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Molecular and Cellular Biology, March 2000, p. 1982-1992, Vol. 20, No. 6
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

An AU-Rich Sequence Element (UUUN[A/U]U) Downstream of the Edited C in Apolipoprotein B mRNA Is a High-Affinity Binding Site for Apobec-1: Binding of Apobec-1 to This Motif in the 3' Untranslated Region of c-myc Increases mRNA Stability

Shrikant Anant1 and Nicholas O. Davidson1,2,*

Departments of Internal Medicine1 and Molecular Biology and Pharmacology,2 Washington University Medical School, St. Louis, Missouri 63110

Received 2 November 1999/Returned for modification 6 December 1999/Accepted 10 December 1999

Apobec-1, the catalytic subunit of the mammalian apolipoprotein B (apoB) mRNA-editing enzyme, is a cytidine deaminase with RNA binding activity for AU-rich sequences. This RNA binding activity is required for Apobec-1 to mediate C-to-U RNA editing. Filter binding assays, using immobilized Apobec-1, demonstrate saturable binding to a 105-nt apoB RNA with a Kd of ~435 nM. A series of AU-rich templates was used to identify a high-affinity (~50 nM) binding site of consensus sequence UUUN[A/U]U, with multiple copies of this sequence constituting the high-affinity binding site. In order to determine whether this consensus site could be functionally demonstrated from within an apoB RNA, circular-permutation analysis was performed, revealing one major (UUUGAU) and one minor (UU) site located 3 and 16 nucleotides, respectively, downstream of the edited base. Secondary-structure predictions reveal a stem-loop flanking the edited base with Apobec-1 binding to the consensus site(s) at an open loop. A similar consensus (AUUUA) is present in the 3' untranslated regions of several mRNAs, including that of c-myc, that are known to undergo rapid degradation. In this context, it is presumed that the consensus motif acts as a destabilizing element. As an independent test of the ability of Apobec-1 to bind to this sequence, F442A cells were transfected with Apobec-1 and the half-life of c-myc mRNA was determined following actinomycin D treatment. These studies demonstrated an increase in the half-life of c-myc mRNA from 90 to 240 min in control versus Apobec-1-expressing cells. Apobec-1 expression mutants, in which RNA binding activity is eliminated, failed to alter c-myc mRNA turnover. Taken together, the data establish a consensus binding site for Apobec-1 embedded in proximity to the edited base in apoB RNA. Binding to this site in other target RNAs raises the possibility that Apobec-1 may be involved in other aspects of RNA metabolism, independent of its role as an apoB RNA-specific cytidine deaminase.

* Corresponding author. Mailing address: Division of Gastroenterology, Campus Box 8124, Washington University Medical School, 660 South Euclid Ave., St. Louis, MO 63110. Phone: (314) 362-2027. Fax: (314) 362-2033. E-mail: NOD{at}IM.WUSTL.EDU.

Molecular and Cellular Biology, March 2000, p. 1982-1992, Vol. 20, No. 6
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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