Formation of tRNA Wobble Inosine in Humans Is Disrupted by a Millennia-Old Mutation Causing Intellectual Disability.

Jillian Ramos,Felix Hagelskamp,Stefanie M Kellner,Lu Han,Dragony Fu,Eric M Phizicky,Fowzan S Alkuraya,Yan Li

doi:10.1128/mcb.00203-19

Abstract

The formation of inosine at the wobble position of eukaryotic tRNAs is an essential modification catalyzed by the ADAT2/ADAT3 complex. In humans, a valine-to-methionine mutation (V144M) in ADAT3 that originated ∼1,600 years ago is the most common cause of autosomal recessive intellectual disability (ID) in Arabia. While the mutation is predicted to affect protein structure, the molecular and cellular effects of the V144M mutation are unknown. Here, we show that cell lines derived from ID-affected individuals expressing only ADAT3-V144M exhibit decreased wobble inosine in certain tRNAs. Moreover, extracts from the same cell lines of ID-affected individuals display a severe reduction in tRNA deaminase activity. While ADAT3-V144M maintains interactions with ADAT2, the purified ADAT2/3-V144M complexes exhibit defects in activity. Notably, ADAT3-V144M exhibits an increased propensity to form aggregates associated with cytoplasmic chaperonins that can be suppressed by ADAT2 overexpression. These results identify a key role for ADAT2-dependent folding of ADAT3 in wobble inosine modification and indicate that proper formation of an active ADAT2/3 complex is crucial for proper neurodevelopment.

Highlights

The formation of inosine at the wobble position of eukaryotic tRNAs is an essential modification catalyzed by the ADAT2/ADAT3 complex
Using absolute quantification by liquid chromatography-mass spectrometry (LC-MS) with calibration standards, we found that the abundance of inosine was reduced by ϳ30% in the tRNAs of both V144M-lymphoblastoid cell lines (LCLs) compared to either wild-type LCLs (WT-LCLs) (Fig. 2B)
While the V144M mutation is a relatively minor change since valine and methionine represent amino acid residues with hydrophobic side chains, our studies uncover severe molecular defects associated with ADAT3-V144M that are likely to underlie the reduced wobble inosine levels detected in the tRNAs of intellectual disability (ID)-affected individuals with ADAT3-V144M mutations

Summary

Introduction

The formation of inosine at the wobble position of eukaryotic tRNAs is an essential modification catalyzed by the ADAT2/ADAT3 complex. ADAT3-V144M exhibits an increased propensity to form aggregates associated with cytoplasmic chaperonins that can be suppressed by ADAT2 overexpression These results identify a key role for ADAT2-dependent folding of ADAT3 in wobble inosine modification and indicate that proper formation of an active ADAT2/3 complex is crucial for proper neurodevelopment. It has been shown that highly translated genes in eukaryotic organisms, including humans, are correlated with an enrichment in wobble inosine tRNA-dependent codons, suggesting a critical role for tRNA inosine modification in maintaining proper levels of protein expression [4, 5]. The mechanistic cause of ADAT3-associated pathogenesis remains unclear

Methods

Results

Conclusion