Deficient methylation and formylation of mt-tRNA(Met) wobble cytosine in a patient carrying mutations in NSUN3

Abstract

Epitranscriptome modifications are required for structure and function of RNA and defects in these pathways have been associated with human disease. Here we identify the RNA target for the previously uncharacterized 5-methylcytosine (m(5)C) methyltransferase NSun3 and link m(5)C RNA modifications with energy metabolism. Using whole-exome sequencing, we identified loss-of-function mutations in NSUN3 in a patient presenting with combined mitochondrial respiratory chain complex deficiency. Patient-derived fibroblasts exhibit severe defects in mitochondrial translation that can be rescued by exogenous expression of NSun3. We show that NSun3 is required for deposition of m(5)C at the anticodon loop in the mitochondrially encoded transfer RNA methionine (mt-tRNA(Met)). Further, we demonstrate that m(5)C deficiency in mt-tRNA(Met) results in the lack of 5-formylcytosine (f(5)C) at the same tRNA position. Our findings demonstrate that NSUN3 is necessary for efficient mitochondrial translation and reveal that f(5)C in human mitochondrial RNA is generated by oxidative processing of m(5)C.

Figure 1. Identification of pathogenic compound heterozygous mutations in NSun3.

(a) OXPHOS complex activity in unfrozen patient muscle homogenate (red line). Grey bars represent the normal range. Note the different scale for complex I–II as compared with complex III–V. (b) Segregation analysis and electropherogram corresponding to the genomic DNA mutations identified in the family of a patient carrying NSun3 mutations. The patient's parental allele carries a c.295C>T transition in exon 3, while the maternal allele has a 3,314 nt deletion spanning exon 3 (c.123-615_466+2155del). (c) Mutation analysis of NSun3 mRNA level in human dermal fibroblasts of wild-type (wt/wt), patient (mut/mut) and patient cells rescued with a NSUN3 construct (mut/mut+NSUN3). Gel electrophoresis of DNA fragments obtained in a reverse transcriptase–PCR using total RNA from the indicated samples. (d) Sanger sequencing of the bands from the mut/mut sample excised from the gel presented in c showing a stop mutation in the full-length NSUN3 mRNA and the lack of exon 3 on the shorter band. (e) Schematic overview of the NSUN3 gene, summarizing the mutations in the patient cells (red) on both genomic DNA (gDNA) and mRNA level. (f) Immunofluorescence labelling of a Flag-tagged NSun3 construct (red) in HeLa cells. Cells were counterstained for the mitochondrial import receptor subunit TOM20 (green) and DAPI (blue). Scale bar, 10 μm. (g) Sub-cellular localization of NSun3 analysed by western blotting with antibodies against NSun3, TOM22 (mitochondrial outer membrane), mtSSB1 (mitochondrial matrix), GAPDH (cytosol), Histone H3 (nucleus). HEK293T cells were fractionated into debris (‘D', lane 2), cytosol (‘C', lane 3) and mitochondria (‘M', lanes 4–6) ‘T' indicates the total cell lysate. ‘fl' indicates full-length TOM22, ‘tr' stands for truncated TOM22.

Figure 2. NSun3 is essential for mitochondrial translation.

(a) Western blot of two different experiments detecting NSun3 protein in mitochondria of human dermal fibroblasts (hDF). wt/wt: wild-type hDF; mut/mut: patient hDF; mut/mut+NSUN3: patient hDF expressing the V5-tagged NSUN3 construct. Lysate of HEK293 cells over-expressing the Flag-tagged NSun3 served as a control. ‘Unspec' indicates a non-specific band detected in human mitochondria by anti-NSun3 antibodies. CBS stands for Coomassie blue staining. (b) Oxygen consumption rate in wild-type (wt/wt) and patient-derived cells (mut/mut) as well as in wt/wt and mut/mut cells expressing a V5-tagged NSun3 construct (+NSUN3). Graph is a representative for three independent replicates, P values obtained in Student's t-test. (c) Relative cell growth rate of wt/wt, mut/mut and mut/mut+NSUN3 human dermal fibroblasts in galactose-containing medium normalized by growth rate in glucose-containing medium. (d) mtDNA copy-number determination by qPCR, performed in triplicate, of mitochondrial DNA fragments relative to the nuclear B2M gene. Statistical analysis was carried out using a two-tailed student's t-test. Error bars represent s.d. of the mean. (e) qPCR determination of mt-rRNA (12S and 16S) and mt-mRNA (CO1 and CO2) levels compared with GAPDH in wt/wt, mut/mut and mut/mut+NSUN3 cells. qPCR was performed in triplicate and error bars indicate the s.d. of the mean. (f) Northern blot analysis of mt-tRNAs in two different wt/wt cells, mut/mut and mut/mut+NSUN3 cells. Cytoplasmic 5S rRNA is used as loading control. (g) Mitochondrial de novo protein synthesis assessed with ³⁵S metabolic labelling. CBS: Coomassie blue stained gel as loading control. (h) Quantification of the bands intensities shown in g using ImageQuant.

Figure 3. Pathogenic mutations in NSUN3 cause loss of m⁵C34 in mitochondrial tRNA^Met.

(a) miCLIP reads mapped to mtDNA (mt-tRNA black, mt-mRNA lime, mt-rRNA yellow). (b) miCLIP (green) and HITS-CLIP read counts (grey) corresponding to the MT-TM gene coding for mt-tRNA^Met. The length of mt-tRNA is indicated on the x axis. The green dashed line rectangle indicates the position of anticodon arm. (c) Methylated (orange) and unmethylated (grey) cytosines (x axis) detected by BS RNA-Seq (individual reads on y axis) for mt-tRNA^Met for wt/wt, mut/mut and mut/mut+NSUN3 cells. (d) Schematic structure of mt-tRNA^Met and the position of m⁵C (orange circle) in the anticodon arm (green dashed line rectangle). (e–g) miCLIP and HITS-CLIP reads, BS RNA-Seq and position of m⁵C of MT-TL1/mt-tRNA^Leu(UUR) (the details as per b–d). (h–j) miCLIP and HITS-CLIP reads, BS RNA-Seq and position of m⁵C of MT-TS2/mt-tRNA^Ser(AGY) (details as per b–d). The red dashed line rectangle (e,h) and the red dashed line square (g,j) indicate the tRNA variable region.

Figure 4. m⁵C34 in mt-tRNA^Met is a necessary precursor for f⁵C34.

(a) Heatmaps of BS RNA-Seq reads for mt-tRNA^Met (MT-TM) for wt/wt and mut/mut cells, showing cytosines of individual reads (on y axis). Methylated (and hydroxymethylated) cytosines are shown in orange, while unmodified or formylated cytosines are shown in grey (x axis). (b) Heatmaps of fCAB RNA-Seq reads for mt-tRNA^Met (MT-TM) for wt/wt and mut/mut cells, showing cytosines of individual reads (on y axis). Purple indicates methylated, hydroxymethylated or formylated, while unmodified cytosines are shown in grey. (c) Heatmaps of RedBS RNA-Seq for mt-tRNA^Met (MT-TM) for wt/wt and mut/mut cells showing cytosines of individual reads (on y axis). Purple indicates methylated, hydroxymethylated or formylated, while unmodified cytosines are shown in grey. (d) Summary of the fCAB RNA-Seq results for wt/wt and mut/mut cells. (e) Summary of the RedBS RNA-Seq results for wt/wt and mut/mut cells. (f) Graphical overview of the mt-tRNA^Met C34 formylation pathway. NSun3 methylates unmodified C34 into m⁵C34, which is then further processed (responsible enzyme(s) unknown) into f⁵C34.