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.
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 K(d) of approximately 435 nM. A series of AU-rich templates was used to identify a high-affinity ( approximately 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.
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Department of Internal Medicine, Washington University Medical School, St. Louis, Missouri 63110, USA.
Davidson
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D020413:3' Untranslated Regions; D000071479:APOBEC-1 Deaminase; D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003564:Cytidine Deaminase; D008969:Molecular Sequence Data; D011485:Protein Binding; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics* / Q000378:metabolism; D017393:RNA Editing; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment
D020413:3' Untranslated Regions; D001055:Apolipoproteins B; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D000071479:APOBEC-1 Deaminase; D003564:Cytidine Deaminase