mt tRFs, New Players in MELAS Disease

Abstract

MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) is an OXPHOS disease mostly caused by the m.3243A>G mutation in the mitochondrial tRNA^Leu(UUR) gene. Recently, we have shown that the mutation significantly changes the expression pattern of several mitochondrial tRNA-derived small RNAs (mt tsRNAs or mt tRFs) in a cybrid model of MELAS and in fibroblasts from MELAS patients versus control cells. Among them are those derived from mt tRNA LeuUUR containing or not the m.3243A>G mutation (mt 5'-tRF LeuUUR-m.3243A>G and mt 5'-tRF LeuUUR), whose expression levels are, respectively, increased and decreased in both MELAS cybrids and fibroblasts. Here, we asked whether mt 5'-tRF LeuUUR and mt 5'-tRF LeuUUR-m.3243A>G are biologically relevant and whether these mt tRFs are detected in diverse patient samples. Treatment with a mimic oligonucleotide of mt tRNA LeuUUR fragment (mt 5'-tRF LeuUUR) showed a therapeutic potential since it partially restored mitochondrial respiration in MELAS cybrids. Moreover, these mt tRFs could be detected in biofluids like urine and blood. We also investigated the participation of miRNA pathway components Dicer and Ago2 in the mt tRFs biogenesis process. We found that Dicer and Ago2 localize in the mitochondria of MELAS cybrids and that immunoprecipitation of these proteins in cytoplasm and mitochondria fractions revealed an increased mt tRF/mt tRNA ratio in MELAS condition compared to WT. These preliminary results suggest an involvement of Dicer and Ago2 in the mechanism of mt tRF biogenesis and action.

Keywords: mitochondrial dysfunction; retrograde signaling; sncRNAs; tRF and tiRNA; tRNA fragment.

Figure 1

Partial localization of Dicer and Ago2 in the mitochondrial fraction of WT and MELAS cybrid cells. (A) Immunofluorescence microscopy analysis of endogenous Dicer and Ago2 subcellular localization in WT and MELAS cybrid cells. Nuclei were stained with DAPI (blue), and mitochondria were immunodetected with anti-ATP5A1 (OXPHOS Complex V subunit) or anti-CLPP (a serine protease located in the mitochondrial matrix). Scale bars, 10 μm. (B) Representative immunoblots of Dicer and Ago2 in subcellular fractions of WT and MELAS cybrid cells. Dicer and Ago2 levels were determined in nucleus and intact cells, cytosol and mitoplasts enriched fractions together with respective markers (Pol II (nucleus), Vinculin (cytoplasm), and SDHA (mitochondria)).

Figure 2

mt tRNAs may recruit Dicer and Ago2. (A and B) RT-qPCR analysis of the expression of mt tRNAs LeuUUR, and GluUUC and mt tRFs, mt 5′-tRF LeuUUR and mt i-tRF GluUUC in Dicer (A) and Ago2 (B) immunoprecipitates from cytosolic and mitochondrial fractions of WT and MELAS cybrids. Data are represented as fold change respect to values from IgG immunoprecipitates. (C) Ratio between fold enrichment values for a mt tRF and its parental mt tRNA in Dicer and Ago2 immunoprecipitates from cytosolic and mitochondrial fractions of WT and MELAS cybrids. Differences from control values were found to be statistically significant at ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 3

Over-expression of mt tRNA LeuUUR fragment (mt 5′-tRF LeuUUR) increases mitochondrial respiration in MELAS cybrids. (A) Analysis of oxygen consumption rate (OCR) of WT and MELAS cybrids, and MELAS cybrids transfected with an oligonucleotide mimic of mt-tRNA LeuUUR fragment (mt 5′-tRF LeuUUR), an oligonucleotide antisense against mt-tRNA LeuUUR-m.3243A>G fragment (mt 5′-tRF LeuUUR-m.3243A>G) and their respective controls. OCR was measured under basal conditions and after sequential addition of different OXPHOS inhibitors: oligomycin, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (CCCP), rotenone, and antimycin A. The scatter plot shows basal OCR (determined as the difference between OCR before oligomycin and OCR after rotenone/antimycin incorporation A), ATP-linked OCR (difference between OCR before and after oligomycin), proton leak (difference between basal OCR and ATP-linked OCR), reserve capacity (difference between the CCCP-stimulated rate and basal OCR), non-mitochondrial OCR (OCR after rotenone and antimycin A treatment), and maximal OCR (difference between OCR after CCCP and non-mitochondrial OCR). (B) RT-qPCR analysis of the expression of ND1, ND2, ND4, COXI, COXIII, and CYTB mRNAs in MELAS cybrids transfected with an oligonucleotide mimic of mt-tRNA LeuUUR fragment (mt 5′-tRF LeuUUR) with respect to negative control-transfected cells. Data are represented as fold change respect to values from control samples. The horizontal bar represents the media value for control samples. Differences from control values were found to be statistically significant at ^*p < 0.05 and ^**p < 0.01.

Figure 4

Expression profile of mt 5′-tRF LeuUUR, mt 5′-tRF LeuUUR-m.3243A>G, mt i-tRF GluUUC, and mt 3′-tRF ValUAC in a small cohort of diverse samples from MELAS patients and controls. RT-qPCR analysis of the expression of mt 5′-tRF LeuUUR, mt 5′-tRF LeuUUR-m.3243A>G, mt i-tRF GluUUC and mt 3′-tRF ValUAC in biofluids (urine and plasma), PBMCs, and muscle tissues from MELAS patients as compared to controls. Data are represented as fold change respect to values from control samples. Each triangle represents an individual and the horizontal bar the media value for control group. Differences from control values were found to be statistically significant at ^*p < 0.05 and ^**p < 0.01.