• Asian Pacific Journal of Cancer Prevention
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• 제14권2호
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• pp.1073-1075
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• 2013

Background: In normal cells, activated epidermal growth factor receptor (EGFR) molecules are subjected to ubiquitination-mediated proteasome degradation pathway by c-Cbl, an ubiquitin ligase that checks uncontrolled proliferation. Hence expression of wild type c-Cbl molecule is essential to keep this degradation machinery in a functional state. Loss of expression or function of c-Cbl may consequently lead to sustained activation of EGFR and promote carcinogenesis, loss of function mutations in the c-Cbl gene already being reported in lung and hematopoietic cancers. However, the genetic status of c-Cbl in oral squamous cell carcinoma (OSCC) is not known. Hence in the present study we investigated the genomic DNA isolated from OSCC tissue biopsy samples for mutations in the RING finger domain coding region of c-Cbl gene, which has also been reported to be most frequently mutated in other cancers. Materials and Methods: Total genomic DNA isolated from thirty two post surgical OSCC tissue samples were amplified using primers flanking the exon 8 of c-Cbl gene that codes for the RING finger domain. The PCR amplicons were then resolved in a 1.2% agarose gel, purified and subjected to direct sequencing to screen for mutations. Results: The sequencing data of the thirty two OSCC samples did not identify mutations in the RING finger domain coding region of c-Cbl gene. Conclusions: To the best of our knowledge, this is the first time that the genetic status of c-Cbl gene in OSCC samples has been investigated. The present data indicates that genetic alteration of RING finger domain coding region of c-Cbl gene is relatively infrequent in OSCC samples.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2017년도 9th Asian Crop Science Association conference
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• pp.149-149
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• 2017

The RING (Really Interesting New Gene) finger proteins are known to play crucial roles in various abiotic stresses in plants. In this study, we report on RING finger E3 ligase, ${\underline{O}ryza}$ ${\underline{s}ativa}$ ${\underline{s}alt$-, ${\underline{A}BA}$- and ${\underline{d}rounght}$ stress-${\underline{i}nduced}$ RING finger ${\underline{p}}rotein{\underline{1}}$ gene (OsSAD1). In vitro ubiquitination assay demonstrated that unlike OsSAD1, a single amino acid substitution ($OsSAD1^{C168A}$) of the RING domain showed no E3 ligase activity, supporting the notion that the activity of most E3s is specified by a RING domain. Result of Yeast-Two hybridization, In vivo protein degradation assay supports that OsSAD1 interacting with 3 substrate, OsSNAC2, OsGRAS44 and OsPIRIN1, and mediates proteolysis of 3 substrates via the 26S proteasome pathway. Subcellular localizations of OsSAD1 while approximately 62% of transient signals were detected in cytosol, 38% of signals were showed nucleus. However, transiently expression of OsSAD1 was detected in cytosol 30% while as 70% of nucleus under 200 mM salt treated rice protoplasts. Results of bimolecular fluorescence complementation (BiFC) showed that two nucleus-localized proteins (OsSNAC2 and OsGRAS44) interacted with OsSAD1 in the both cytosol and nucleus. Heterogeneous overexpression of OsSAD1 Heterogeneous overexpresssion of OsSAD1 in Arabidopsis exhibited sensitive phenotypes with respect to Salt-, mannitol-responsive seed germination, seedling growth. In ABA conditions, OsSAD1 overexpression plants showed highly tolerance phenotypes, such as root length and stomatal closure. Our findings suggest that the OsSAD1 may play a negative regulator in salt stress response by modulating levels of its target proteins.

• Asian-Australasian Journal of Animal Sciences
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• 제30권8호
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• pp.1183-1189
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• 2017

Objective: Owing to the public availability of complete genome sequences, including avian species, massive bioinformatics analyses may be conducted for computational gene prediction and the identification of gene regulatory networks through various informatics tools. However, to evaluate the biofunctional activity of a predicted target gene, in vivo and in vitro functional genomic analyses should be a prerequisite. Methods: Due to a lack of quail genomic sequence information, we first identified the partial genomic structure and sequences of the quail SH3 domain containing ring finger 2 (SH3RF2) gene. Subsequently, SH3RF2 was knocked out using clustered regularly interspaced short palindromic repeat/Cas9 technology and single cell-derived SH3RF2 mutant sublines were established to study the biofunctional activity of SH3RF2 in quail myoblast (QM7) cells during muscle differentiation. Results: Through a T7 endonuclease I assay and genotyping analysis, we established an SH3RF2 knockout (KO) QM7#4 subline with 61 and 155 nucleotide deletion mutations in SH3RF2. After the induction of myotube differentiation, the expression profiles were analyzed and compared between regular QM7 and SH3RF2 KO QM7#4 cells by global RNA sequencing and bioinformatics analysis. Conclusion: We did not detect any statistically significant role of SH3RF2 during myotube differentiation in QM7 myoblast cells. However, additional experiments are necessary to examine the biofunctional activity of SH3RF2 in cell proliferation and muscle growth.

• 대한의생명과학회지
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• 제13권1호
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• pp.1-10
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• 2007

Nebulin, a giant modular protein from muscle, is thought to act as molecular ruler in sarcomere assembly. In skeletal muscle, the C-terminal ${\sim}50 kDa$ region of nebulin extends into the Z-line lattice. The most recent studies implicated highlighting its extensive isoform diversity and exciting reports revealed its expression in cardiac and non-muscle tissues containing brain. Also these novel findings are indicating that nebulin is actually a multifunctional filament system, perhaps playing roles in signal transduction, contractile regulation, and myofibril force generation, as well as other not yet defined functions. However the binding protein of nebulin and function in brain is still unknown. A novel binding partner of nebulin C-terminal region was identified by screening a human brain cDNA library using yeast two-hybrid system. Nebulin C-terminus binding protein 51 (NCBP51) was contained a RING-finger domain and identified a new isoform of RING finger protein 125 (RNF125). The interaction was confirmed using the GST pull-down assay. NCBP51 belongs to a family of the RING finger proteins and its function remains to be identified in brain. The role of nebulin and NCBP51 will be studied by loss-of-function using siRNA technique in brain.

• Journal of Plant Biotechnology
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• 제35권3호
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• pp.177-183
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• 2008

본 연구는 동진벼 유래의 Ac/Ds 삽입변이집단의 GUS 분석을 통하여 뿌리 및 미숙종자에서 강하게 GUS가 발현한 개체를 선발하여 FST(flanking sequence tag) 분석 한 결과 Ds 전이 인자가 3번 염색체 zinc finger RING-H2 관련 Oszinc626 유전자의 첫 번째 exon 부위에 single copy로 삽입되어 있었으며, 선발변이체는 뿌리 및 종자 발달이 정상인 동진벼에 비해 매우 낮은 것으로 나타났다. Oszinc626 유전자는 RING-H2 type(C3-H2-C3)으로 $Cys-X_2-Cys-X_{28}-Cys-X-His-X_2-His-X_2-Cys-X_{14}-Cys-X_2-Cys$ 배열이 C-terminus 가장 말단에 위치하며, 49 kDa의 분자량을 가지고 있다. 또한 Southern blot 분석에서 Oszinc626 유전자는 벼 게놈상에 single copy로 존재하였다. RT-PCR을 통한 돌연변이 유전자의 발현분석 결과 250 mM의 염과, $4^{\circ}C$ 저온등과 같은abiotic stress에 의해 발현이 증가함을 보였고, 호르몬처리에 있어서 ABA와 IAA의 식물호르몬을 처리했을 경우 24시간까지 계속해서 발현양이 증가하는 것을 보이는 반면, 2,4-D 처리의 경우 30분 후에 발현이 일시적으로 증가되었으나 이후 발현이 급속히 감소한 것을 보였다. 벼의 조직 별 발현 검정에서 미성숙한 종자, 뿌리 분열조직 및 신초 등 주로 생장점 부위에서 강하게 발현되는 것을 보임에 따라 Oszinc626 유전자의 경우 식물의 생장에 관여하는 주동 유전자의 하나로 판단된다.

• Journal of Microbiology
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• 제42권1호
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• pp.32-36
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• 2004

We have previously isolated three synthetic lethal mutants from Schizosaccharomyces pombe in order to identify mutations in the genes that are functionally linked to spmex67 with respect to mRNA export. A novel rsm1 gene was isolated by complementation of the growth defect in one of the synthetic lethal mutants, SLMex1. The rsml gene contains no introns and encodes a 296 amino-add-long protein with the RING finger domain, a C3HC4 in the N-terminal half. The Δrsm1 null mutant is viable, but it showed a slight poly(A)$\^$＋/ RNA accumulation in the nucleus. Also, the combination of Δrsm1 and Δspmex67 mutations confers synthetic lethality that is accompanied by the severe poly(A)$\^$＋/ RNA export defect. These results suggest that rsm1 is involved in mRNA export from the nucleus.

• BMB Reports
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• 제40권2호
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• pp.270-276
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• 2007

We have cloned a novel mouse zinc finger protein gene Znf313 by rapid amplification of cDNA ends (RACE) according to the homologue of human ZNF313 gene. The cDNA is 2,163 base pairs (bp) in length and encodes a 229 amino acids (aa) protein with a $C_3HC_4$ ring finger domain and three $C_2H_2$ domains. 89% and 93% nucleotide (nt) and aa sequence identity is observed with its human homologue. Revealed by Northern blot and RT-PCR, full mRNA consists of 2.16 kb and widely expresses in tissues as a single transcript, most abundantly in heart, liver, kidney and testis. The expression of Znf313 in testis is detected in all development stages. Western blot analysis also reveals that Znf313 is expressed in the tissues. Immunohistochemical staining and subcellular localization demonstrate that Znf313 is expressed both in the cytoplasm and nucleus whereas predominantly localized in the nucleus. Present data suggests that Znf313 gene might play a fundamental role in gene transcription and regulation in organism and relates to spermatogenesis.

• BMB Reports
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• 제53권2호
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• pp.112-117
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• 2020

A recent study suggested that methylation of ubiquitin-like with PHD and RING finger domain 1 (UHRF1) is regulated by SET7 and lysine-specific histone demethylase 1A (LSD1) and is essential for homologous recombination (HR). The study demonstrated that SET7-mediated methylation of UHRF1 promotes polyubiquitination of proliferating cell nuclear antigen (PCNA), inducing HR. However, studies on mediators that interact with and recruit UHRF1 to damaged lesions are needed to elucidate the mechanism of UHRF1 methylation-induced HR. Here, we identified that poly [ADP-ribose] polymerase 1 (PARP1) interacts with damage-induced methylated UHRF1 specifically and mediates UHRF1 to induce HR progression. Furthermore, cooperation of UHRF1-PARP1 is essential for cell viability, suggesting the importance of the interaction of UHRF1-PARP1 for damage tolerance in response to damage. Our data revealed that PARP1 mediates the HR mechanism, which is regulated by UHRF1 methylation. The data also indicated the significant role of PARP1 as a mediator of UHRF1 methylation-correlated HR pathway.

• Asian Pacific Journal of Cancer Prevention
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• 제13권10호
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• pp.5137-5142
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• 2012

UHRF2 is a member of the ubiquitin plant homeo domain RING finger family, which has been proven to be frequently up-regulated in colorectal cancer cells and play a role as an oncogene in breast cancer cells. However, the role of UHRF2 in glioma cells remains unclear. In this study, we performed real-time quantitative PCR on 32 pathologically confirmed glioma samples (grade I, 4 cases; grade II, 11 cases; grade III, 10 cases; and grade IV, 7 cases; according to the 2007 WHO classification system) and four glioma cell lines (A172, U251, U373, and U87). The expression of UHRF2 mRNA was significantly lower in the grade III and grade IV groups compared with the noncancerous brain tissue group, whereas its expression was high in A172, U251, and U373 glioma cell lines. An in vitro assay was performed to investigate the functions of UHRF2. Using a lentivirus-based RNA interference (RNAi) approach, we down-regulated UHRF2 expression in the U251 glioma cell line. This down-regulation led to the inhibition of cell proliferation, an increase in cell apoptosis, and a change of cell cycle distribution, in which S stage cells decreased and G2/M stage cells increased. Our results suggest that UHRF2 may be closely related to tumorigenesis and the development of gliomas.

• Asian Pacific Journal of Cancer Prevention
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• 제16권4호
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• pp.1343-1348
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• 2015

Human UHRF1 (ubiquitin-like PHD and RING finger domain-containing 1) has been reported to be over-expressed in many cancers, but its role in ovarian cancer remains elusive. Here, we determined whether knockdown of UHRF1 by lentivirus-mediated shRNA could inhibit ovarian cancer cell growth. Lentivirus-mediated short hairpin RNAs (lv-shRNAs-UHRF1) were designed to trigger the gene silencing RNA interference (RNAi) pathway. The efficiency of lentivirus-mediated shRNA infection into HO-8910 and HO-8910 PM cells was determined using fluorescence microscopy to observe lentivirus-mediated GFP expression and was confirmed to be over 80 percent. UHRF1 expression in infected HO-8910 and HO-8910 PM was evaluated by real-time PCR and Western blot analysis. The Cell Counting Kit-8 (CCK-8) assay was used to measure cell viability; flow cytometry and Hoechst 33342 assay was applied to measure cell cycle arrest and apoptosis. Cell invasion was assessed using transwell chambers. Our results demonstrated that the loss of UHRF1 promoted HO-8910 and HO-8910 PM cell apoptosis, while inhibiting cell proliferation. In addition, UHRF1 knockdown significantly inhibited the invasion of human ovarian cancer cells. In the present study, we also showed that depleting HO-8910 cells of UHRF1 caused activation of the DNA damage response pathway, with the cell cycle arrested in G2/M-phase. The DNA damage response in cells depleted of UHRF1 was illustrated by phosphorylation of CHK (checkpoint kinase) 2 on Thr68, phosphorylation of CDC25 (cell division control 25) on Ser 216 and phosphorylation of CDK1 (cyclin-dependent kinase 1) on Tyr 15.