Figure 4
From: TDP-43 stabilises the processing intermediates of mitochondrial transcripts
TDP-43 overexpression increases polycistronic transcripts of L- and H-strand mtDNA. (a) Relative band intensities of mt-tRNAs in total RNA extracted from DAP-TDP-43–expressing T-REx 293 cells (harvested at 0, 24, and 48 h after induction with doxycycline, Dox) were quantified by northern blotting after polyacrylamide gel electrophoresis. mt-AAA represents mt-tRNAAAA; e.g., mt-Pro, mt-tRNAPro, etc. 5S rRNA stained with SYBR Gold served as the loading control. Data are mean ± SD, n = 3–6. **P < 0.01; *P < 0.05 (paired t test). (b) Overexposed views of representative northern blots from (a) showing precursor bands of each mt-tRNA. 5S rRNA stained with SYBR Gold served as the loading control. (c) Total RNA extracted from DAP-TDP-43–expressing T-REx 293 cells harvested at 0, 24, or 48 h after Dox induction were reverse transcribed (+RT) or not reverse transcribed (−RT) with the indicated gene-specific primers for L-strand-specific RT-PCR analysis. The PCR products were separated by PAGE and detected with SYBR Gold staining. The primers amplified the following: a 140-nt region in GAPDH mRNA and a 300-nt region of the L-strand transcript containing mt-tRNATyr, mt-tRNACys, mt-tRNAAsn, and mt-tRNAAla (Y - A, top left), a 400-nt region between mirrorND2 and mt-tRNAGln (Q) of the L-strand transcript (bottom left), a 300-nt region between mirrorCOXI and mt-tRNATyr (Y) of the L-strand transcript (top right), and a 400-nt region between mirrorCYB and mt-tRNAGlu (E) of the L-strand transcript (bottom right). Schematic diagrams of the corresponding PCR fragments are shown (upper diagram). (d) Recombinant TDP-43 fused with trigger factor (TF) and FLAG tag (FL) (TF-TDP43-FL) was mixed with synthesised mirrorCOXI-YCNA (mt-tRNATyr-mt-tRNACys-mt-tRNAAsn -mt-tRNAAla; 2000 nt) RNA and pulled down with anti-FLAG beads. mirrorCOXI-YCNA was detected by northern blotting, and TF-FL or TF-TDP43-FL by western blotting with anti-FLAG. (e) mirrorCOXI-YCNA was incubated with mitochondrial extract from 293 T cells for the indicated times in the presence of TF-FL or TF-TDP43-FL. TFAM (transcription factor A mitochondrial protein) served as a loading control. Data in the graph represent the mean ± SEM of three independent experiments.
