Detection of tRNA-specific adenosine deaminase activity and wobble inosine modification in human cell lysates

The wobble inosine modification plays a central role in translation by enabling a single tRNA to decode multiple synonymous codons. In eukaryotes, the formation of wobble inosine is catalyzed by a heterodimeric adenosine deaminase complex comprised of the ADAT2 and ADAT3 subunits. Notably, pathogenic variants in the ADAT3 subunit have been identified as the cause of autosomal recessive intellectual disability in the human population by impacting wobble inosine levels. Here, we describe approaches for monitoring adenosine deaminase activity and inosine modification status at the wobble position of cellular tRNAs. To detect adenosine deaminase activity, we provide protocols for preparing extracts from human cells followed by enzymatic assays with in vitro transcribed tRNA substrates. Furthermore, we describe a method to monitor wobble inosine status of individual tRNAs using cDNA sequencing. These assays can be used to decipher the molecular basis for neurodevelopmental disorders linked to wobble inosine deficiency and disease-associated ADAT2/3 variants.