A deafness-associated tRNA mutation caused pleiotropic effects on the m1G37 modification, processing, stability and aminoacylation of tRNAIle and mitochondrial translation

Abstract

Defects in the posttranscriptional modifications of mitochondrial tRNAs have been linked to human diseases, but their pathophysiology remains elusive. In this report, we investigated the molecular mechanism underlying a deafness-associated tRNAIle 4295A>G mutation affecting a highly conserved adenosine at position 37, 3′ adjacent to the tRNA’s anticodon. Primer extension and methylation activity assays revealed that the m.4295A>G mutation introduced a tRNA methyltransferase 5 (TRMT5)-catalyzed m1G37 modification of tRNAIle. Molecular dynamics simulations suggested that the m.4295A>G mutation affected tRNAIle structure and function, supported by increased melting temperature, conformational changes and instability of mutated tRNA. An in vitro processing experiment revealed that the m.4295A>G mutation reduced the 5′ end processing efficiency of tRNAIle precursors, catalyzed by RNase P. We demonstrated that cybrid cell lines carrying the m.4295A>G mutation exhibited significant alterations in aminoacylation and steady-state levels of tRNAIle. The aberrant tRNA metabolism resulted in the impairment of mitochondrial translation, respiratory deficiency, decreasing membrane potentials and ATP production, increasing production of reactive oxygen species and promoting autophagy. These demonstrated the pleiotropic effects of m.4295A>G mutation on tRNAIle and mitochondrial functions. Our findings highlighted the essential role of deficient posttranscriptional modifications in the structure and function of tRNA and their pathogenic consequence of deafness.

Novel m.4268T>C mutation in the mitochondrial tRNAIle gene is associated with hearing loss in two Chinese families

“…TRIzol was used to isolate total mitochondrial RNA from knockdown or control cell lines, as previously described[ 20 ]. Briefly, 2 μg of total mitochondrial RNA was electrophoresed through a 10% polyacrylamide/7 M urea gel in Tris–borate–Ethylenediaminetetraacetic acid (EDTA) buffer (after heating the sample at 65 °C for 10 min), and then electroblotted onto a positively charged nylon membrane for the hybridization analysis with oligodeoxynucleotide probes.…”

tRNA Metabolism and Lung Cancer: Beyond Translation

“…Normally, tRNAs are considered to be housekeeping molecules with no additional functions. However, more and more evidence shows that tRNAs are related to various physiological and pathological processes ( Lord et al, 2017 ; Liu et al, 2020 ; Zhu et al, 2020 ; Meng et al, 2021 ). As mentioned above, tRNA-associated metabolism plays a vital role in the development of lung cancer ( Table 1 ).…”

Autophagy: A Novel Horizon for Hair Cell Protection

“…Recently, the molecular mechanism of deafness-related tRNAIle4295A>G mutations was deeply studied. It was found that abnormal tRNA metabolism may lead to mitochondrial translation disorders, respiratory deficiency, increased level of reactive oxygen species, and autophagy promotion [ 127 ]. However, whether the upregulation of autophagy in HCs is protective or not was not demonstrated in this study.…”