Selective ADAR editing and the coordination with splicing
Abstract: Maturation of precursor messenger RNAs (pre-mRNA) in eukaryotes includes processes like capping, 3’ end formation and splicing. Some premRNAs undergo an additional process called RNA editing where a single nucleotide is modified to generate a new nucleotide identity. One such RNA editing event is the hydrolytic deamination of adenosine (A) that results in inosine (I). Because of its base pairing properties, inosine is recognized as guanosine (G) by cellular machineries, like the spliceosome or the ribosome. The enzymes catalyzing A to I editing are the ADARs (Adenosine deaminases that act on RNA). In vivo, these enzymes selectively locate and deaminate individual adenosines within a few substrates, all with long double stranded helixes interrupted by bulges and internal loops. It is not yet fully understood how ADARs discriminate between sites for selective editing and other adenosines in a double stranded context. Edited sites are often located near exon/intron borders where editing depends on intron sequences to form the double stranded structure required for ADAR recognition. Thus editing has to occur prior to splicing. In the work described in this thesis we have investigated the mechanism of selective editing and the possible coordination of editing and splicing.We have found that ADAR1 and ADAR2 display different editing specificities for a natural substrate, in vitro. ADAR2, but not ADAR1 perform site selective editing, similar to what occurs in vivo. Further, we have found that the selective editing is not determined by mismatches in the vicinity of the edited site. It is rather the immediate structure surrounding the adenosine that affects editing selectivity by ADAR2 in vitro.The coordination of editing and splicing was investigated in vivo. We found that the C-terminal domain (CTD) of the RNA polymerase II is required for efficient ADAR2 editing during ongoing transcription, while splicing of substrates for editing is independent of the CTD.
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