Characterization of the DWNN domain and ring finger-like motif within the DCM of the Drosophila melanogaster SNAMA protein

Abstract

SNAMA is a 139 kDa Drosophila melanogaster protein belonging to the Retinoblastoma Binding Protein 6 (RbBP6) superfamily. Mammalian RbBP6 interacts with both p53 and Rb and plays a critical role in embryonic development and tumorigenesis as a negative regulator of p53. All RbBP6 members contain a 5´ DWNN Catalytic Domain (DCM) consisting of a DWNN (domain with no name) domain followed by a zinc finger and RING (really interesting new gene) finger motif. RING finger and U-box ubiquitin ligases are dependent on their RING finger conformation for their ubiquitin ligase activity. The main focus of this work was to further elucidate the functional characteristics of the DCM within SNAMA. This was achieved by examinining transcription regulation of the SNAMA gene and determining whether SNAMA exhibits ubiquitin ligase activity. 5´ Rapid amplification of cDNA ends (RACE) was used to determine the core promoter elements of SNAMA through the discovery of the transcription initiation site. Ubiquitin conjugation assays using pure recombinant SNAMA as the ubiquitin ligase were performed to analyze the ubiquitin ligase activity of the protein. Transcriptional regulation of the SNAMA gene is achieved through a single TATA-less promoter that gives rise to a single transcript of 3.9 kb. Furthermore, this work shows that SNAMA is a unique RING finger ubiquitin ligase that is capable of autoubiquitination in the absence of zinc ions. This activity is dependent on its RING finger-like motif, which has greatest specificity to the UbcH5a conjugating enzyme. Furthermore, SNAMA relies on E1, E2, ATP, and ubiquitin for its E3 activity. Thus, SNAMA is the first member of the RbBP6 family to portray RING finger ubiquitin ligase activity and represents a “drugable” target for pharmaceutical intervention.