A SEQUENCE-SPECIFIC RNA-BINDING PROTEIN COMPLEMENTS APOBEC-1 TO EDIT APOLIPOPROTEIN-B MESSENGER-RNA

Citation
A. Mehta et Dm. Driscoll, A SEQUENCE-SPECIFIC RNA-BINDING PROTEIN COMPLEMENTS APOBEC-1 TO EDIT APOLIPOPROTEIN-B MESSENGER-RNA, Molecular and cellular biology, 18(8), 1998, pp. 4426-4432
Citations number
36
Categorie Soggetti
Biology,"Cell Biology
ISSN journal
02707306
Volume
18
Issue
8
Year of publication
1998
Pages
4426 - 4432
Database
ISI
SICI code
0270-7306(1998)18:8<4426:ASRPCA>2.0.ZU;2-E
Abstract
The editing of apolipoprotein B (apo-B) mRNA involves the site-specifi c deamination of cytidine to uracil. The specificity of editing is con ferred by an 11-nucleotide mooring sequence located downstream from th e editing site. Apobec-1, the catalytic subunit of the editing enzyme, requires additional proteins to edit apo-B mRNA in vitro, but the fun ction of these additional factors, known as complementing activity, is not known. Using RNA affinity chromatography, we show that the comple menting activity binds to a 280-nucleotide apo-B RNA in the absence of apobec-1. The activity did not bind to the antisense strand or to an RNA with three mutations in the mooring sequence. The eluate from the wild-type RNA column contained a 65-kDa protein that UV cross-linked t o apo-B mRNA but not to the triple-mutant RNA. This protein was not de tected in the eluates from the mutant or the antisense RNA columns. In troduction of the mooring sequence into luciferase RNA induced cross-l inking of the 65-kDa protein. A 65-kDa protein that interacted with ap obec-1 was also detected by far-Western analysis in the eluate from th e wild-type RNA column but not from the mutant RNA column. For purific ation, proteins were precleared on the mutant RNA column prior to chro matography on the wild-type RNA column. Silver staining of the affinit y-purified fraction detected a single prominent protein of 65 kDa. Our results suggest that the complementing activity may function as the R NA-binding subunit of the holoenzyme.