Fig 1.
Secondary structure of wild type and recombinant 5S rRNAs.
(a) Human 5S rRNA secondary structure, modified from [26]. (b) 2D structure of rec.5S rRNA versions, mfold predictions corrected manually. Sequences of the anti-replicative insertions (shown in red) correspond to boundaries of the KSS deletion in mtDNA; arrows indicate the deletion point.
Fig 2.
Structure and melting temperatures predictions for duplexes between rec.5S rRNA and mutant or wild-type mtDNA regions.
Upper part of each duplex corresponds to the anti-replicative insertion of rec.5S rRNA indicated at the left. Nucleotides complementary to the 5’ boundary of the KSS deletion are shown in orange; those complementary to the 3’ deletion boundary are in green. 5S-KSS-13H and 5S-KSS-15H annealed to the L-strand of mtDNA; 5S-KSS-14L and 5S-KSS-15L annealed to the H-strand of mtDNA.
Fig 3.
In vivo test of rec.5S rRNA import into human mitochondria.
Northern blot analysis of rec.5S rRNA variants in total (T) and mitoplast (M) RNA preparations from cells transfected with various RNA, as indicated above the panels; NT, non-transfected cells; tr, 5ng of corresponding T7 transcript used for cell transfection. Gels stained with Ethidium bromide (EthBr) and hybridized with probes indicated at the left are shown. Rec 5S, probes corresponding to the insertion sequence specific for each rec.5S rRNA variant; 5.8S and Vmt, probes to cytosolic 5.8S rRNA and mitochondrial tRNAVal. 5S, probe to 5S rRNA, can hybridize with endogenous 5S rRNA and with rec.5S rRNA versions (which are 5–8 nucleotides shorter), thus giving double bands clearly visible in 13H-M sample. The long strip marked by * on the panel “tr13H, EthBr” is due to an artifact; 5ng of the transcript (tr13H) can be visible only by probing.
Table 1.
Relative efficiencies of rec.5S rRNA mitochondrial import.
Fig 4.
Quantification of 5S-KSS-14L RNA expression by semi-quantitative RT-PCR.
On the left, PAGE analysis of the RT-PCR reactions performed on the total cellular RNA or known amounts of purified T7-transcripts, as indicated above the panel. On the right, an example of the calibration curve used for quantification of 5S-KSS-14L RNA (in red) in cybrid cell line.
Table 2.
Amounts of anti-replicative molecules in rec.5S rRNA expressing cell lines.
Fig 5.
Effect of rec.5S rRNA expression on the heteroplasmy level of KSS mutation.
(a) Proportion of KSS mtDNA (Y axis) in cybrids cell lines with low expression of rec.5S rRNA versions (see Table 1) after 8 weeks of cultivation in high glucose or glucose-free media, as indicated. (b) Time-depended decrease of KSS mutation load in cells expressing high amounts of rec.5S rRNAs 5S-KSS-13H(1) and 5S-KSS-14L(1) in glucose-free medium. (c) MtDNA copy numbers normalized to nuclear gene TST1, measured in the same cell populations as in (b). Cells issued from independent transfections were cultivated separately; each population was divided into 4 parallel cultures. Total DNA was isolated from a portion of each cell cultures every 2 weeks and analysed by 2–3 independent qPCR experiments, each measure performed in triplicates. Standard deviations are calculated from qPCR data obtained on the independently cultured cells (n = 4). Statistical differences were determined with a two-tailed Student’s t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).