Hypertension-associated mitochondrial DNA 4401A>G mutation caused the aberrant processing of tRNAMet, all 8 tRNAs and ND6 mRNA in the light-strand transcript

Abstract

Mitochondrial tRNA processing defects were associated with human diseases but their pathophysiology remains elusively. The hypertension-associated m.4401A>G mutation resided at a spacer between mitochondrial tRNAMet and tRNAGln genes. An in vitro processing experiment revealed that the m.4401A>G mutation caused 59% and 69% decreases in the 5′ end processing efficiency of tRNAGln and tRNAMet precursors, catalyzed by RNase P, respectively. Using human umbilical vein endothelial cells-derived cybrids, we demonstrated that the m.4401A>G mutation caused the decreases of all 8 tRNAs and ND6 and increases of longer and uncleaved precursors from the Light-strand transcript. Conversely, the m.4401A>G mutation yielded the reduced levels of tRNAMet level but did not change the levels of other 13 tRNAs, 12 mRNAs including ND1, 12S rRNA and 16S rRNA from the Heavy-strand transcript. These implicated the asymmetrical processing mechanisms of H-strand and L-strand polycistronic transcripts. The tRNA processing defects play the determined roles in the impairing mitochondrial translation, respiratory deficiency, diminishing membrane potential, increasing production of reactive oxygen species and altering autophagy. Furthermore, the m.4401A>G mutation altered the angiogenesis, evidenced by aberrant wound regeneration and weaken tube formation in mutant cybrids. Our findings provide new insights into the pathophysiology of hypertension arising from mitochondrial tRNA processing defects.

Asymmetrical effects of deafness-associated mitochondrial DNA 7516delA mutation on the processing of RNAs in the H-strand and L-strand polycistronic transcripts

“…Transcripts were purified by denaturing polyacrylamide gel electrophoresis (PAGE) [8 M urea, 8% polyacrylamide/bisacrylamide (19:1)] and were dissolved in 1 mM EDTA. Mitochondrial RNase P was reconstituted from purified recombinant proteins MRPP1, MRPP2 and MRPP3 as described previously ( 15 , 26 ). The reaction mixture was incubated in 40 μl assay buffer containing 20 mM HEPES (pH 7.6), 20 mM KCl, 2 mM MgCl 2 , 2 mM DTT, 0.1 mg/ml bovine serum albumin, 80 μM S -adenosyl methionine, 1 U RiboLock RNase Inhibitor (Thermo Fisher Scientific), 300 ng pre-tRNAs, 800 nM MRPP1/2 and 100 nM MRPP3.…”

“…For tRNA Northern blot analysis, 6 μg of total cellular RNA was electrophoresed through a 10% polyacrylamide/8 M urea gel in 1× TBE buffer after heating the samples at 65°C for 10 min, and then electroblotted onto a positively charged Nylon membrane (Roche) for the hybridization analysis with digoxigenin (DIG)-labeled oligodeoxynucleotide probes, respectively. A set of DIG-labeled probes of 22 mitochondrial tRNAs and 5S rRNA was described elsewhere ( 26 , 40 , 41 ). The hybridization and quantification of density in each band were performed as detailed previously ( 40–43 ).…”

“…For mRNA Northern blot analysis, 6 μg of total cellular RNA was fractionated by electrophoresis through a 2% agarose–formaldehyde gel, transferred onto a positively charged membrane (Roche) and hybridized with a set of DIG-labeled RNA probes: COX2, CYTB, ND1, ND3, ND6, COX1, 12S rRNA, 16S rRNA and β - actin as a control, respectively. Probes were synthesized as described previously ( 13 , 26 , 44 ). The hybridization and quantification of density in each band were performed as detailed previously ( 26 , 44 ).…”

“…The tRNA Ile 4263A>G and tRNA Ala 5655A>G mutations at the 5′ end (conventional position 1) of corresponding tRNAs altered the 5′ end processing of tRNA Ile and tRNA Ala precursors, respectively ( 24 , 25 ). Strikingly, the m.4401A>G mutation at the junction of tRNA Met and tRNA Gln genes caused the 5′ end aberrant processing of tRNA Met in the H-strand transcripts and tRNA Gln as well as other seven tRNAs and ND6 mRNA in the L-strand transcripts ( 26 ). These suggested the asymmetrical processing mechanisms of H-strand and L-strand polycistronic transcripts ( 26 ).…”

“…Strikingly, the m.4401A>G mutation at the junction of tRNA Met and tRNA Gln genes caused the 5′ end aberrant processing of tRNA Met in the H-strand transcripts and tRNA Gln as well as other seven tRNAs and ND6 mRNA in the L-strand transcripts ( 26 ). These suggested the asymmetrical processing mechanisms of H-strand and L-strand polycistronic transcripts ( 26 ). However, the pathogenic mechanisms underlying these tRNA processing defects remain elusive.…”