Abstract
In the accompanying paper (Zaidi, S. H. E., Denman, R., and Malter, J. S. (1994) J. Biol. Chem. 269, 24000-24006) we demonstrate that in tumor and normal cells, multiple cytosolic proteins interact with a 29-base sequence in the 3'-untranslated region of amyloid precursor protein (APP) mRNA. These data suggested that APP gene expression may be modulated by regulated APP mRNA decay. We have investigated this prediction by measuring the decay rates of APP mRNA in resting and mitogen-treated peripheral blood mononuclear cells and H4 and K562 tumor cell lines. In resting peripheral blood mononuclear cells, APP mRNA decayed with a half-life of 4 h. Under these conditions, the activity of APP mRNA-binding proteins was not detectable. After activation, binding protein activities were induced, and APP mRNA decay was blocked with a half-life of > 12 h. In log phase neuronal or lymphoid tumor cell lines, binding activity was constitutively present and APP mRNA displayed a half-life of > 12 h. Protein synthesis inhibition by cycloheximide had no effect on APP mRNA decay in normal or tumor cells. Transfected wild type or mutant APP mRNAs that lacked the 29-base region were stable (t1/2 > 10 h) in K562 tumor cells. Therefore, we conclude that the 29-base region functions in cis to destabilize APP mRNA in resting, normal cells. Upon activation APP mRNA-binding proteins are induced, interact with the 29-base region, and likely participate in stabilization of the mRNA.
Highlights
From the Department of Pathology and Laboratory Medicine, Neuroscience Program and Instituteof Aging, University of Wisconsin, Madison, Wisconsin 53792-2472
The mutant cDNA was decay machinery [15].We examined if APP mRNA decay was in vitro transcribed and the radiolabeled APP mRNAs influenced by protein synthesis inhibition
42-kDa APP RNA-protein complex than that seen with acti- tion of APP mRNA decay to thisprocess in normalPBMC and vated PBMC
Summary
Was size separated on denaturing formaldehyde-agarose gels contain- Decay Rate of APP mRNA in Resting Human PBMC-APP ing 5 pg of ethidium bromide/sample. Autoradiograms were quantitatedby laser densitometry and APP-specific signals normalized to ribosomal RNA or the signal obtained with the control probe for glyceraldehyde-3-phosphate creased steady-state levels in normal PBMC and neuronal and glial tumor cell lines. Leftmost lane, APP mRNA was detected by Northern blotting in total RNA isolated from resting PBMC. 0, 3, 6, and 9 h post-actinomycin D addition inTPAE'HA-treated cells, mRNA-RNA was isolated from PBMC from the same cells were recovered from culture, and total RNA was isolated and donors (as in Fig. 1)after stimulation with a combination of
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