br Occurrence of Inosine in RNA Inosine is widespread

Occurrence of Inosine in RNA Inosine is widespread among various types of RNAs including transfer RNA (tRNA), ribosomal RNA (rRNA), messenger RNA (mRNA), long noncoding RNA (lncRNA), and microRNA (miRNA). Within these RNAs, inosine can appear in different locations as well. Table 1 lists examples of the occurrence of inosine in various human RNAs. The functional consequence of the inosine modification depends on both the type of RNA and nucleotide position modified. In this section, we discuss inosine occurrence in different RNAs and methods for detection of inosine at specific locations in an RNA.
Adenosine Deaminases That Act on RNA (ADARs) As can be appreciated from the discussion earlier, inosine is widespread in the human transcriptome. The vast majority of inosine sites in human RNAs are products of Adenosine Deaminases that act on RNA (ADARs) [88]. These enzymes catalyze the hydrolytic deamination of adenosine and require duplex structure in the substrate for reaction. The conversion of A-to-I in an RNA duplex changes an A–U ketorolac toradol pair to the less stable I•U mismatch. Indeed, ADARs were first discovered as RNA duplex unwinding enzymes [72,89,90]. In humans, two active ADARs are known: ADAR1 and ADAR2 (ADARB1). A third member of the ADAR family (ADAR3/ADARB2) is expressed but not catalytically active [91]. The ADAR1 gene is located on chromosome 1 in the human genome. There are two abundant isoforms of ADAR1. ADAR1 p150 is induced by interferon, and ADAR1 p110 is constitutively expressed [92]. An upstream interferon inducible promoter allows the generation of the p150 isoform and p110 is generated from multiple promoters [93]. Alternative splicing results in the generation of additional ADAR1 isoforms [94]. ADAR2 is located on human chromosome 21. There is one abundant form of ADAR2 expressed in human. However, alternative splicing can create multiple ADAR2 spliced isoforms. Several ADAR2 splice variants have been identified with altered enzymatic activity [20,95]. The ADAR3 gene is located on chromosome 10 and may have originated from an ADAR2 gene duplication, given the fact that ADAR2 and ADAR3 are similar in sequence and domain organization [91,96]. Even though ADAR3 is catalytically inactive, it has the capability to bind both dsRNA and ssRNA [91]. It has been proposed that ADAR3 could modulate ADAR editing by binding to potential substrates without editing them [91].
ADAR Substrate Recognition ADARs have multiple domains capable of binding RNA substrates. ADAR-reactive adenosines are located in duplex structures consistent with the presence of dsRBDs in the ADAR proteins. However, the ADAR deaminase domains also require duplex structure [150]. No high-resolution structures of full-length ADARs bound to RNA have been reported to date. However, there is a wealth of knowledge in the literature on dsRBD–RNA interactions, including NMR structures of human ADAR2 dsRBDs bound to RNA [107]. In addition, our laboratory recently reported crystal structures of the human ADAR2 deaminase domain bound to RNA providing a detailed picture of ADAR interactions near an editing site [121]. In this section, we describe our current understanding of ADAR-substrate recognition with a particular focus on the reported high-resolution structures of dsRBD–dsRNA and deaminase domain–dsRNA complexes.
Catalytic Mechanism ADARs catalyze a hydrolytic deamination reaction on dsRNA substrates similar to that catalyzed by ADA and CDA on nucleoside substrates [164,165]. In analogy to the mechanisms of ADA and CDA, a mechanism for ADAR2 has been proposed (Fig. 6) [4,166]. The zinc ion is chelated by H394, C451, and C516 and is also bound to a water molecule. Once the substrate adenosine is flipped into the ADAR2 active site, E396 (as glutamate) deprotonates the water molecule bound to zinc. The resulting hydroxide then attacks the purine at C6 with protonation at N1 via the acid form of E396. These steps form a high energy intermediate with a tetrahedral center at C6. Next, with the participation of E396, a proton is transferred from the newly formed C6 hydroxyl to the adenosine amino group residing on the same carbon. Once protonated, the adenosine amino group can leave as ammonia (NH3) and at the same time the product inosine is formed.