Figure 5. 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 and D) Box-plot representing 3' extensions to 12S (C) and 16S (D) mt-rRNAs via circularisation RT-PCR for PDE12+/+ and PDE12−/− cells, and from PDE12−/− cells expressing either wild-type PDE12 or E351A. *p<0.05, ***p<0.001 with Student t-test relative to PDE12+/+. (p values: 12S: [+/+] vs. [−/−] p=0.013, [+/+] vs. [−/− + WT] p=0.32, [+/+] vs. [−/− + E351A] p=0.84; 16S: [+/+] vs. [−/−] p=4.6×10⁻⁷, [+/+] vs. [−/− + WT] p=0.53, [+/+] vs. [−/− + E351A] p=5.3×10⁻⁴) (E) Steady-state levels of 12S and 16S mt-rRNAs for PDE12+/+, PDE12+/− and PDE12−/− determined via northern blotting. (F) Radioactive MPAT assay for 16S mt-rRNA extracted from sucrose-gradient fractions for free mt-LSU (fractions 9 and 10) and monosome (12-15) for PDE12+/+ and PDE12−/− cells.

DOI: http://dx.doi.org/10.7554/eLife.27596.013

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 PDE12+/+, PDE12+/− and PDE12−/− cell lines.

DOI: http://dx.doi.org/10.7554/eLife.27596.014