Journal
RNA
Volume 10, Issue 9, Pages 1399-1411Publisher
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1261/rna.7490704
Keywords
RNA editing; apoB; APOBEC1; ACF; kinetics
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Funding
- NIGMS NIH HHS [NIGMS 48519] Funding Source: Medline
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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 45degreesC. We have used these optimized conditions, to assess steady-state kinetic parameters for APOBIEC1 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.
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