Characterization of the apolipoprotein B mRNA editing enzyme: no similarity to the proposed mechanism of RNA editing in kinetoplastid protozoa.
Intestinal apolipoprotein B mRNA is edited at nucleotide 6666 by a C to U transition resulting in a translational stop codon. The enzymatic properties of the editing activity were characterised in vitro using rat enterocyte cytosolic extract. The editing activity has no nucleotide or ion cofactor requirement. It shows substrate saturation with an apparent Km for the RNA substrate of 2.2 nM. The editing enzyme requires no lag period prior to catalysis, and does not assemble into a higher order complex on the RNA substrate. In crude cytosolic extract editing activity is completely abolished by treatment with micrococcal nuclease or RNAse A. Partially purified editing enzyme is no longer sensitive to nucleases, but is inhibited in a dose dependent manner by nuclease inactivated crude extract. The buoyant density of partially purified editing enzyme is 1.3 g/ml, that of pure protein. Therefore, the apolipoprotein B mRNA editing activity consists of a well defined enzyme with no RNA component. The nuclease sensitivity in crude cytosolic extract is explained by the generation of inhibitors for the editing enzyme. The editing of apo B mRNA has little similarity to complex mRNA processing events such as splicing and unlike editing in kinetoplastid protozoa does not utilise guide RNAs.
Greeve
J
J
Division of Molecular Medicine, MRC Clinical Research Centre, Harrow, Middlesex, UK.
Navaratnam
N
N
Scott
J
J
D000071479:APOBEC-1 Deaminase; D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics*; D001483:Base Sequence; D003564:Cytidine Deaminase / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D004722:Endoribonucleases / Q000378:metabolism; D004847:Epithelial Cells; D004848:Epithelium / Q000201:enzymology; D056890:Eukaryota / Q000378:metabolism; D007422:Intestines / Q000201:enzymology; D007700:Kinetics; D008297:Male; D008836:Micrococcal Nuclease / Q000378:metabolism; D008969:Molecular Sequence Data; D012323:RNA Processing, Post-Transcriptional; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D016914:Ribonuclease H; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D001055:Apolipoproteins B; D012333:RNA, Messenger; D004722:Endoribonucleases; D016914:Ribonuclease H; D012259:Ribonuclease, Pancreatic; D008836:Micrococcal Nuclease; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D000071479:APOBEC-1 Deaminase; C000605091:Apobec1 protein, rat; D003564:Cytidine Deaminase