DDX20 positively regulates the interferon pathway to inhibit viral infection.

The DEAD-box (DDX) family comprises RNA helicases characterized by the conserved sequence D(Asp)-E(Glu)-A(Ala)-D(Asp), participating in various RNA metabolism processes. Some DDX family members have been identified for their crucial roles in viral infections. In this study, RNAi library screening of the DDX family unveiled the antiviral activity of DDX20. Knockdown of DDX20 enhanced the replication of viruses such as vesicular stomatitis virus (VSV) and herpes simplex virus type I (HSV-1), while overexpression of DDX20 significantly diminished the replication level of these viruses. Mechanistically, DDX20 elevated the phosphorylation level of IRF3 induced by external stimuli by facilitating the interaction between TBK1 and IRF3, thereby promoting the expression of IFN-ß. The increased IFN-ß production, in turn, upregulated the expression of interferon-stimulated genes (ISGs), including Cig5 and IFIT1, thereby exerting the antiviral effect. Finally, in an in vivo infection study, Ddx20 gene-deficient mice exhibited increased susceptibility to viral infection. This study provides new evidence that DDX20 positively modulates the interferon pathway and restricts viral infection.

DDX20: A Multifunctional Complex Protein.

DEAD-box decapping enzyme 20 (DDX20) is a putative RNA-decapping enzyme that can be identified by the conserved motif Asp-Glu-Ala-Asp (DEAD). Cellular processes involve numerous RNA secondary structure alterations, including translation initiation, nuclear and mitochondrial splicing, and assembly of ribosomes and spliceosomes. DDX20 reportedly plays an important role in cellular transcription and post-transcriptional modifications. On the one hand, DDX20 can interact with various transcription factors and repress the transcriptional process. On the other hand, DDX20 forms the survival motor neuron complex and participates in the assembly of snRNP, ultimately affecting the RNA splicing process. Finally, DDX20 can potentially rely on its RNA-unwinding enzyme function to participate in microRNA (miRNA) maturation and act as a component of the RNA-induced silencing complex. In addition, although DDX20 is not a key component in the innate immune system signaling pathway, it can affect the nuclear factor kappa B (NF-κB) and p53 signaling pathways. In particular, DDX20 plays different roles in tumorigenesis development through the NF-κB signaling pathway. This process is regulated by various factors such as miRNA. DDX20 can influence processes such as viral replication in cells by interacting with two proteins in Epstein-Barr virus and can regulate the replication process of several viruses through the innate immune system, indicating that DDX20 plays an important role in the innate immune system. Herein, we review the effects of DDX20 on the innate immune system and its role in transcriptional and post-transcriptional modification processes, based on which we provide an outlook on the future of DDX20 research in innate immunity and viral infections.

Integration of RNAi and RNA-seq uncovers the regulation mechanism of DDX20 on vitellogenin expression in Scylla paramamosain.

Vitellogenesis in crustaceans is controlled by several steroid hormones. In humans, the expression of SF-1, a gene that regulates gonadal development and the synthesis of steroid hormones, is affected by DDX20. However, how the homologous gene FTZ-F1 is regulated by DDX20 and its association with vitellogenesis remains unknown in the mud crab Scylla paramamosain. In this study, SpDDX20 and SpFTZ-F1 were identified in the transcriptome of mature ovarian tissue from the mud crab. qRT-PCR results revealed that the expression levels of SpFTZ-F1 and SpVTG in the ovaries of crab in the experimental group injected with dsDDX20 (EO) were significantly higher (P < 0.05) than those in the negative control group injected with dsEGFP (NO) and the blank control group injected with SPSS (BO). The differentially expressed genes (DEGs) identified by comparative transcriptome analysis of the EO group and NO group were enriched into five pathways related to ovarian steroidogenesis. The expression of CYP17, CYP3A4, CYP1A1 and 3ß-HSD were up-regulated in pathways related to steroid hormone production and biosynthesis. The expression of the INSR, IRS and PI3K genes in the insulin signaling pathway were significantly increased (P < 0.05). The expression level of the TGF-ß gene was up-regulated (P < 0.05) in the transforming growth factor pathway, whereas the expression level of the Smad2 gene was down-regulated (P < 0.05). The expression of GnRHR, GS, AC and PKA genes in the gonadotropin-releasing hormone signaling pathway were up-regulated. Our data provide a foundation for investigating the relationship between DDX20 and FTZ-F1 in the regulation of vitellogenin expression in S. paramamosain.

A Macrophage Differentiation-Mediated Gene: DDX20 as a Molecular Biomarker Encompassing the Tumor Microenvironment, Disease Staging, and Prognoses in Hepatocellular Carcinoma.

Background: DDX20 involves the mechanism of cell proliferate, mitogenic Ets transcriptional suppressor (METS), which can arrest the cell cycle of macrophages. However, little is known about DDX20 expression, clinical values, and the relationship with tumor microenvironment in HCC. Methods: We mined the transcriptional, protein expression and survival data of DDX20 in HCC from online databases. The immunological effects of DDX20 were estimated by bioinformatic algorithms. The RNAi and CRISPR screening were used to assess the gene effect of DDX20 for the EGFR gene in liver tumor cell. Results: We found that the DDX20 was highly expressed in HCC. The qRT-PCR result shows a significantly upregulated DDX20 expression in HCC samples from the West China Hospital. The high mRNA expression of DDX20 is associated with a poor survival. DDX20 expression is positively correlated with MDSCs in HCC tissues. Moreover, DDX20 has a high predicted ability for the response to immunotherapy. Furthermore, hsa-mir-324-5p could regulate the macrophage differentiation by interacting with DDX20. Meanwhile, the EGFR gene gets a high dependency score for DDX20. Conclusion: In sum, DDX20 may serve as a prognostic marker for worse clinical outcomes with HCC and potentially enable more precise and personalized immunotherapeutic strategies in the future.

The combined detection of Amphiregulin, Cyclin A1 and DDX20/Gemin3 expression predicts aggressive forms of oral squamous cell carcinoma.

BACKGROUND: Large-scale genetic and epigenetic deregulations enable cancer cells to ectopically activate tissue-specific expression programmes. A specifically designed strategy was applied to oral squamous cell carcinomas (OSCC) in order to detect ectopic gene activations and develop a prognostic stratification test. METHODS: A dedicated original prognosis biomarker discovery approach was implemented using genome-wide transcriptomic data of OSCC, including training and validation cohorts. Abnormal expressions of silent genes were systematically detected, correlated with survival probabilities and evaluated as predictive biomarkers. The resulting stratification test was confirmed in an independent cohort using immunohistochemistry. RESULTS: A specific gene expression signature, including a combination of three genes, AREG, CCNA1 and DDX20, was found associated with high-risk OSCC in univariate and multivariate analyses. It was translated into an immunohistochemistry-based test, which successfully stratified patients of our own independent cohort. DISCUSSION: The exploration of the whole gene expression profile characterising aggressive OSCC tumours highlights their enhanced proliferative and poorly differentiated intrinsic nature. Experimental targeting of CCNA1 in OSCC cells is associated with a shift of transcriptomic signature towards the less aggressive form of OSCC, suggesting that CCNA1 could be a good target for therapeutic approaches.

Human bone mesenchymal stem cells-derived exosomal miRNA-361-5p alleviates osteoarthritis by downregulating DDX20 and inactivating the NF-κB signaling pathway.

Osteoarthritis (OA) is a chronic disease featured by joint hyperplasia, deterioration of articular cartilage, and progressive degeneration. Abnormal expression of microRNAs (miRNAs) has been found to be implicated in the pathological process of OA. In this study, the role of miR-361-5p transferred by exosomes derived from human bone mesenchymal stem cells (hBMSCs) in OA was investigated. The expression of Asp-Glu-Ala-Asp-box polypeptide 20 (DDX20) and miR-361-5p in interleukin-1ß (IL-1ß)-treated chondrocytes was determined by reverse transcription quantitative polymerase chain reaction. DDX20 was knocked down by transfection of short hairpin RNA targeting DDX20, and the effects of DDX20 downregulation on IL-1ß-induced damage of chondrocytes were detected. The interaction between DDX20 and miR-361-5p was tested by luciferase report assay. hBMSCs-derived exosomes loaded with miR-361-5p were co-incubated with chondrocytes followed by detection of cell viability, proliferation and inflammatory response. An OA rat model was established to further explore the role of miR-361-5p in vivo. Western blot, luciferase reporter and immunofluorescence staining assays were used to evaluate the activation of the nuclear factor kappa-B (NF-κB) signaling pathway. We found that DDX20 was upregulated, while miR-361-5p was underexpressed in IL-1ß-treated chondrocytes. Downregulation of DDX20 inhibits levels of matrix metalloproteinases (MMPs) and suppresses inflammation induced by IL-1ß. Mechanistically, miR-361-5p was verified to directly target DDX20. In addition, hBMSC-derived exosomes-transferred miR-361-5p alleviates chondrocyte damage and inhibits the NF-κB signaling pathway via targeting DDX20. Inhibition of NF-κB signaling reverses the effect of overexpressed DDX20 on IL-1ß-induced chondrocyte damage. Moreover, exosomal miR-361-5p alleviates OA damage in vivo. Overall, hBMSC-derived exosomal miR-361-5p alleviates OA damage by targeting DDX20 and inactivating the NF-κB signaling pathway.

Death-associated protein kinase 1 suppresses hepatocellular carcinoma cell migration and invasion by upregulation of DEAD-box helicase 20.

Death-associated protein kinase 1 (DAPK) is a calcium/calmodulin kinase that plays a vital role as a suppressor gene in various cancers. Yet its role and target gene independent of p53 is still unknown in hepatocellular carcinoma (HCC). In this study, we discovered that DAPK suppressed HCC cell migration and invasion instead of proliferation or colony formation. Using a proteomics approach, we identified DEAD-box helicase 20 (DDX20) as an important downstream target of DAPK in HCC cells and critical for DAPK-mediated inhibition of HCC cell migration and invasion. Using integrin inhibitor RGD and GTPase activity assays, we discovered that DDX20 suppressed HCC cell migration and invasion through the CDC42-integrin pathway, which was previously reported as an important downstream pathway of DAPK in cancer. Further research using cycloheximide found that DAPK attenuates the proteasomal degradation of DDX20 protein, which is dependent on the kinase activity of DAPK. Our results shed light on new functions and regulation for both DAPK and DDX20 in carcinogenesis and identifies new potential therapeutic targets for HCC.

The multiple lives of DEAD-box RNA helicase DP103/DDX20/Gemin3.

Gemin3, also known as DDX20 or DP103, is a DEAD-box RNA helicase which is involved in more than one cellular process. Though RNA unwinding has been determined in vitro, it is surprisingly not required for all of its activities in cellular metabolism. Gemin3 is an essential gene, present in Amoeba and Metazoa. The highly conserved N-terminus hosts the helicase core, formed of the helicase- and DEAD-domains, which, based on crystal structure determination, have key roles in RNA binding. The C-terminus of Gemin3 is highly divergent between species and serves as the interaction site for several accessory factors that could recruit Gemin3 to its target substrates and/or modulate its function. This review article focuses on the known roles of Gemin3, first as a core member of the survival motor neuron (SMN) complex, in small nuclear ribonucleoprotein biogenesis. Although mechanistic details are lacking, a critical function for Gemin3 in this pathway is supported by numerous in vitro and in vivo studies. Gene expression activities of Gemin3 are next underscored, mainly messenger ribonucleoprotein trafficking, gene silencing via microRNA processing, and transcriptional regulation. The involvement of Gemin3 in abnormal cell signal transduction pathways involving p53 and NF-κB is also highlighted. Finally, the clinical implications of Gemin3 deregulation are discussed including links to spinal muscular atrophy, poliomyelitis, amyotrophic lateral sclerosis, and cancer. Impressive progress made over the past two decades since the discovery of Gemin3 bodes well for further work that refines the mechanism(s) underpinning its multiple activities.

High expression of DDX20 enhances the proliferation and metastatic potential of prostate cancer cells through the NF-κB pathway.

DEAD (Asp-Glu-Ala-Asp) box polypeptide 20 (DDX20), a member of the DEAD box protein family, encodes an RNA helicase. Previous research has revealed that DDX20 may act as a tumor suppressor in hepatocellular carcinoma and as a tumor promoter in breast cancer. These conflicting reports prompted us to explore the role of DDX20 in prostate cancer (PCa). To gain insight into the functions of DDX20 in PCa, we examined DDX20 expression patterns in a PCa tissue microarray with 99 PCa tissue samples. The results of immunohistochemical staining revealed that DDX20 expression is frequently upregulated in PCa tissues compared with that in the adjacent tissues and further clinicopathological analysis showed that the expression level of DDX20 closely correlates with tumor size, TNM stage (positive correlation) and patient prognosis (negative correlation). Both gain­of- and loss­of­function assays were performed in vitro; the overexpression of DDX20 enhanced the proliferation and metastatic potential of cancer cells and this was examined by performing a cell counting kit-8 (CCK-8) assay, wound healing assay and Transwell migration assay. Furthermore, we found that there is a positive correlation between the expression of matrix metallopeptidase 9 (MMP9) and DDX20 expression. These findings led us to examine whether DDX20 may exert effects through the NF­κB pathway. Luciferase reporter assays suggested that DDX20 altered the activity of NF-κB. Taken together, these findings show that DDX20 may promote the progression of PCa through the NF-κB pathway.

Intracellular Bacterial Pathogens Trigger the Formation of U Small Nuclear RNA Bodies (U Bodies) through Metabolic Stress Induction.

Invasive bacterial pathogens induce an amino acid starvation (AAS) response in infected host cells that controls host defense in part by promoting autophagy. However, whether AAS has additional significant effects on the host response to intracellular bacteria remains poorly characterized. Here we showed that Shigella, Salmonella, and Listeria interfere with spliceosomal U snRNA maturation in the cytosol. Bacterial infection resulted in the rerouting of U snRNAs and their cytoplasmic escort, the survival motor neuron (SMN) complex, to processing bodies, thus forming U snRNA bodies (U bodies). This process likely contributes to the decline in the cytosolic levels of U snRNAs and of the SMN complex proteins SMN and DDX20 that we observed in infected cells. U body formation was triggered by membrane damage in infected cells and was associated with the induction of metabolic stresses, such as AAS or endoplasmic reticulum stress. Mechanistically, targeting of U snRNAs to U bodies was regulated by translation initiation inhibition and the ATF4/ATF3 pathway, and U bodies rapidly disappeared upon removal of the stress, suggesting that their accumulation represented an adaptive response to metabolic stress. Importantly, this process likely contributed to shape the host response to invasive bacteria because down-regulation of DDX20 expression using short hairpin RNA (shRNA) amplified ATF3- and NF-κB-dependent signaling. Together, these results identify a critical role for metabolic stress and invasive bacterial pathogens in U body formation and suggest that this process contributes to host defense.

Reconstitution of the human U snRNP assembly machinery reveals stepwise Sm protein organization.

The assembly of spliceosomal U snRNPs depends on the coordinated action of PRMT5 and SMN complexes in vivo. These trans-acting factors enable the faithful delivery of seven Sm proteins onto snRNA and the formation of the common core of snRNPs. To gain mechanistic insight into their mode of action, we reconstituted the assembly machinery from recombinant sources. We uncover a stepwise and ordered formation of distinct Sm protein complexes on the PRMT5 complex, which is facilitated by the assembly chaperone pICln. Upon completion, the formed pICln-Sm units are displaced by new pICln-Sm protein substrates and transferred onto the SMN complex. The latter acts as a Brownian machine that couples spontaneous conformational changes driven by thermal energy to prevent mis-assembly and to ensure the transfer of Sm proteins to cognate RNA. Investigation of mutant SMN complexes provided insight into the contribution of individual proteins to these activities. The biochemical reconstitution presented here provides a basis for a detailed molecular dissection of the U snRNP assembly reaction.

Pseudophosphorylated αB-crystallin is a nuclear chaperone imported into the nucleus with help of the SMN complex.

The human small heat shock protein αB-crystallin (HspB5) is a molecular chaperone which is mainly localized in the cytoplasm. A small fraction can also be found in nuclear speckles, of which the localization is mediated by successional phosphorylation at Ser-59 and Ser-45. αB-crystallin does not contain a canonical nuclear localization signal sequence and the mechanism by which αB-crystallin is imported into the nucleus is not known. Here we show that after heat shock pseudophosphorylated αB-crystallin mutant αB-STD, in which all three phosphorylatable serine residues (Ser-19, Ser-45 and Ser-59) were replaced by negatively charged aspartate residues, is released from the nuclear speckles. This allows αB-crystallin to chaperone proteins in the nucleoplasm, as shown by the ability of αB-STD to restore nuclear firefly luciferase activity after a heat shock. With the help of a yeast two-hybrid screen we found that αB-crystallin can interact with the C-terminal part of Gemin3 and confirmed this interaction by co-immunoprecipitation. Gemin3 is a component of the SMN complex, which is involved in the assembly and nuclear import of U-snRNPs. Knockdown of Gemin3 in an in situ nuclear import assay strongly reduced the accumulation of αB-STD in nuclear speckles. Furthermore, depletion of SMN inhibited nuclear import of fluorescently labeled recombinant αB-STD in an in vitro nuclear import assay, which could be restored by the addition of purified SMN complex. These results show that the SMN-complex facilitates the accumulation of hyperphosphorylated αB-crystallin in nuclear speckles, thereby creating a chaperone depot enabling a rapid chaperone function in the nucleus in response to stress.

MicroRNA-140 acts as a liver tumor suppressor by controlling NF-κB activity by directly targeting DNA methyltransferase 1 (Dnmt1) expression.

UNLABELLED: MicroRNAs (miRNAs) are small RNAs that regulate the expression of specific target genes. While deregulated miRNA expression levels have been detected in many tumors, whether miRNA functional impairment is also involved in carcinogenesis remains unknown. We investigated whether deregulation of miRNA machinery components and subsequent functional impairment of miRNAs are involved in hepatocarcinogenesis. Among miRNA-containing ribonucleoprotein complex components, reduced expression of DDX20 was frequently observed in human hepatocellular carcinomas, in which enhanced nuclear factor-κB (NF-κB) activity is believed to be closely linked to carcinogenesis. Because DDX20 normally suppresses NF-κB activity by preferentially regulating the function of the NF-κB-suppressing miRNA-140, we hypothesized that impairment of miRNA-140 function may be involved in hepatocarcinogenesis. DNA methyltransferase 1 (Dnmt1) was identified as a direct target of miRNA-140, and increased Dnmt1 expression in DDX20-deficient cells hypermethylated the promoters of metallothionein genes, resulting in decreased metallothionein expression leading to enhanced NF-κB activity. MiRNA-140-knockout mice were prone to hepatocarcinogenesis and had a phenotype similar to that of DDX20 deficiency, suggesting that miRNA-140 plays a central role in DDX20 deficiency-related pathogenesis. CONCLUSION: These results indicate that miRNA-140 acts as a liver tumor suppressor, and that impairment of miRNA-140 function due to a deficiency of DDX20, a miRNA machinery component, could lead to hepatocarcinogenesis.

FUS-SMN protein interactions link the motor neuron diseases ALS and SMA.

Mutations in the RNA binding protein FUS cause amyotrophic lateral sclerosis (ALS), a fatal adult motor neuron disease. Decreased expression of SMN causes the fatal childhood motor neuron disorder spinal muscular atrophy (SMA). The SMN complex localizes in both the cytoplasm and nuclear Gems, and loss of Gems is a cellular hallmark of fibroblasts in patients with SMA. Here, we report that FUS associates with the SMN complex, mediated by U1 snRNP and by direct interactions between FUS and SMN. Functionally, we show that FUS is required for Gem formation in HeLa cells, and expression of FUS containing a severe ALS-causing mutation (R495X) also results in Gem loss. Strikingly, a reduction in Gems is observed in ALS patient fibroblasts expressing either mutant FUS or TDP-43, another ALS-causing protein that interacts with FUS. The physical and functional interactions among SMN, FUS, TDP-43, and Gems indicate that ALS and SMA share a biochemical pathway, providing strong support for the view that these motor neuron diseases are related.

A miRNA machinery component DDX20 controls NF-κB via microRNA-140 function.

Hepatocellular carcinoma is the third leading cause of cancer mortality worldwide, but the molecular mechanisms in tumorigenesis remain largely unknown. Previously, a DEAD-box protein DDX20, a component of microRNA-containing ribonucleoprotein complexes, was identified as a liver tumor suppressor candidate in an oncogenomics-based in vivo RNAi screen. However, the molecular mechanisms were unknown. Here, we show that deficiency of DDX20 results in the enhancement of NF-κB activity, a crucial intracellular signaling pathway closely linked with hepatocarcinogenesis. While DDX20 normally suppresses NF-κB activity by regulating NF-κB-suppressing miRNA-140 function, this suppressive effect was lost in DDX20-deficient cells. The impairment of miRNA function due to DDX20 deficiency appears to be miRNA species-specific at the point of loading miRNAs into the RNA-induced silencing complex. These results indicate that DDX20 deficiency enhances NF-κB activity by impairing the NF-κB-suppressive action of microRNAs, and suggest that dysregulation of the microRNA machinery components may also be involved in pathogenesis in various human diseases.

Epstein-Barr virus nuclear antigen 3C stabilizes Gemin3 to block p53-mediated apoptosis.

The Epstein-Barr nuclear antigen 3C (EBNA3C), one of the essential latent antigens for Epstein-Barr virus (EBV)-induced immortalization of primary human B lymphocytes in vitro, has been implicated in regulating cell proliferation and anti-apoptosis via interaction with several cellular and viral factors. Gemin3 (also named DDX20 or DP103) is a member of DEAD RNA helicase family which exhibits diverse cellular functions including DNA transcription, recombination and repair, and RNA metabolism. Gemin3 was initially identified as a binding partner to EBNA2 and EBNA3C. However, the mechanism by which EBNA3C regulates Gemin3 function remains unclear. Here, we report that EBNA3C directly interacts with Gemin3 through its C-terminal domains. This interaction results in increased stability of Gemin3 and its accumulation in both B lymphoma cells and EBV transformed lymphoblastoid cell lines (LCLs). Moreover, EBNA3C promotes formation of a complex with p53 and Gemin3 which blocks the DNA-binding affinity of p53. Small hairpin RNA based knockdown of Gemin3 in B lymphoma or LCL cells remarkably attenuates the ability of EBNA3C to inhibit the transcription activity of p53 on its downstream genes p21 and Bax, as well as apoptosis. These findings provide the first evidence that Gemin3 may be a common target of oncogenic viruses for driving cell proliferation and anti-apoptotic activities.

NANOS1 and PUMILIO2 bind microRNA biogenesis factor GEMIN3, within chromatoid body in human germ cells.

Nanos and pumilio bind each other to regulate translation of specific mRNAs in germ cells of model organisms, such as D. melanogaster or C. elegans. Recently described human homologues NANOS1 and PUMILIO2 form a complex similar to their ancestors. This study was aimed to identify the proteins interacting with NANOS1-PUMILIO2 complex in the human spermatogenic cells. Here, using the yeast two-hybrid system we found that NANOS1 and PUMILIO2 proteins interact with RNA DEAD-box helicase GEMIN3, a microRNA biogenesis factor. Moreover, GEMIN3 coimmunoprecipitates with NANOS1 and PUMILIO2 in transfected mammalian cells. By double immunofluorescence staining, we observed that complexes built of NANOS1, PUMILIO2 and GEMIN3 are located within cytoplasmic region of germ cells. These proteins condense to form a compact aggregate in the round spermatids of the human and mouse germ cells. This aggregate was reminiscent of the chromatoid body (CB), a perinuclear structure present in the mammalian male germ line. This structure is considered evolutionary remnant of germ plasm, a hallmark structure of germ cells in lower metazoan. Using a CB marker VASA protein, we demonstrated that CBs are present in the human round spermatids, as they are in the mouse. Moreover, NANOS1, PUMILIO2 and GEMIN3 colocalize with VASA protein. We demonstrated for the first time that a mammalian Nanos-Pumilio complex functions within CB, a center of RNA storing and processing, involving microRNAs. NANOS1-PUMILIO2 complex, together with GEMIN3 and small noncoding RNAs, possibly regulate mRNA translation within CB of the human germ cells.

[Gemin3 inhibits cell apoptosis through suppression of p53 expression].

OBJECTIVE: To investigate the role of Gemin3 in cell proliferation and its regulation pathway. METHODS: Using co-immunoprecipitation and GST pull-down assay to determine the domain of Gemin3 and p53 binding and interaction in vitro and in vivo. To check the effect of Gemin3 on p53 by luciferase reporter assay. Stable Gemin3 knock-down cell lines were generated by lentivirus-delivered small hairpin RNA then puromycin selection. Real-time PCR was used to confirm the effect of Gemin3 level on p53 and its downstream genes, and flow cytometry was used to analyze the effect of Gemin3 on apoptosis. RESULTS: The C-terminal of Gemin3 interacted with the DNA binding domain of p53. The p53 reporter gene, PA3M-p53 and increasing amount of GFP-Gemin3 were co-transfected into Saos-2 cells. Gemin3 repressed p53 expression at transcription level. Real-time PCR indicated that the expression of p53, p21 and Bax in Gemin3 knock-down cells was higher than that in the control cells. Western blot showed Gemin3 knock-down cells had a higher p53 espression. Flow cytometric assay showed that knock-down Gemin3 expression led to an increased cell apoptosis. CONCLUSION: Gemin3 binds with p53 forming a complex and plays an anti-apoptotic role by repressing the p53 expression.

SMN, Gemin2 and Gemin3 associate with beta-actin mRNA in the cytoplasm of neuronal cells in vitro.

Childhood spinal muscular atrophy is caused by a reduced expression of the survival motor neuron (SMN) protein. SMN has been implicated in the axonal transport of beta-actin mRNA in both primary and transformed neuronal cell lines, and loss of this function could account, at least in part, for spinal muscular atrophy onset and pathological specificity. Here we have utilised a targeted screen to identify mRNA associated with SMN, Gemin2 and Gemin3 in the cytoplasm of a human neuroblastoma cell line, SHSY5Y. Importantly, we have provided the first direct evidence that beta-actin mRNA is present in SMN cytoplasmic complexes in SHSY5Y cells.

Abnormal interaction of motor neuropathy-associated mutant HspB8 (Hsp22) forms with the RNA helicase Ddx20 (gemin3).

A number of missense mutations in the two related small heat shock proteins HspB8 (Hsp22) and HspB1 (Hsp27) have been associated with the inherited motor neuron diseases (MND) distal hereditary motor neuropathy and Charcot-Marie-Tooth disease. HspB8 and HspB1 interact with each other, suggesting that these two etiologic factors may act through a common biochemical mechanism. However, their role in neuron biology and in MND is not understood. In a yeast two-hybrid screen, we identified the DEAD box protein Ddx20 (gemin3, DP103) as interacting partner of HspB8. Using co-immunoprecipitation, chemical cross-linking, and in vivo quantitative fluorescence resonance energy transfer, we confirmed this interaction. We also show that the two disease-associated mutant HspB8 forms have abnormally increased binding to Ddx20. Ddx20 itself binds to the survival-of-motor-neurons protein (SMN protein), and mutations in the SMN1 gene cause spinal muscular atrophy, another MND and one of the most prevalent genetic causes of infant mortality. Thus, these protein interaction data have linked the three etiologic factors HspB8, HspB1, and SMN protein, and mutations in any of their genes cause the various forms of MND. Ddx20 and SMN protein are involved in spliceosome assembly and pre-mRNA processing. RNase treatment affected the interaction of the mutant HspB8 with Ddx20 suggesting RNA involvement in this interaction and a potential role of HspB8 in ribonucleoprotein processing.