Molecular mechanisms underlying selective degradation of mitochondrial tRNA leading to HUPRA syndrome

On November 9, the international academic journal Nucleic Acids Research published online the latest collaborative research results of the Zhou Xiaolong Research Group of the Center for Excellence in Molecular and Cell Sciences (Institute of Biochemistry and Cell Biology) of the Chinese Academy of Sciences and the Wang Jian Research Group of Shanghai Children's Medical Center, "Selective degradation of tRNA^Ser" (AGY) is the primary driver for mitochondrial seryl-tRNA synthetase-related disease”
。

Mammalian cells have two sets of protein synthesis systems, namely cytoplasmic and mitochondrial protein synthesis systems
.
The human mitochondrial genome contains 37 genes, including 2 rRNAs (12S and 16S), 22 tRNAs, and 13 protein-coding genes
.
All nucleic acid components (mRNA, rRNA, tRNA) of the mitochondrial translation machine are encoded by the mitochondrial genome, and all protein components are encoded by nuclear genes, synthesized in the cytoplasm, and transported to the mitochondria by mitochondrial localization signal peptides to play a function
.
Mitochondrial protein synthesis produces only 13 proteins encoded by the mitochondrial genome, which are all core subunits of mitochondrial oxidative phosphorylation complexes I, III, IV, V, and all of which are transmembrane proteins, which play a crucial role
in the assembly and function of oxidative phosphorylation complexes.
The rate and fidelity of mitochondrial protein synthesis directly control the accuracy of mitochondrial genetic information transmission, the correct assembly and function of oxidative respiratory chain complexes, and then control mitochondrial metabolism and major cellular life activities
.

Mitochondrial aminoacyl-tRNA synthetase (mt-aaRS) is a key protein synthesis factor that provides raw materials
for protein synthesis within mitochondria by catalyzing aminoacylation of mitochondrial tRNA.
mt-aaRS gene mutations can lead to autosomal recessive diseases, and its clinical phenotype is most significant in the central nervous system, and can also involve muscles, hearts and other tissues and organs
.
Such diseases are often difficult to diagnose clinically, the pathogenesis is unknown, and the treatment methods are extremely limited, which urgently needs in-depth research
.

Mitochondrial seryl-tRNA synthase (SARS2) catalyzes the aminoacylation of two mitochondrial tRNAs Ser [hmtRNA Ser (AGY) & hmtRNA^Ser (UCN)], in particular, hmtRNA^Ser (AGY) due to lack of D-stem/loop structure, It is the only tRNA
in human cells that does not have an inverted L-type tertiary structure.
At present, cases of this gene mutation are very rare, and the phenotypes of patients reported are mainly divided into two categories, one is fatal HUPRA syndrome, characterized by hyperuricemia, pulmonary hypertension, renal failure and alkalosis; The other type is mainly manifested as progressive spastic quadriparesis
.

This study reported a patient with SARS2 gene mutation, which mainly showed pulmonary hypertension, gross motor development delay, recurrent seizures, brain atrophy, etc.
, and the patient's phenotype and the known SARS2 gene mutation caused by the disease overlapped and different
.
Whole exome sequencing analysis showed that the SARS2 gene had compound heterozygous mutations, including non-classical splicing site mutations (c.
654-14T>A) and frameshift mutations (c.
1519dupC), which were newly discovered mutations
。 c.
654-14T>A leads to the retention of 12 bases in the intron region, which in turn leads to the insertion of 4 amino acids in the SARS2 active center (the mutant is named Ins12), while the c.
1519dupC mutation leads to deletion and abnormal extension of the SARS2 C-terminus, replacing the key C-terminal tRNA binding domain (the mutant is named dupC).

In this study (1), the effects of SARS2 mutant on SARS2 amino acid activation, aminoacylation and tRNA binding ability were studied in detail by biochemical methods, and it was found that Ins12 could not catalyze the aminoacylation of two mitochondrial tRNA Ser, but it could activate^Ser and bind tRNA, indicating that the insertion of 4 amino acids in the active center could not correctly guide the CCA end of tRNA to the catalytic active site
。 The aminoacylation activity of dupC on both mitochondrial tRNA^Ser was significantly impaired.
At the same time, dupC's affinity for the two mitochondrial tRNA Ser was significantly impaired, indicating that the C-terminus of the SARS2 protein is critical
for the binding of tRNA^Ser.
(2) The structural characteristics of wild-type SARS2 and Ins12 were analyzed and compared by structural biology, and combined with Alpha fold structural simulation, it was revealed that the insertion of four amino acids in the active center of Ins12 mutant destroyed the dimerization ability
of SARS2.
(3) By establishing multiple induced pluripotent stem cell models, it was revealed that the homeostatic level of hmtRNA Ser (AGY) in patient-derived cells was significantly reduced, and hmtRNA Ser (UCN) was not affected, suggesting that the aminoacylation defect of hmtRNA Ser(AGY) led to hmtRNA ^Ser (AGY) is more likely to degrade; In addition, SARS2 may also act as a "molecular chaperone" for tRNA, acting as
a structural stabilizing factor by binding to hmtRNA^Ser (AGY), which lacks a D stem/loop.
The insufficient content of aminoacylated hmtRNA^Ser (AGY) further caused a significant downregulation of the mitochondrial translation system of patients' cells, and led to defects in mitochondrial respiratory chain oxidative phosphorylation capacity, abnormal glycolysis process, abnormal increase of reactive oxygen species in cells, increased apoptosis level, active mitochondrial autophagy and dysbalance of mitochondria, resulting in homeostatic imbalance and dysfunction
of mitochondria 。 (4) By constructing Ins12 and dupC mouse models, it was found that homozygous and compound heterozygous mice with two mutations had early embryonic lethality, while the skeletal muscle tissues of Sars2 Ins12 and dupC heterozygous mutant mice had significant downregulation of two tRNA^Ser content, impaired mitochondrial translation and abnormal mitochondrial morphology
.

In this study, a new SARS2 point mutation leading to mitochondrial HUPRA syndrome was identified, extending the clinical phenotype associated with SARS2 deficiency.
Using molecular, structural, cellular and genetic methods and technical methods, the comprehensive effects of SARS2 point mutations on the structure and function, mitochondrial translation and function of SARS2 were systematically analyzed, and it was revealed that the selective degradation of hmtRNA^Ser(AGY) led to a significant decrease in homeostatic level was the pathogenic mechanism of mitochondrial diseases caused by SARS2 gene mutations, which significantly deepened our understanding of the molecular mechanism of mitochondrial diseases associated with mt-aaRS functional defects
。 This study provides a theoretical basis
for the clinical diagnosis, disease intervention and development of treatment strategies for such diseases.

Associate Professor Yu Tingting of Shanghai Children's Medical Center and Zhang Yi, a doctoral student jointly trained by Shanghai Children's Medical Center-Molecular Cell Center of Excellence, are co-first authors of this paper, and Professor Wang Jian of Shanghai Children's Medical Center and researcher Zhou Xiaolong of Molecular Cell Excellence Center are co-corresponding authors
of this paper.
Fang Pengfei, a researcher from the Institute of Organic Studies, Chinese Academy of Sciences, participated in this study
.
I would like to thank Professor Wang Enduo of the Center of Excellence for Molecular Cells and Professor Li Yanxin of Shanghai Children's Medical Center for their help
in the research process.
This research was supported by the Ministry of Science and Technology, the Foundation of China, the Chinese Academy of Sciences and the Shanghai Municipality
.

Article link: https://academic.
oup.
com/nar/advance-article/doi/10.
1093/nar/gkac1028/6814471

The pathogenic mechanism of SARS2 mutations leading to mitochondrial diseases