title: Characterization of the mouse ubiquitin-conjugating enzyme gene UbcM4.
abstract: The ubiquitination pathway targets not only normal (short-lived) intracellular eukaryotic proteins for degradation when appropriate, but also serves to eliminate mutant/misfolded proteins from the cell. An understanding of the molecular basis of the interaction between the ubiquitin-conjugating enzymes (E2s), ubiquitin protein ligases (E3s), and target proteins is essential to explain the process in normal cellular function and in disease. UbcM4 is the mouse ortholog of the human E2, UbcH7, which can participate in the in vitro degradation of many proteins including p53. We describe the characterization of the mouse UbcM4 gene and the identification of a UbcM4 pseudogene. Four UbcM4 transcripts of approximately 0.7, 1.5, 2.1, and 2.6 kb, observed on Northern blots, are differentiated by their utilization of alternative UbcM4 polyadenylation sites. A single alternative splice variant cDNA, termed UbcM4Deltaex2, was also identified. The polypeptide encoded by UbcM4Deltaex2 is incapable of forming an ubiquitin-thioester in contrast to UbcM4, despite retaining the key cysteine residue essential for ubiquitin thioester formation and the active site consensus sequence that defines the ubiquitin-conjugating enzyme class. These observations are of particular relevance for analysis of UbcM4 function in vivo as our studies indicate that the targeted deletion of the coding exon absent in UbcM4Deltaex2 would produce an inactive UbcM4 protein and presents an alternative to disruption of its transcriptional initiation site/promoter region. Furthermore, it suggests that a similar strategy may be applicable to disrupt the function of other ubiquitin-conjugating enzymes in vivo.
authors.affiliation: Molecular Medicine Unit, Clinical Sciences Building, St. James's University Hospital, Leeds LS9 7TF, UK.
authors.forename: T P
authors.initials: TP
authors.lastname: Moynihan
authors.forename: U
authors.initials: U
authors.lastname: Nuber
authors.forename: H C
authors.initials: HC
authors.lastname: Ardley
authors.forename: S A
authors.initials: SA
authors.lastname: Rose
authors.forename: A F
authors.initials: AF
authors.lastname: Markham
authors.forename: M
authors.initials: M
authors.lastname: Scheffner
authors.forename: P A
authors.initials: PA
authors.lastname: Robinson
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D008025:Ligases / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D011544:Pseudogenes; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D011994:Recombinant Proteins; D014452:Ubiquitins; C117188:Ube2l3 protein, mouse; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases