Direct RNA sequencing of firefly luciferase transcripts containing inosine

Inosine (I) is a regulated base modification occurring as a result of A-to-I editing of certain mRNA and tRNA species through the activity of Adenosine Deaminase RNA Specific (ADAR) and Adenosine Deaminase tRNA Specific (ADAT) enzymes. As a nucleotide, inosine monophosphate (IMP) serves as a precursor during de novo synthesis of adenosine monophosphate (AMP) and guanosine monophosphate (GMP) but has the potential to be phosphorylated into inosine triphosphate (ITP), which can be aberrantly incorporated into RNA. Inosine triphosphate pyrophosphatase (ITPase, coded for by the ITPA gene), catalyzes the hydrolysis of ITP and dITP to prevent accumulation and misincorporation of inosine into RNA and DNA. Individuals with ITPA null mutations harbor inosine misincorporation within RNA of their cells and develop a fatal multisystem disorder with encephalopathy and infantile dilated cardiomyopathy. It is unclear, however, how inosine misincorporation in RNA is related to the symptoms of ITPase deficiency. The study is focused on investigating the misincorporation of inosine in a tractable in vitro transcription system.