E3 ubiquitin ligases target proteins for degradation by adding ubiquitin residues. We characterized full-length cDNAs for human and mouse UBE3B, a novel HECT-domain E3 ligase, and analyzed the structure of human UBE3B on chromosome 12q24.1. Alternative splicing of exon 20 of UBE3B generated two major transcripts. The 5.7-kb mRNA lacked exon 20 and encoded a full-length protein ligase, variant 1 (UBE3B_v1). A second transcript contained a 97-bp insertion encoded by exon 20 that introduced an in-frame stop codon. The predicted protein (UBE3B_v2) would lack the HECT domain and would be nonfunctional, since the HECT domain constitutes the active site for ubiquitin transfer. No alternative splicing was observed in this region of mouse UBE3B. Elimination of the HECT domain by alternative splicing has not been reported in any genes encoding HECT domain ligases and may represent a novel mechanism in regulating intracellular levels of functional HECT-domain ligases.
[Show abstract][Hide abstract] ABSTRACT: The post-translational modification of proteins by the ubiquitination pathway is an important regulatory mechanism in eukaryotes. To date, however, studies on the evolutionary history of the proteins involved in this pathway have been restricted to E1 and E2 enzymes, while E3 studies have been focused mainly in metazoans and plants. To have a wider perspective, here we perform a genomic survey of the HECT family of E3 ubiquitin-protein ligases, an important part of this post-translational pathway, in genomes from representatives of all major eukaryotic lineages. We classify eukaryotic HECTs and reconstruct, by phylogenetic analysis, the putative repertoire of these proteins in the last eukaryotic common ancestor (LECA). Furthermore, we analyse the diversity and complexity of protein domain architectures of HECTs along the different extant eukaryotic lineages. Our data show that LECA had six different HECTs and that protein expansion and N-terminal domain diversification shaped HECT evolution. Our data reveal that the genomes of animals and unicellular holozoans considerably increased the molecular and functional diversity of their HECT system compared to other eukaryotes. Other eukaryotes, such as the Apusozoa Thecanomas trahens or the Heterokonta Phytophthora infestans independently expanded their HECT repertoire. In contrast, plant, excavate, rhodophyte, chlorophyte and fungal genomes have a more limited enzymatic repertoire. Our genomic survey and phylogenetic analysis clarifies the origin and evolution of different HECT families among eukaryotes and provides a useful phylogenetic framework for future evolutionary studies of this regulatory pathway.
"Transcripts downregulated in blastomere- relative to the whole embryo-derived lines include UBE3B, that encodes a HECT-domain E3 ubiquitin ligase which targets proteins for degradation ; QK1 which product is RNA binding protein important for normal development in vertebrates and regulates protein translation, RNA splicing, export from the nucleus, and stability , ; PASK that encodes a PAS domain-containing protein kinase involved in regulation of many intracellular signaling pathways in response to both extrinsic and intrinsic stimuli  and RAB3B that encodes one of the Ras-associated GTP-binding protein 3 family members that regulates secretory vesicle transport between the Golgi apparatus and the plasma membrane . "
[Show abstract][Hide abstract] ABSTRACT: We have derived hESC from biopsied blastomeres of cleavage stage embryos under virtually the same conditions we used for the derivation of hESC lines from inner cell mass of blastocyst stage embryos. Blastomere-derived hESC lines exhibited all the standard characteristics of hESC including undifferentiated proliferation, genomic stability, expression of pluripotency markers and the ability to differentiate into the cells of all three germ layers both in vitro and in vivo. To examine whether hESC lines derived from two developmental stages of the embryo differ in gene expression, we have subjected three blastomere-derived hESC lines and two ICM-derived hESC lines grown under identical culture conditions to transcriptome analysis using gene expression arrays. Unlike previously reported comparisons of hESC lines which demonstrated, apart from core hESC-associated pluripotency signature, significant variations in gene expression profiles of different lines, our data show that hESC lines derived and grown under well-controlled defined culture conditions adopt nearly identical gene expression profiles. Moreover, blastomere-derived and ICM-derived hESC exhibited very similar transcriptional profiles independent of the developmental stage of the embryo from which they originated. Furthermore, this profile was evident in very early passages of the cells and did not appear to be affected by extensive passaging. These results suggest that during derivation process cells which give rise to hESC acquire virtually identical stable phenotype and are not affected by the developmental stage of the starting cell population.
PLoS ONE 10/2011; 6(10):e26570. DOI:10.1371/journal.pone.0026570 · 3.23 Impact Factor
"Additionally, alternative splicing of exons 24 and 25 in HERC4 may result in altered binding to the ubiquitin conjugase, a further possibility for interfering with ubiquitin ligase activity. Alternative splicing was also recently reported for the HECT ubiquitin ligase UBE3B , but no functional consequences are known so far. It will be interesting to elucidate further the possible regulation of ubiquitin ligases by alternative splicing and find out its functional con- sequences. "
[Show abstract][Hide abstract] ABSTRACT: The HERC family of ubiquitin ligases is characterized by the presence of a HECT domain and one or more RCC1-like domains. We report the identification of two novel members, HERC4 and HERC6, and subdivide the family into one group of two large and one group of four small members according to protein size and domain structure. The small members share a similar genomic organization, three of them mapping to chromosomal region 4q22, indicating strong evolutionary cohesions. Phylogenetic analysis reveals that the HERC ancestor emerged in nematodes and that the family expanded throughout evolution. The mRNA expression pattern of the small human members was found to be diverse in selected tissues and cells; overexpressed proteins display a similar cytosolic distribution. These data indicate that the HERC family members exhibit similarities in many aspects, but also sufficient differences indicating functional diversity.
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