Regulation of RNF144A E3 Ubiquitin Ligase Activity by Self-association through Its Transmembrane Domain

RNF144A, an E3 ubiquitin ligase for DNA-dependent protein kinase catalytic subunit (DNA-PKcs), can promote DNA damage-induced cell apoptosis. Here we characterize an important regulation of RNF144A through its transmembrane (TM) domain. The TM domain of RNF144A is highly conserved among species. Deletion of the TM domain abolishes its membrane localization and also significantly reduces its ubiquitin ligase activity. Further evidence shows that the TM domain is required for RNF144A self-association and that the self-association may be partially mediated through a classic GXXXG interaction motif. A mutant RNF144A-G252L/G256L (in the G²⁵²XXXG²⁵⁶ motif) preserves membrane localization but is defective in self-association and ubiquitin ligase activity. On the other hand, a membrane localization loss mutant of RNF144A still retains self-association and E3 ligase activity, which can be blocked by additional G252L/G256L mutations. Therefore, our data demonstrate that the TM domain of RNF144A has at least two independent roles, membrane localization and E3 ligase activation, to regulate its physiological function. This regulatory mechanism may be applicable to other RBR (RING1-IBR-RING2) E3 ubiquitin ligases because, first, RNF144B also self-associates. Second, all five TM-containing RBR E3 ligases, including RNF144A and RNF144B, RNF19A/Dorfin, RNF19B, and RNF217, have the RBR-TM(GXXXG) superstructure. Mutations of the GXXXG motifs in RNF144A and RNF217 have also be found in human cancers, including a G252D mutation of RNF144A. Interestingly, RNF144A-G252D still preserves self-association and ubiquitin ligase activity but loses membrane localization and is turned over rapidly. In conclusion, both proper membrane localization and self-association are important for RNF144A function.     

Cloning and characterization of a gene encoding the human putative ubiquitin conjugating enzyme E2Z (UBE2Z)

Ubiquitination, the covalent attachment of the protein ubiquitin (Ub) to other cellular proteins, has been implicated in a number of important physiological processes. ubiquitin conjugating enzyme (E2) plays an important role in the ubiquitin system. Here we report the research about a human putative ubiquitin conjugating enzyme gene, UBE2Z. The length of UBE2Z cDNA is 3054 base pairs and contains an open reading frame encoding 246 amino acids. The UBE2Z gene was mapped to human chromosome 17q21.32 and consisted of 6 exons. RT-PCR showed that UBE2Z was widely expressed in human tissues, especially high in placenta, pancreas, spleen and testis. The UBE2Z-GFP fusion protein was located in both nucleus and cytoplasm of AD293 cells.     

食管癌细胞抗失巢凋亡基因UBCH7的发现与鉴定

失巢凋亡(Anoikis)是细胞失去与细胞外基质(Extra-cellular matrix, ECM)粘附时发生的特殊形式的凋亡, 是机体维持组织稳态的关键机制之一。抗失巢凋亡能力的获得是肿瘤细胞发生远处转移的前提条件之一。为了鉴定与食管癌细胞抗失巢凋亡相关的基因, 文章首先构建食管癌细胞系的逆转录病毒文库, 感染对失巢凋亡敏感的NIH3T3细胞, 利用感染病毒cDNA文库的混合细胞系进行软琼脂集落形成实验, 挑取在悬浮条件下仍可生长成为较大集落的细胞单克隆(潜在具有抗失巢凋亡能力的细胞), 通过逆转录病毒载体特异的引物PCR扩增失巢凋亡抗性克隆基因组中的插入cDNA片段, 以此获得食管癌细胞系cDNA文库中潜在的具有失巢凋亡抗性的基因。经测序发现其中一个失巢凋亡克隆中整合的cDNA片段包括人UBCH7/UBE2L3基因全长的编码序列(开放阅读框)。利用携带pMSCV-UBCH7的逆转录病毒感染NIH3T3细胞进行验证, 结果显示细胞失巢凋亡抗性增强, 并且在具有高转移潜能的食管癌细胞系MLuC1中降调UBCH7表达可减弱其失巢凋亡抗性。这些结果表明, UBCH7/UBE2L3是一个与食管癌失巢凋亡抗性相关的基因。     

食管癌细胞抗失巢凋亡基因UBCH7的发现与鉴定

失巢凋亡(Anoikis)是细胞失去与细胞外基质(Extra-cellular matrix,ECM)粘附时发生的特殊形式的凋亡,是机体维持组织稳态的关键机制之一。抗失巢凋亡能力的获得是肿瘤细胞发生远处转移的前提条件之一。为了鉴定与食管癌细胞抗失巢凋亡相关的基因,文章首先构建食管癌细胞系的逆转录病毒文库,感染对失巢凋亡敏感的NIH3T3细胞,利用感染病毒cDNA文库的混合细胞系进行软琼脂集落形成实验,挑取在悬浮条件下仍可生长成为较大集落的细胞单克隆(潜在具有抗失巢凋亡能力的细胞),通过逆转录病毒载体特异的引物PCR扩增失巢凋亡抗性克隆基因组中的插入cDNA片段,以此获得食管癌细胞系cDNA文库中潜在的具有失巢凋亡抗性的基因。经测序发现其中一个失巢凋亡克隆中整合的cDNA片段包括人UBCH7/UBE2L3基因全长的编码序列(开放阅读框)。利用携带pMSCV-UBCH7的逆转录病毒感染NIH3T3细胞进行验证,结果显示细胞失巢凋亡抗性增强,并且在具有高转移潜能的食管癌细胞系MLuC1中降调UBCH7表达可减弱其失巢凋亡抗性。这些结果表明,UBCH7/UBE2L3是一个与食管癌失巢凋亡抗性相关的基因。     

Characterization of a Moniezia expansa ubiquitin-conjugating enzyme E2 cDNA

Ubiquitin and other ubiquitin-like proteins play important roles in post-translational modification. They are phylogenetically well-conserved in eukaryotes. Here we report a new ubiquitin-conjugating enzyme E2 cDNA, which containing a ubiquitin-conjugating enzyme UBCc-domain named UBE2AM. Its cDNA is 899 base pairs in length and contains an open reading frame from nucleotide 171 to 632 encoding 153 amino acids. The result of real time RT-PCR showed that UBEA2 M is expressed in most of M. expansa proglottides and over-expressed in the mature proglottides. Comparison of predicted UBE2AM with UBCc (protein) homologues/orthologous from other species revealed identities between species varying from 97.5 to 99.4% at the amino acid level. Phylogenetic analysis showed the UBE2AM is a member of the eukaryotic UBCc superfamily, which have diverged from a common ancestor and the gene is clustered in the same group with the ubiquitin-conjugating enzyme E2A-like protein from Taenia asiatica.     

Isolation of a cDNA for a novel human RING finger protein gene, RNF18, by the virtual transcribed sequence (VTS) approach(1)

We have recently developed a novel database system, designated as the virtual transcribed sequence (VTS) which efficiently extracts many genes from public human genome databases, and tested the feasibility of this novel computational approach (N. Miyajima, C. Burge, T. Saito, Biochem. Biophys. Res. Commun. 272 (2000) 801; http://host45.maze.co.jp/vts/). In this study, using the VTS approach, we isolated a cDNA for a novel human gene with RING finger motif (C(3)HC(4)), which is not deposited in public EST databases. The isolated cDNA clone is 2163 bp in length, and contains an open reading frame of 452 amino acids. We designated the novel gene as RNF18. A database search showed that the RNF18 gene had the moderate similarity to SS-A/Ro52 protein, which is a ribonucleoprotein reactive with autoantibodies in patients with Sj?gren's syndrome and systemic lupus erythematosus. Tissue distribution analyses by Northern blot and RT-PCR methods demonstrated that the RNF18 messenger RNA was preferentially expressed in testis. The exon-intron boundaries of RNF18 gene were determined by aligning the cDNA sequence with the corresponding genome sequence. The isolated cDNA consists of eight exons that span about 11 kb of the genome DNA. The precise chromosomal location of the RNF18 gene was determined by PCR-based radiation hybrid mapping, and the gene was located to centromere region of chromosome 11 between markers NIB1900 and D11S1350. Taken together, the VTS approach should provide a novel cDNA cloning strategy for isolating unidentified genes, which are not found even in EST databases but are detectable computationally.     

Molecular characterization and expression analysis of ubiquitin-activating enzyme E1 gene in Citrus reticulata

Ubiquitin-activating enzyme E1 (UBE1) catalyzes the first step in the ubiquitination reaction, which targets a protein for degradation via a proteasome pathway. UBE1 plays an important role in metabolic processes. In this study, full-length cDNA and DNA sequences of UBE1 gene, designated CrUBE1, were obtained from ‘Wuzishatangju’ (self-incompatible, SI) and ‘Shatangju’ (self-compatible, SC) mandarins. 5 amino acids and 8 bases were different in cDNA and DNA sequences of CrUBE1 between ‘Wuzishatangju’ and ‘Shatangju’, respectively. Southern blot analysis showed that there existed only one copy of the CrUBE1 gene in genome of ‘Wuzishatangju’ and ‘Shatangju’. The temporal and spatial expression characteristics of the CrUBE1 gene were investigated using semi-quantitative RT-PCR (SqPCR) and quantitative real-time PCR (qPCR). The expression level of the CrUBE1 gene in anthers of ‘Shatangju’ was approximately 10-fold higher than in anthers of ‘Wuzishatangju’. The highest expression level of CrUBE1 was detected in pistils at 7 days after self-pollination of ‘Wuzishatangju’, which was approximately 5-fold higher than at 0 h. To obtain CrUBE1 protein, the full-length cDNA of CrUBE1 genes from ‘Wuzishatangju’ and ‘Shatangju’ were successfully expressed in Pichia pastoris. Pollen germination frequency of ‘Wuzishatangju’ was significantly inhibited with increasing of CrUBE1 protein concentrations from ‘Wuzishatangju’.     

Molecular characterization, polymorphism and association of porcine SPATA19 gene

Spermatogenesis associated 19 (SPATA19) is an important reproduction related gene. In this study, we cloned the full-length cDNA sequence of porcine SPATA19 gene through the rapid amplification of cDNA ends (RACE) method. The porcine SPATA19 gene encodes a protein of 154 amino acids which shares high homology with the SPATA19 of ten species: giant panda (87?%), dog (86?%), cattle (84?%), rabbit (78?%), sumatran orangutan (72?%), human (71?%), rhesus monkey (71?%), chimpanzee (70?%), mouse (71?%) and rat (69?%). The phylogenetic analysis revealed that the porcine SPATA19 gene has a closer genetic relationship with the SPATA19 gene of dog. This gene is structured in six exons and five introns as revealed by computer-assisted analysis. PCR-RFLP was established to detect the GU475012:c.515T>C substitution of porcine SPATA19 gene mRNA and association of this mutation with litter size traits was assessed in Large White (n?=?100) and Landrace (n?=?100) pig populations. Results demonstrated that this polymorphic locus was significantly associated with the litter size of all parities in Large White sows and Landrace sows. Therefore, SPATA19 gene could be an useful candidate gene in selection for increasing litter size in pigs. These data serve as a foundation for further insight into this novel porcine gene.