Download or read book Analysis of the Structural and Sequential Preferences of Human ADAR2 A-to-I Editing written by Tristan Thomas Eifler and published by . This book was released on 2013 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: ADARs (adenosine deaminases acting on RNA) are a family of RNA editing enzymes that deaminate adenosines (A) in double-stranded RNA (dsRNA), generating inosine, which is read by the cellular machinery as guanosine. Consequently, ADAR editing of messenger RNA can result in amino acid substitutions, thereby increasing the diversity of an organism's proteome. It is still not entirely clear why particular adenosines in dsRNA molecules are selected for ADAR editing. Furthermore, very little is known regarding how ADAR catalytic domains interact with substrate dsRNA during deamination. This dissertation describes experiments that probe the structural and sequential determinants of ADAR2 editing in dsRNA to reach a better understanding of ADAR2 selectivity.We have observed that overexpressing human ADAR2 (hADAR2) in the budding yeast Saccharomyces cerevisiae reduces cell growth to a degree proportional to the deaminase activity of the enzyme. We hypothesized that this is a product of hADAR2 editing endogenous yeast RNA. As described in Chapter 2, we employed next-generation sequencing to sequence the transcriptomes of S. cerevisiae expressing catalytically active and inactive hADAR2 and identified 19 candidate hADAR2 editing sites. Aligning the edited regions revealed both canonical and novel preferred flanking sequences for hADAR2 editing. Specifically, we observed a preference for A two nucleotides downstream of the edited A in addition to previously noted preferences for U and G 5' and 3' of the editing site, respectively. Follow-up analysis of the predicted hADAR2 editing sites in yeast was necessary to confirm editing and test the novel preferred nucleotide neighbor observed in the sequence alignment. We Sanger sequenced reverse-transcription polymerase chain reaction (RT-PCR) products of yeast total RNA and performed in vitro deamination assays on RNA substrates. We confirmed editing at 8 of the 19 candidate sites and determined that mutating the A 2 nucleotides downstream of the edited A in the yeast hADAR2 substrate BDF2 decreases editing efficacy at that site both in vivo and in vitro. This work is described in Chapter 3. Not only did we discover novel hADAR2 substrates but a novel preferred neighbor for hADAR2 editing. This expands understanding of hADAR2 selectivity and provides us with new editing substrates for future study.It has been understood that ADAR binding with dsRNA is mediated by its double-stranded RNA binding domains (dsRBDs). However, as described in Chapter 4, we determined the yeast hADAR2 substrate BDF2 is rapidly edited by the isolated ADAR2 deaminse domain. We also examined an established human hADAR2 substrate with structural and sequential similarities to BDF2, GLI1, and found it too was rapidly edited independently of the hADAR2 dsRBDs. Not only does this have implications in how hADAR2 recognizes, binds, and edits RNA but suggests the hADAR2 deaminase domain could used to form a covalently-linked heterocomplex with substrate RNA suitable for X-ray crystallography. Finally, Chapter 5 describes our attempts to incorporate the unnatural amino acid p-azidophenylalanine into a hADAR2 truncation using an orthogonal tRNA/tRNA synthetase pair for site-specific ligation of probe molecules to the protein and the generation of a hADAR2 quadruple cysteine mutant. We determined that the hADAR2 quadruple cysteine mutant retains deaminase activity and may therefore be useful in linking the protein with probe molecules via disulfide bonds for structural studies.