Maturation of selected human mitochondrial tRNAs requires deadenylation
- University of Cambridge, United Kingdom
- University of Porto, Portugal
Figures
Figure 1 with 1 supplement
PDE12 is essential for efficient mitochondrial translation.
(A) Western blotting for steady-state levels of OxPhos components in PDE12+/+, PDE12 ± and PDE12−/− cells, and in PDE12−/− expressing either wild-type PDE12 or the E351A catalytic mutant at …
Figure 1—figure supplement 1
Gene targeting of PDE12 with ZFNs and complementation.
(A) Schematic of PDE12 gene locus indicating target sites of ZFNs. The indels present in clones 71 (PDE12−/−) or 118 (PDE12+/−) are indicated. (B) Western to confirm knockout of PDE12 in biallelic …
Figure 2 with 1 supplement
Mitochondrial mRNAs are unaffected by absence of PDE12.
(A) Radioactive MPAT assay for ND1 and COII transcripts for PDE12+/+ and PDE12−/− with densitometric profile of tail length determined via ImageQuant. (B) Box plot representing length of 3' …
Figure 2—figure supplement 1
mtDNA copy-number and mt-mRNA are unaffected in PDE12−/−.
(A) mtDNA copy-number determination using qPCR of mitochondrial DNA fragments relative to the nuclear B2M gene. (mean ± SEM; n = 3). PDE12−/− not significant with Student t-test relative to …
Figure 3 with 1 supplement
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 …
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 …
Figure 4 with 3 supplements
A subset of mt-tRNAs are affected by spurious 3’ adenylation in the absence of PDE12, leading to reduced aminoacylation of specific mt-tRNAs.
(A) Schematic of mt-tRNA structure depicting 3' extension determined via the radioactive MPAT (b) and MPAT-Seq (c). DB, discriminator base (B) Radioactive MPAT assay for a subset of mt-tRNAs …
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Figure 4—source data 1
Read count for mitochondrial poly(A) (MPAT) next generation RNA sequencing (MPAT-Seq).
- https://doi.org/10.7554/eLife.27596.009
Figure 4—figure supplement 1
Radioactive MPAT assay for mt-tRNALys.
(A) Radioactive MPAT assay for mt-tRNALys for PDE12+/+, PDE12−/−, PDE12−/− cell lines expressing PDE12 wild-type cDNA or E351A, and PDE12+/+ and PDE12−/− expressing mtPAP. (B) Quantification of MPAT …
Figure 4—figure supplement 2
Northern blotting for mt-tRNAs.
(A) High-resolution northern blots for indicated mt-tRNAs for PDE12+/+, PDE12−/−, PDE12−/− cell lines expressing PDE12 wild-type cDNA or E351A. M indicates mature mt-tRNA species. (B) and (C) …
Figure 4—figure supplement 3
Aminoacylation analysis by sodium periodate oxidation.
High-resolution northern blot analysis of total RNA extracted from PDE12+/+, PDE12−/−, PDE12−/− cell lines expressing PDE12 wild-type cDNA or E351A. Following extraction, RNA samples were either …
Figure 5 with 1 supplement
Spurious adenylation to the 3' terminus of 16S mt-rRNA in the absence of PDE12 does not affect mitochondrial monosome formation.
(A and B) Radioactive MPAT assay for 12S (a) and 16S (b) mt-rRNAs extracted from PDE12+/+, PDE12+/− and PDE12−/− cells, and for PDE12−/− cells expressing either wild-type PDE12 or E351A for 24 hr. (C…
Figure 5—figure supplement 1
Structural and western blot analysis of mtLSU.
(A) Structure of the human mt-LSU indicating proteins adjacent to the 3’ terminus of 16S mt-rRNA. (B) western blotting of steady-state levels of protein subunits of the human mitoribosome in …
Figure 6 with 1 supplement
Increased adenylation activity is detrimental to mitochondrial gene expression.
(A) Western blotting for steady-state levels of OxPhos components and mtPAP (top). Mitochondrial translation products were labelled with [35S]-Met in PDE12+/+ and PDE12−/− cells with either …
Figure 6—figure supplement 1
The role of PDE12 in maturation of mt-tRNATyr.
(A) Adjacent organization of mt-tRNATyr and mt-tRNACys genes in human mtDNA indicating overlapping ‘A’ residue (red) required for both transcripts. This residue is required as the discriminator base …
Additional files
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Supplementary file 1
Distribution of non-encoded extensions on the mature 3’ terminus of mt-rRNA in control cells (+/+), PDE12−/− cells, and PDE12−/− cells expressing either wild-type PDE12 or PDE12 E351A mutant determined by cRT-PCR.
- https://doi.org/10.7554/eLife.27596.017
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Supplementary file 2
Antibodies and vectors.
- https://doi.org/10.7554/eLife.27596.018
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Supplementary file 3
Oligonucleotide sequences.
Sequences of DNA oligonucleotides for use in radioactive MPAT assay
- https://doi.org/10.7554/eLife.27596.019
