RESUMO
Site-specific editing of apolipoprotein B (apoB) mRNA by the cytidine deaminase, APOBEC-1 is proposed to require interactions of auxiliary protein(s) with an eleven nucleotide element, the mooring sequence, located 3' of the C --> U editing site. An analysis of the RNA sequence dependence for protein-RNA interactions and editosome assembly in rat liver and the small intestine demonstrated that the mooring sequence was a minimal requirement for these interactions. Sequences 3' of the mooring sequence either interacted with 66 kDa and 44 kDa proteins or enhanced the interactions of these proteins with the mooring sequence. The data also suggested tissue-specific differences in the relative importance of the 3' cis-acting 'enhancer' elements in the efficiency or stability of editosome assembly. We propose that the previously demonstrated differences in apoB mRNA editing efficiency and its regulation in liver and intestine may in part be due to differences in auxiliary protein interactions with apoB mRNA 3' of the mooring sequence.
Assuntos
Apolipoproteínas B/genética , Edição de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Sequência de Bases , Elementos Facilitadores Genéticos , Técnicas In Vitro , Intestino Delgado/metabolismo , Fígado/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Ribonucleoproteínas/metabolismo , Distribuição TecidualRESUMO
A protein complex involved in apolipoprotein B (apoB) RNA editing, referred to as AUX240 (auxiliary factor containing p240), has been identified through the production of monoclonal antibodies against in vitro assembled 27S editosomes. The 240-kDa protein antigen of AUX240 colocalized with editosome complexes on immunoblots of native gels. Immunoadsorbed extracts were impaired in their ability to assemble editosomes beyond early intermediates and in their ability to edit apoB RNA efficiently. Supplementation of adsorbed extract with AUX240 restored both editosome assembly and editing activities. Several proteins, in addition to p240, ranging in molecular mass from 150 to 45 kDa coimmunopurify as AUX240 under stringent wash conditions. The activity of the catalytic subunit of the editosome APOBEC-1 and mooring sequence RNA binding proteins of 66 and 44 kDa could not be demonstrated in AUX240. The data suggest that p240 and associated proteins constitute an auxiliary factor required for efficient apoB RNA editing. We propose that the role of AUX240 may be regulatory and involve mediation or stabilization of interactions between APOBEC-1 subunits and editing site recognition proteins leading the assembly of the rat liver C/U editosome.
