The long term goal is to understand RNA editing as a posttranscriptional mechanism for regulating gene expression.
The aim of this proposal is to investigate the editing of apolipoprotein-B (apo-B) mRNA at the molecular level. A novel mechanism modifies apo-B mRNA in the mammalian intestine by converting cytosine to uracil which produces two proteins with different functions in lipid metabolism. The proposed research will identify with mRNA sequences and cellular factors involved in the editing of apo-B mRNA using an in vitro system previously developed for this reaction. To define the sequence requirements for editing, the nucleotides in apo-B mRNA which interact with the editing protein in vitro will be identified by hydroxy radical footprinting. Synthetic apo-b mRNAs containing mutations at these sites will be assayed for their ability to bind protein and to undergo editing in vitro. The editing protein will be purified by column chromatography from extracts of baboon intestinal cells which have high levels of activity. A partial peptide sequence will be obtained from the purified protein. To isolate cDNA clones for the editing protein, a radiolabeled apo-B mRNA will be used to ligand-screen a cDNA expression library to identify clones which encode binding activity. Alternatively, an oligonucleotide containing a sequence predicted from the peptide sequence will be used to enrich for the editing protein cDNA in a polymerase chain reaction. To confirm the identity of the clone, synthetic RNA transcribed from a full-length cDNA will be translated in vitro using reticulocyte lysate and the translation products will be tested for editing activity. The structure and function of the protein will be analyzed by deletion analysis of the cloned cDNA. The truncated proteins will be synthesized in vitro and assayed for protein binding and editing activity. Other genes which may be edited by this mechanism will be identified on the basis of their homology to apo-B mRNA. It is anticipated that these studies will provide the basis for understanding RNA editing at the molecular level.
