Phylogenetic analysis of the apolipoprotein B mRNA-editing region - Evidence for a secondary structure between the mooring sequence and the 3 ' efficiency element

Apolipoprotein (apo) B mRNA editing is the deamination of C-6666 to uridine , which changes the codon at position 2153 from a genomically encoded gluta mine (CAA) to an in-frame stop codon (UAA), The apoB mRNA-editing enzyme co mplex recognizes the editing region of the apoB pre-mRNA with exquisite pre cision. Four sequence elements spanning 139 nucleotides (nt) on the apoB mR NA have been identified that specify this precision, In cooperation with th e indispensable mooring sequence and spacer element, a 5' efficiency elemen t and a 3' efficiency element enhance editing in vitro, A phylogenetic comp arison of 32 species showed minor differences in the apoB mRNA sequence, an d the apoB mRNA from 31 species was robustly edited in vitro. However, guin ea pig mRNA was poorly edited. Compared with the consensus sequences of the se 31 species, guinea pig apoB mRNA has three variations in the 3' efficien cy element, and the conversion of these to the consensus sequence increased editing to the levels in the other species. From this information, a model for the secondary structure was formulated in which the mooring sequence a nd the 3' efficiency element form a double-stranded stem. Thirty-one mammal ian apoB mRNA sequences are predicted to form this stem positioning C-6666 two nucleotides upstream of the stem, However, the guinea pig apoB mRNA has a mutation in the 3' efficiency element (C-6743 to U) that predicts an ext ension of the stem and hence the lower editing efficiency. A test of this m odel demonstrated that a single substitution at 6743 (U to C) in the guinea pig apoB mRNA, that should reduce the stem, enhanced editing, and mutation s in the 3' efficiency element that extended the stem for three base pairs dramatically reduced editing. Furthermore, the addition of a 20-nucleotide 3' efficiency element RNA to a Fis-nucleotide guinea pig apoB mRNA lacking the 3' efficiency element more than doubled the in vitro editing activity. Based on these results, a model is proposed in which the mooring sequence a nd the 3' efficiency element form a double-stranded stem, thus suggesting a mechanism of how the 3' efficiency element enhances editing.