Figure 3. Knockout of PDE12 leads to increased stalling events on selected codons on mtDNA-encoded mRNAs.

(A) Sedimentation of mitochondrial ribosomes on 10–30% isokinetic sucrose gradients for PDE12+/− and PDE12−/− cell lines. Fractions obtained for PDE12+/+, and PDE12−/− were simultaneously analysed by western blotting with antibodies to bL12 (mt-LSU) and mS18b (mt-SSU). Northern blotting was performed using RNA extracted from each fraction with the indicated probes. Quantification of relative RNA content in each fraction was performed using ImageQuant. (B) Mean A-site codon occupancy of mitochondrial ribosomal protected fragments (mtRPFs) in the mitochondrial transcriptome of PDE12+/+ and PDE12−/−. See Materials and methods for further explanation. (C) mtRPF profile for the ND4L/ND4 transcript in PDE12+/+ and PDE12−/− as an example of increased stalling of mitoribosomes at lysine codons (Note: The ND4L ORF does not contain any lysine codons).

DOI: http://dx.doi.org/10.7554/eLife.27596.006

Figure 3—figure supplement 1. Increased stalling events on lysine codons in ΔFLP cell line.

(A) Mean A-site occupancy of mitochondrial ribosome protected fragments (mtRPFs) centred on each codon of the mitochondrial mRNA transcriptome of WT and ΔFLP. The ΔFLP cell line has been described in Gilkerson et al. (2008). (B) mtRPF profile for the ND4L/ND4 transcript in WT and ΔFLP as an example of increased stalling of mitoribosomes at lysine codons.

DOI: http://dx.doi.org/10.7554/eLife.27596.007