Fig. 5. ATFS-1 limits mtDNA-encoded transcript accumulation and directly binds to the non-coding region of mtDNA.

A. ChIP-seq profiles of the entire mitochondrial genome (mtDNA) of wild-type worms raised on spg-7(RNAi), using ATFS-1 antibody or no antibody. The y-axis is the number of sequence reads and the x-axis is the 13,794 nucleotide mitochondrial genome. The protein coding genes are marked with blue arrows, the rRNA genes with purple arrows, the non-coding region in green, and the putative UPR^mtE is marked with a red box.

B. ChIP of the mtDNA non-coding region in the presence or absence of ATFS-1 antibody from wild-type worms raised on control or spg-7(RNAi) as measured by qPCR (N = 3, ± SD, p* (student t-test) < 0.05).

C. mtDNA non-coding region sequence with the putative UPR^mtE in red.

D. EMSA using recombinant ATFS-1 and wild-type D-loop oligonucleotides or D-loop oligonucleotides with the 9 base pair UPR^mtE scrambled. Unbound oligos (black square), ATFS-1-DNA complex (black arrow) and the loading well (gray arrow) are indicated.

E. Immunoblots of lysates from worms raised on control or spg-7(RNAi) following fractionation into total lysate (T), postmitochondrial supernatant (S) and mitochondrial pellet (M) (Nargund et al., 2012). Endogenous NDUFS3 serves as a mitochondrial marker and hsp-60_pr∷gfp as a stress-induced cytosolic marker. Full-length (black arrow) and the mitochondrial forms (gray arrows) of ATFS-1 are indicated.

F. Relative mtDNA copy number in wild-type and atfs-1(tm4525) worms raised on control or spg-7(RNAi) as determined by qPCR (N = 3, ± SD, p* (student t-test) < 0.05).

G-I. Expression levels of the mtDNA-encoded mRNAs nd6, cox2 and atp6 in wild-type or atfs-1(tm4525) worms raised on control or spg-7(RNAi) determined by qRT-PCR (N = 3, ± SD, p* (student t-test) < 0.05).

J. Expression level of the mtDNA-encoded ctb-1 mRNA in wild-type or atfs-1(tm4525) worms expressing no transgenes (-) or transgenic wild-type ATFS-1, ATFS-1^Δ1-32.myc or ATFS-1^ΔNLS raised on control or spg-7(RNAi) determined by qRT-PCR (N = 3, ± SD, p* (student t-test) < 0.05).

K. Schematics of wild-type ATFS-1, ATFS-1 with an impaired mitochondrial targeting sequence (MTS) (ATFS-1^Δ1-32.myc), and ATFS-1 with a mutated nuclear localization sequence (NLS) (ATFS-1^ΔNLS).

L. Fluorescent photomicrographs of atfs-1(tm4525);hsp-60_pr∷gfp worms expressing transgenic ATFS-1, ATFS-1^Δ1-32.myc, or ATFS-1^ΔNLS raised at 20°C. Scale bar, 0.5 mm.

See also Figure S5.