Sequence requirements for the editing of apolipoprotein B mRNA

Apolipoprotein (apo) B48 is produced in the mammalian intestine by a tissue-specific RNA-editing mechanism, which mediates a C to U conversion at position 6666 in apoB mRNA. This generates an inframe translation stop codon (UAA) in place of glutamine (CAA) at position 2153. To establish the sequences required for editing we have used an in vitro conversion assay to monitor the editing of synthetic RNAs by rat intestinal extracts. Transcripts containing 55 nucleotides (positions 6649-6703) or more of human apoB mRNA sequence were edited in vitro. Transcripts containing 42 nucleotides (positions 6648-6689) and 26 nucleotides (positions 6662-6687) were edited at 62 and 24% efficiency, respectively, of the 55-nucleotide sequence. To delineate the precise sequence requirements for editing, mutants were generated where 6-nucleotide sections of the 55-base region were changed to anti-sense sequence. Mutation of the 12-nucleotide region immediately downstream of C-6666 abolished editing, and mutation of 6-base sequences immediately 3' and 5' of this 12-nucleotide region significantly reduced editing. Having identified the key region of interest, a panel of 46 mutant RNAs carrying single base substitutions or deletions between nucleotide positions 6657 and 6685 was constructed. Mutagenesis in the sequence 5'-TGATCAGTATA-3' (positions 6671-6681) downstream of C-6666 had the most dramatic effect, since almost all mutations abolished or greatly reduced conversion in vitro. These results suggest that editing is a highly sequence-specific process. We propose that this downstream region is a recognition and/or binding site for the editing enzyme. A search for this sequence in other genes may help to reveal other RNAs that undergo editing.