Mtu1-Mediated Thiouridine Formation of Mitochondrial tRNAs Is Required for Mitochondrial Translation and Is Involved in Reversible Infantile Liver Injury

<div><p>Reversible infantile liver failure (RILF) is a unique heritable liver disease characterized by acute liver failure followed by spontaneous recovery at an early stage of life. Genetic mutations in <i>MTU1</i> have been identified in RILF patients. MTU1 is a mitochondrial enzyme that catalyzes the 2-thiolation of 5-taurinomethyl-2-thiouridine (τm⁵s²U) found in the anticodon of a subset of mitochondrial tRNAs (mt-tRNAs). Although the genetic basis of RILF is clear, the molecular mechanism that drives the pathogenesis remains elusive. We here generated liver-specific knockout of Mtu1 (Mtu1^LKO) mice, which exhibited symptoms of liver injury characterized by hepatic inflammation and elevated levels of plasma lactate and AST. Mechanistically, Mtu1 deficiency resulted in a loss of 2-thiolation in mt-tRNAs, which led to a marked impairment of mitochondrial translation. Consequently, Mtu1^LKO mice exhibited severe disruption of mitochondrial membrane integrity and a broad decrease in respiratory complex activities in the hepatocytes. Interestingly, mitochondrial dysfunction induced signaling pathways related to mitochondrial proliferation and the suppression of oxidative stress. The present study demonstrates that Mtu1-dependent 2-thiolation of mt-tRNA is indispensable for mitochondrial translation and that Mtu1 deficiency is a primary cause of RILF. In addition, Mtu1 deficiency is associated with multiple cytoprotective pathways that might prevent catastrophic liver failure and assist in the recovery from liver injury.</p></div

Serum biochemical data of Mtu1^Flox and Mtu1^LKO mice.

<p>Serum biochemical data of Mtu1^Flox and Mtu1^LKO mice.</p

Mtu1 deficiency impaired mitochondrial translation and respiratory activities in hepatocytes.

<p>(A) Autoradiogram of mitochondrial translation in primary hepatocytes isolated from control (Flox) and hepatocyte-specific Mtu1 knockout (LKO) mice. (B) Protein levels of representative mitochondrial proteins were examined by western blotting. CI~CV indicates Complexes I ~ V. (C) Representative blue-native gel image of respiratory complexes purified from Flox and LKO mice. (D) Mitochondrial proteins incorporated in Complexes I ~ V were examined by blue-native PAGE followed by western blotting. (E) The activities of Complexes I ~ IV in the livers of Flox and LKO mice are shown. Individual complex activity was normalized to the activity of citrate synthase (CS). n = 3 each. **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Mtu1 is essential for embryonic development.

<p>(A) Breeding strategy to generate constitutive Mtu1 knockout mice. ATG indicates the start codon. (B) Total number of wild-type (Mtu1^+/+), heterozygous (Mtu1^+/-) and homozygous (Mtu1^-/-) animals obtained from parental Mtu1^+/- mice. (C) Morphology of Mtu1^+/+, Mtu1^+/- and Mtu1^-/- embryos at stage E9. Bar = 5 mm. Note that the development of Mtu1^-/- embryos was severely delayed. (D) Genotyping analysis of embryos. DNA fragments corresponding to the wild-type (WT) allele and deleted (KO) allele are shown. Asterisks indicate the genotypes of the embryos shown in (C). (E) Blood vessel networks in embryos shown in (C) were examined by immunostaining against PECAM-1.</p

Loss of s² modification in Mtu1-deficient liver.

<p>(A-C) Representative mass chromatograms of mt-tRNA^Gln (A), mt-tRNA^Glu (B) and mt-tRNA^Lys (C) purified from the livers of control (Flox) and knockout (LKO) mice. Arrowheads indicate traces of fragments containing s² modification in LKO mice. Note that the levels of s²-containing mt-tRNAGln, mt-tRNAGlu and mt-tRNALys were almost absent in LKO mice. n.d.: not detected. n.d.: not detected.</p

Aberrant mitochondrial morphology in Mtu1-deficient hepatocytes.

<p>(A) Representative electron microscopy images of hepatocytes in control (Flox) and hepatocyte-specific Mtu1 knockout (LKO) mice. Bar = 5 μm. (B) The mitochondrial area in Flox and LKO hepatocytes was examined by histogram and scatter plot (Insert). n = 300 and 330 for Flox and LKO, respectively. ***<i>P</i> < 0.001. (C) Representative images of hepatic mitochondria at high magnification. Bar = 1 μm. Boxes with dashed and solid lines indicate aberrant structures magnified in lower panels. (D) Examination of the amounts of proteins related to mitochondrial proteostasis by western blotting. Total mitochondrial proteins were stained and used as a loading control. Arrow indicates bands corresponding to AFG3L2.</p

Hepatocyte-specific Mtu1 knockout mice exhibited liver injury and altered metabolism.

<p>(A) Relative expression levels of <i>Mtu1</i> in liver tissues of control (Flox) and Mtu1^LKO (LKO) mice. n = 3 each. ****<i>P</i><0.0001. (B) The body weight of male mice at 3 weeks of age. n = 3 each. (C-E) Plasma lactate (C), aspartate aminotransferase (AST) (D) and alanine aminotransferase (ALT) (E) of Mtu1^LKO and control male mice at 3 weeks of age. n = 6 each. *<i>P</i> < 0.05, ****<i>P</i> < 0.0001. (F) Expression levels of selected metabolism-related genes in the livers of Mtu1^LKO and control male mice at 3 weeks of age were examined by quantitative PCR (n = 4 each). The relative expression levels are shown as a heat map. *<i>P</i> <0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001, ****<i>P</i> < 0.0001.</p

Enhancement of mitochondrial biogenesis and oxidative stress response in Mtu1-deficient hepatocytes.

<p>(A) Relative expression of <i>Pgc1α</i> and <i>Fgf21</i> in the livers of control (Flox) and knockout (LKO) mice. n = 4 each. **<i>P</i> < 0.01. (B) The ratios of mitochondrial DNA (mtDNA) levels to nuclear DNA (nDNA) levels in the livers of Flox and LKO mice are shown. n = 4 each. ***<i>P</i> < 0.001. (C) Relative expression of mitochondrial DNA-encoded mitochondrial genes in the livers of Flox and LKO mice. n = 4 each. *<i>P</i> < 0.05, **<i>P</i> < 0.01. (D) Western blotting revealing an increase in MTOR, phospho-MTOR (p-MTOR), ERK1/2 and phospho-ERK1/2 levels in primary hepatocytes. (E) Examination of protein carbonylation by Oxyblot in mitochondria isolated from Flox and LKO livers. Total mitochondrial proteins were stained with CBB and used as a loading control. Asterisks indicate negative control samples. (F) Expression levels of genes related to the oxidative stress response were examined by quantitative PCR (n = 4 each). The relative expression levels are shown in a heat map. *<i>P</i> <0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p