Apolipoprotein B (apoB) mRNA is edited in rat liver and intestine to convert a CAA glutamine codon to a UAA translational stop codon by the direct conversion of cytidine to uridine at nucleotide 6666. We have proposed the 'mooring sequence' model for apoB RNA editing, in which editing complexes (editosomes) assemble on specific apoB mRNA flanking sequences to direct this site-specific editing event. One sequence element (approx. nts 6671-81, the presumed 'mooring sequence') has been previously identified as necessary for editing. We have identified two additional sequence elements which are necessary for efficient editing: (1) a 5' 'Regulator' region which modulates editing efficiency and (2) a 'Spacer' region between the editing site and the 3' mooring sequence, whose distance is critical for efficient editing. Utilizing this data, we have induced editing at a cryptic site and have defined a 22 nucleotide 'cassette' of specific apoB sequence which is sufficient to support wild-type levels of editing in vitro in a background of distal apoB RNA sequence.
Apolipoprotein B (apoB) mRNA is edited in rat liver and intestine through the direct conversion of cytidine to uridine at nucleotide 6666. Recently, we have proposed the 'Mooring Sequence' model, in which editing complexes (editosomes) assemble on specific apoB mRNA flanking sequences to direct this site-specific editing event. To test this model, apoB mRNA deletion and translocation mutants were constructed and analyzed. Specific sequences 3' of the editing site were absolutely required for editing, while specific sequences and bulk RNA 5' of the editing site were required for efficient editing. Translocation of apoB 3' flanking sequences induced editing of an upstream cytidine, demonstrating that 3' sequences are necessary and sufficient to direct editing in vitro. 3' flanking sequences were also shown to be necessary and sufficient for editosome complex assembly. These data provide strong support for a 'Mooring Sequence' model in which 3' apoB flanking sequences direct editosome assembly and subsequent editing in vitro, while 5' flanking sequences enhance these functions.
Specific apolipoprotein B (apoB) mRNA editing can be performed in vitro on apoB RNA substrates. Native gels and glycerol gradient sedimentation have been used to determine the physical properties of the in vitro editing activity in rat liver cytosolic S100 extracts. ApoB RNA substrates were progressively assembled as 27S complexes for 3 hr with similar kinetics as seen for the accumulation of edited RNA. Assembly was not observed on RNAs from apoB deletion constructs that did not support editing. The 27S complex contained both edited and unedited RNA sequences. Inhibition of 27S complex assembly by vanadyl-ribonucleoside complexes was accompanied by inhibition of editing. Based on these data, we propose that the 27S complex is the in vitro "editosome." A "mooring sequence" model for RNA recognition and editosome assembly has been proposed involving RNA sequences flanking the edited nucleotide.
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