Optimization of apolipoprotein B mRNA editing by APOBEC1 apoenzyme and the role of its auxiliary factor, ACF.
Expression and purification to homogeneity of the apolipoprotein B mRNA editing subunit, APOBEC1, has allowed the demonstration that this apoenzyme has considerable residual enzymatic activity on a minimal apoB mRNA substrate, even in the absence of any auxiliary factors. Assay of this activity as a function of various experimental conditions has led to substantial optimization of assay conditions through the use of incomplete factorial and response surface experiments. Surprisingly, the apoenzyme is thermostable, and has a temperature optimum near 45 degrees C. We have used these optimized conditions, to assess steady-state kinetic parameters for APOBEC1 mRNA editing activity with and without the auxiliary factor, ACF. An important effect of the auxiliary factor is to broaden the temperature range of APOBEC1 activity, lowering the optimal temperature and enabling it to function optimally at lower temperatures. A model consistent with this observation is that at lower temperatures ACF promotes a conformational transition in the RNA substrate that occurs spontaneously at higher temperature. Notably, the substantial RNA editing activity of APOBEC1 alone may be responsible for the "hyperediting" observed upon overexpression of APOBEC1 in transgenic mice.
Chester
Ann
A
RNA Editing Group, MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College, Hammersmith Campus, London, UK.
Weinreb
Violetta
V
Carter
Charles W
CW
Navaratnam
Naveenan
N
D000071479:APOBEC-1 Deaminase; D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics*; D016367:Baculoviridae / Q000235:genetics; D020029:Base Pairing; D001483:Base Sequence; D003067:Coenzymes / Q000378:metabolism*; D003564:Cytidine Deaminase / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017393:RNA Editing; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
C411647:A1CF protein, human; D001055:Apolipoproteins B; D003067:Coenzymes; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D000071479:APOBEC-1 Deaminase; C000605089:APOBEC1 protein, human; C000605090:Apobec1 protein, mouse; D003564:Cytidine Deaminase