood in both first-episode (n = 30) and chronic (n = 30) SCZ patients compared to controls (n = 60). Next, we targeted-sequenced three single nucleotide polymorphisms (SNPs) in SART3 and six SNPs in RNF4 for association with SCZ using the genomic DNA extracted from peripheral blood leukocytes from SCZ participants (n = 392) and controls (n = 572). We observed a combination of SNPs that included rs1203860, rs2282765 (both in RNF4), and rs2287550 (in SART3) was associated with increased risk of SCZ, suggesting common pathogenic mechanisms between these two genes. We then conducted experiments in HEK293T cells to better understand the interaction between RNF4 and SART3. We observed that SART3 lowered the expression of RNF4 through ubiquitination and downregulated the expression of nuclear factor E2-related factor 2 (NRF2), a downstream factor of RNF4, implicating the existence of a possible shared regulatory mechanism for RNF4 and SART3. In conclusion, our study provides evidence that the interaction between RNF4 and SART3 contributes to the risk of SCZ. The findings shed light on the underlying molecular mechanisms of SCZ and may lead to the development of new therapies and interventions for this disorder.

Ring finger protein 216 loss-of-function induces white matter hyperintensities by inhibiting oligodendroglia proliferation.

White matter hyperintensities(WMHs) refer to a group of diseases with numerous etiologies while oligodendrocytes remain the centerpiece in the pathogenesis of WMHs. Ring Finger Protein 216 (RNF216) encodes a ubiquitin ligase, and its mutation begets WMHs, ataxia, and cognitive decline in patients. Yet no study has revealed the function of RNF216 in oligodendroglia and WHIs before. In this study, we summarized the phenotypes of RNF216-mutation cases and explored the normal distribution of RNF216 in distinct brain regions and neuronal cells by bioinformatic analysis. Furthermore, MO3.13, a human oligodendrocyte cell line, was applied to study the function alteration after RNF216 knockdown. As a result, WMHs were the most common symptom in RNF216-mutated diseases, and RNF216 was indeed relatively enriched in corpus callosum and oligodendroglia in humans. The downregulation of RNF216 in oligodendroglia remarkably hampered cell proliferation by inhibiting the Akt pathway while having no significant effect on cell injury and oligodendrocyte maturation. Combining clinical, bioinformatical, and experimental evidence, our study implied the pivotal role of RNF216 in WMHs which might serve as a potent target in the therapy of WMHs.

Lysosome-Related Genes and RNF19B as Prognostic Markers for Survival and Immunotherapy Efficacy in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) poses a considerable worldwide health concern due to its associated high risk of death. The heterogeneity of HCC poses challenges in developing practical risk stratification tools and identifying prognostic markers for personalized targeted treatments. Recently, lysosomes were shown to be crucial contributors to numerous cellular activities, including tumor initiation and immune response regulation. We aimed to construct a reliable prognostic signature based on lysosome-related genes and determine its association with the immune microenvironment. We comprehensively analyzed lysosome-related genes in HCC to investigate their influence on patient survival and the tumor immune microenvironment. A prognostic signature comprising 14 genes associated with lysosomes was created to estimate the survival outcomes of individuals with HCC. In addition, we verified the prognostic importance of Ring Finger Protein 19B (RNF19B) in patients with HCC through multiplex immunohistochemistry analysis. Our constructed lysosome-related prediction model could significantly discriminate between HCC patients with good and poor survival outcomes ( P < 0.05). We also found that elevated RNF19B expression was linked to unfavorable prognostic outcomes and showed a connection with specific clinicopathological characteristics. Moreover, it was observed that RNF19B could facilitate the transformation of macrophages into M2-polarized macrophages and showed a significant positive correlation with PD-1 and CTLA-4. In summary, our study proposes that the expression of lysosome-related genes is associated with the immune microenvironment, serving as a predictor for HCC patient survival. Meanwhile, RNF19B was identified as a novel prognostic marker for predicting overall survival and immunotherapy effects in patients with HCC.

RNF213 Polymorphisms in Intracranial Artery Dissection.

The ring finger protein 213 gene (RNF213) is involved in several vascular diseases, both intracranial and systemic ones. Some variants are common in the Asian population and are reported as a risk factor for moyamoya disease, intracranial stenosis and intracranial aneurysms. Among intracranial vascular diseases, both moyamoya disease and intracranial artery dissection are more prevalent in the Asian population. We performed a systematic review of the literature, aiming to assess the rate of RNF213 variants in patients with spontaneous intracranial dissections. Four papers were identified, providing data on 53 patients with intracranial artery dissection. The rate of RNF213 variants is 10/53 (18.9%) and it increases to 10/29 (34.5%), excluding patients with vertebral artery dissection. All patients had the RNF213 p.Arg4810Lys variant. RNF213 variants seems to be involved in intracranial dissections in Asian cohorts. The small number of patients, the inclusion of only patients of Asian descent and the small but non-negligible coexistence with moyamoya disease familiarity might be limiting factors, requiring further studies to confirm these preliminary findings and the embryological interpretation.

Knockdown the moyamoya disease susceptibility gene, RNF213, upregulates the expression of basic fibroblast growth factor and matrix metalloproteinase-9 in bone marrow derived mesenchymal stem cells.

Moyamoya disease (MMD) is a chronic, progressive cerebrovascular occlusive disease. Ring finger protein 213 (RNF213) is a susceptibility gene of MMD. Previous studies have shown that the expression levels of angiogenic factors increase in MMD patients, but the relationship between the susceptibility gene RNF213 and these angiogenic mediators is still unclear. The aim of the present study was to investigate the pathogenesis of MMD by examining the effect of RNF213 gene knockdown on the expression of matrix metalloproteinase-9 (MMP-9) and basic fibroblast growth factor (bFGF) in rat bone marrow-derived mesenchymal stem cells (rBMSCs). Firstly, 40 patients with MMD and 40 age-matched normal individuals (as the control group) were enrolled in the present study to detect the levels of MMP-9 and bFGF in serum by ELISA. Secondly, Sprague-Dawley male rat BMSCs were isolated and cultured using the whole bone marrow adhesion method, and subsequent phenotypic analysis was performed by flow cytometry. Alizarin red and oil red O staining methods were used to identify osteogenic and adipogenic differentiation, respectively. Finally, third generation rBMSCs were transfected with lentivirus recombinant plasmid to knockout expression of the RNF213 gene. After successful transfection was confirmed by reverse transcription-quantitative PCR and fluorescence imaging, the expression levels of bFGF and MMP-9 mRNA in rBMSCs and the levels of bFGF and MMP-9 protein in the supernatant of the culture medium were detected on the 7th and 14th days after transfection. There was no significant difference in the relative expression level of bFGF among the three groups on the 7th day. For the relative expression level of MMP-9, there were significant differences on the 7th day and 14th day. In addition, there was no statistically significant difference in the expression of bFGF in the supernatant of the RNF213 shRNA group culture medium, while there was a significant difference in the expression level of MMP-9. The knockdown of the RNF213 gene affects the expression of bFGF and MMP-9. However, further studies are needed to determine how they participate in the pathogenesis of MMD. The findings of the present study provide a theoretical basis for clarifying the pathogenesis and clinical treatment of MMD.

ERLIN1/2 scaffolds bridge TMUB1 and RNF170 and restrict cholesterol esterification to regulate the secretory pathway.

Complexes of ERLIN1 and ERLIN2 (ER lipid raft-associated 1 and 2) form large ring-like cup-shaped structures on the endoplasmic reticulum (ER) membrane and serve as platforms to bind cholesterol and E3 ubiquitin ligases, potentially defining functional nanodomains. Here, we show that ERLIN scaffolds mediate the interaction between the full-length isoform of TMUB1 (transmembrane and ubiquitin-like domain-containing 1) and RNF170 (RING finger protein 170). We identify a luminal N-terminal conserved region in TMUB1 and RNF170, which is required for this interaction. Three-dimensional modelling shows that this conserved motif binds the stomatin/prohibitin/flotillin/HflKC domain of two adjacent ERLIN subunits at different interfaces. Protein variants that preclude these interactions have been previously linked to hereditary spastic paraplegia. Using omics-based approaches in combination with phenotypic characterization of HeLa cells lacking both ERLINs, we demonstrate a role of ERLIN scaffolds in limiting cholesterol esterification, thereby favouring cholesterol transport from the ER to the Golgi apparatus and regulating Golgi morphology and the secretory pathway.

A Full-Length Transcriptome and Analysis of the NHL-1 Gene Family in Neocaridina denticulata sinensis.

Neocaridina denticulata sinensis has emerged as a promising model organism for basic studies in Decapod. However, the current transcriptome information on this species is based on next-generation sequencing technologies, which are limited by a short read length. Therefore, the present study aimed to generate a full-length transcriptome assembly of N. denticulata sinensis utilizing the PacBio Sequel Ⅱ platform. The resulting transcriptome assembly comprised 5831 transcripts with an N50 value of 3697 bp. Remarkably, 90.5% of these transcripts represented novel isoforms of known genes. The transcripts were further searched against the NR, SwissProt, KEGG, KOG, GO, NT, and Pfam databases. A total of 24.8% of the transcripts can be annotated across all seven databases. Additionally, 1236 alternative splicing events, 344 transcription factors, and 124 long non-coding RNAs (LncRNAs) were predicted. Based on the alternative splicing annotation results, a RING finger protein NHL-1 gene from N. denticulata sinensis (NdNHL-1) was identified. There are 15 transcripts in NdNHL-1. The longest transcript is 4995 bp in length and encodes a putative protein of 1665 amino acids. A phylogenetic analysis showed its close relationship with NHL-1 from other crustacean species. This report represents the full-length transcriptome of N. denticulata sinensis and will facilitate research on functional genomics and environmental adaptation in this species.

Effect of RNF113A deficiency on oxidative stress-induced NRF2 pathway

ABSTRACT The ring finger protein 113A (RNF113A) serves as an E3 ubiquitin ligase and a subunit of the spliceosome. Mutations in the RNF113A gene are associated with X-linked trichothiodystrophy (TTD). However, the cellular roles of RNF113A remain largely unknown. In this study, we performed transcriptome profiling of RNF113A knockout (KO) HeLa cells using RNA sequencing and revealed the upregulation of NRF2 pathway-associated genes. Further analysis confirmed that the KO of RNF113A promotes nuclear localization of the NRF2 protein and elevates the mRNA levels of NRF2 target genes. RNF113A KO cells showed high levels of intracellular reactive oxygen species (ROS) and decreased resistance to cell death following H2O2 treatment. Additionally, RNF113A KO cells more sensitively formed stress granules (SGs) under arsenite-induced oxidative stress. Moreover, RNF113A KO cells exhibited a decrease in glutathione levels, which could be attributed to a reduction in GLUT1 expression levels, leading to decreased glucose uptake reactions and lower intracellular glucose levels. These alterations potentially caused a reduction in ROS scavenging activity. Taken together, our findings suggest that the loss of RNF113A promotes oxidative stress-mediated activation of the NRF2 pathway, providing novel insights into RNF113A-associated human diseases.

Ring finger protein 138 inhibits transcription factor C/EBPα protein turnover leading to differentiation arrest in acute myeloid leukemia.

E3 ubiquitin ligase, ring finger protein 138 (RNF138) is involved in several biological processes; however, its role in myeloid differentiation or tumorigenesis remains unclear. RNAseq data from TNMplot showed that RNF138 mRNA levels are highly elevated in acute myeloid leukemia (AML) bone marrow samples as compared with bone marrow of normal volunteers. Here, we show that RNF138 serves as an E3 ligase for the tumor suppressor CCAAT/enhancer binding protein (C/EBPα) and promotes its degradation leading to myeloid differentiation arrest in AML. Wild-type RNF138 physically interacts with C/EBPα and promotes its ubiquitin-dependent proteasome degradation while a mutant RNF-138 deficient in ligase activity though interacts with C/EBPα, fails to down-regulate it. We show that RNF138 depletion enhances endogenous C/EBPα levels in peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers. Our data further shows that RNF138-mediated degradation of C/EBPα negatively affects its transactivation potential on its target genes. Furthermore, RNF138 overexpression inhibits all-trans-retinoic acid-induced differentiation of HL-60 cells whereas RNF138 RNAi enhances. In line with RNF138 inhibiting C/EBPα protein turnover, we also observed that RNF138 overexpression inhibited β-estradiol (E2)-induced C/EBPα driven granulocytic differentiation in C/EBPα inducible K562-p42C/EBPα-estrogen receptor cells. Furthermore, we also recapitulated these findings in PBMCs isolated from AML patients where depletion of RNF138 increased the expression of myeloid differentiation marker CD11b. These results suggest that RNF138 inhibits myeloid differentiation by targeting C/EBPα for proteasomal degradation and may provide a plausible mechanism for loss of C/EBPα expression often observed in myeloid leukemia. Also, targeting RNF138 may resolve differentiation arrest by restoring C/EBPα expression in AML.