Intellectual disability-associated gene ftsj1 is responsible for 2'-O-methylation of specific tRNAs.

tRNA modifications at the anti-codon loop are critical for accurate decoding. FTSJ1 was hypothesized to be a human tRNA 2'-O-methyltransferase. tRNAPhe (GAA) from intellectual disability patients with mutations in ftsj1 lacks 2'-O-methylation at C32 and G34 (Cm32 and Gm34). However, the catalytic activity, RNA substrates, and pathogenic mechanism of FTSJ1 remain unknown, owing, in part, to the difficulty in reconstituting enzymatic activity in vitro. Here, we identify an interacting protein of FTSJ1, WDR6. For the first time, we reconstitute the 2'-O-methylation activity of the FTSJ1-WDR6 complex in vitro, which occurs at position 34 of specific tRNAs with m1 G37 as a prerequisite. We find that modifications at positions 32, 34, and 37 are interdependent and occur in a hierarchical order in vivo. We also show that the translation efficiency of the UUU codon, but not the UUC codon decoded by tRNAPhe (GAA), is reduced in ftsj1 knockout cells. Bioinformatics analysis reveals that almost 40% of the high TTT-biased genes are related to brain/nervous functions. Our data potentially enhance our understanding of the relationship between FTSJ1 and nervous system development.

Peripheral insertion modulates the editing activity of the isolated CP1 domain of leucyl-tRNA synthetase.

A large insertion domain called CP1 (connective peptide 1) present in class Ia aminoacyl-tRNA synthetases is responsible for post-transfer editing. LeuRS (leucyl-tRNA synthetase) from Aquifex aeolicus and Giardia lamblia possess unique 20 and 59 amino acid insertions respectively within the CP1 that are crucial for editing activity. Crystal structures of AaLeuRS-CP1 [2.4 Å (1 Å=0.1 nm)], GlLeuRS-CP1 (2.6 Å) and the insertion deletion mutant AaLeuRS-CP1Δ20 (2.5 Å) were solved to understand the role of these insertions in editing. Both insertions are folded as peripheral motifs located on the opposite side of the proteins from the active-site entrance in the CP1 domain. Docking modelling and site-directed mutagenesis showed that the insertions do not interact with the substrates. Results of molecular dynamics simulations show that the intact CP1 is more dynamic than its mutant devoid of the insertion motif. Taken together, the data show that a peripheral insertion without a substrate-binding site or major structural role in the active site may modulate catalytic function of a protein, probably from protein dynamics regulation in two respective LeuRS CP1s. Further results from proline and glycine mutational analyses intended to reduce or increase protein flexibility are consistent with this hypothesis.