TY - JOUR
T1 - 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
T2 - Binding of Apobec-1 to this motif in the 3' untranslated region of c-myc increases mRNA stability
AU - Anant, Shrikant
AU - Davidson, Nicholas O.
PY - 2000/3
Y1 - 2000/3
N2 - 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 ~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.
AB - 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 ~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.
UR - http://www.scopus.com/inward/record.url?scp=0033998026&partnerID=8YFLogxK
U2 - 10.1128/MCB.20.6.1982-1992.2000
DO - 10.1128/MCB.20.6.1982-1992.2000
M3 - Article
C2 - 10688645
AN - SCOPUS:0033998026
SN - 0270-7306
VL - 20
SP - 1982
EP - 1992
JO - Molecular and cellular biology
JF - Molecular and cellular biology
IS - 6
ER -