INDUCTION OF RNA EDITING AT HETEROLOGOUS SITES BY SEQUENCES IN APOLIPOPROTEIN-B MESSENGER-RNA

An RNA editing mechanism modifies apolipoprotein B (apo-B) mRNA in the intestine by converting cytosine at nucleotide (nt) 6666 to uracil. T o define the sequence requirements for editing, mutant apo-B RNAs were analyzed for the ability to be edited in vitro by enterocyte extracts . Editing was detected by a sensitive and linear primer extension assa y. An upstream region (nt 6648 to 6661) which affected the efficiency of editing was identified. RNAs with mutations in this efficiency sequ ence were edited at 22 to 160% of wild-type levels. Point mutations in a downstream 11-nt mooring sequence (nt 6671 to 6681) abolished editi ng, confirming previous studies (R. R. Shah, T. J. Knott, J. E. Legros , N. Navaratnam, J. C. Greeve, and J. Scott, J. Biol. Chem. 266:16301- 16304, 1991). The optimal distance between the editing site and the mo oring sequence is 5 nt, but a C positioned 8 nt upstream is edited eve n when nt 6666 contains U. The efficiency and mooring sequences were i nserted individually and together adjacent to a heterologous C in apo- B mRNA. The mooring sequence alone induced editing of the C at nt 6597 both in vitro and in transfected rat hepatoma cells. Editing at nt 65 97 was specific, was independent of editing at nt 6666, and was stimul ated to wild-type levels when the efficiency sequence was also inserte d. Introduction of the mooring sequence into a heterologous mRNA, luci ferase mRNA, induced editing of an upstream cytidine. Although UV cros s-linking studies have previously shown that proteins of 60 to 66 kDa cross-link to apo-B mRNA, these proteins did not cross-link to the luc iferase translocation mutants.