Apolipoprotein B mRNA editing is associated with UV crosslinking of proteins to the editing site.
Apolipoprotein (apo) B100 mRNA undergoes editing of C-6666 to a U residue, which generates a stop-translation codon and defines the carboxyl terminus of apoB48. To aid purification of the editing enzyme we have undertaken UV crosslinking of a 32P-labeled substrate for apoB mRNA editing in vitro to proteins in an enterocyte editing extract. Proteins of 60 (p60) and 43 (p43) kDa, prominent among crosslinking bands, were competed for by unlabeled substrate, but not by nonspecific RNA, and did not crosslink to antisense RNA. Editing in vitro and UV crosslinking were inhibited by NaCl and vanadyl ribonucleoside complexes and by chemical modification of sulfhydryl, imidazolium, and guanidinium groups on the protein. The editing activity copurified predominantly with p60. To define the binding site for p60 on the substrate RNA, a series of scanning and point mutant RNAs, previously used to define nucleotides 6671-6681 as essential for editing, were used in competition studies with wild-type substrate. Results demonstrated that p60 binding is centered on nucleotides 6671-6674. We suggest that p60 contains the RNA-recognition component of the apoB mRNA-editing enzyme.
Navaratnam
N
N
Medical Research Council Molecular Medicine Group, Royal Postgraduate Medical School, London, United Kingdom.
Shah
R
R
Patel
D
D
Fay
V
V
Scott
J
J
D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics*; D001483:Base Sequence; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003198:Computer Simulation; D004247:DNA / Q000235:genetics; D004848:Epithelium / Q000502:physiology; D007422:Intestines / Q000502:physiology; D008961:Models, Structural; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D016601:RNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000528:radiation effects; D051381:Rats; D013698:Templates, Genetic; D014466:Ultraviolet Rays
D001055:Apolipoproteins B; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D004247:DNA