| Description |
This dissertation reports three major findings. First, that an enzyme that converts adenosine to inosine within double-stranded RNA (dsRNA) does do by hydrolytic deamination. A double-stranded RNA substrate in which all adenosines were uniformly labeled with 13C was synthesized. A combined high-performance liquid chromatography-mass spectrometry (LC/MS) analysis of the nucleoside products from the modified, labeled substrate showed that no carbons were exchanged during the reaction. Including H2(18)O in the reaction showed that water served efficiently as the oxygen donor in vitro. These results indicate a hydrolytic deamination mechanism, and the enzyme is now referred to as double-stranded RNA adenosine deaminase (dsRAD or DRADA). Second, mapping the sites of adenosine to inosine conversion in short RNA duplexes showed that dsRNA had preferences for certain adenosines. dsRAD had a 5' nearest neighbor preference (A + U > C > G) and no 3' nearest neighbor preference. Also, adenosines close to the 3' termini of a duplex were disfavored for modification. Further, dsRAD had selectivity, that is, with appropriate substrates, dsRAD could modify a minimal number of preferred adenosines at reaction completion. These data were useful in identifying potential biological roles for dsRAD. Finally, it was demonstrated that dsRAD can edit hepatitis delta virus (HDV) antigenomic RNA. HDV is a subviral pathogen that has a circular RNA genome that is about 70% double-stranded and is replicated through a RNA intermediate, the antigenome. An adenosine in the antigenome is edited, converting a stop codon to a tryptophan codon, resulting in a longer form of the hepatitis delta antigen. Purified dsRAD was able to edit the HDV antigenomic RNA in vitro. This in vitro edition system, which consisted solely of purified components, parelled in vivo edition in the extent of editing and in the edition of HDV mutants. Previously, several RNAs were suggest as in vivo substrates of dsRAD. However, in most of these cases the evidence that dsRAD modifies the substrates in vivo was limited. The demonstration that in vitro editing of HDV antigenomic RNA by dsRAD was similar to in vivo edition provided strong evidence that dsRAD edits HDV RNA in vivo. |
