| Hepatitis delta virus (HDV) is edited at a single site in its 1.7kb RNA genome, the amber/W site. The level at which this site is edited determines the relative amounts at which two functionally opposing proteins, HDAg-S and HDAg-L, are synthesized. Therefore, in order for HDV to control protein levels, it has to control editing levels. The method by which HDV genotype III (HDV-3) regulates editing was not previously known. Here, using in vitro analyses of the RNA in the vicinity of the HDV editing site, I show that HDV-3 RNA folds into two secondary structures following transcription: a metastable branched double hairpin structure required for editing, and the unbranched rod structure characteristic of HDV that is required for replication. The fraction of RNA that folds into these two structures, combined with how efficiently the amber/W site is edited, determines overall editing levels in cells. This is the first study to show that the amount of RNA folding into a specific secondary structure regulates the functional outcome of that RNA. In another novel finding, I show that the RNA editing enzyme, ADAR 1, binds to a stem loop structure on the HDV-3 branched RNA substrate at least 30 base pairs downstream of the target adenosine. That ADAR 1 binds to a structure far removed from the editing site demonstrates a new discovery in the field of RNA editing, as binding of the ADAR protein to its RNA substrate was thought to occur in the base paired region in the immediate vicinity of the editing site. |
