Mitoribosome profiling captures elongating ribosomes.

(A) Schematic representation of mitoribosome footprinting and separation from cytoplasmic ribosomes by differential centrifugation. (B) Coverage of MRPFs on MT-CYB from mitoribosome profiling generated by either RNase I or MNase footprinting. (C) Distribution of read lengths of MRPFs mapping to the mitochondrial genome. (D) Read phasing analysis of MRPFs, where the 5’ ends of reads are grouped by their subcodon position. Each dot represents an individual mitochondrial gene. (E) Average coverage of mitochondrial genes, calculated by dividing the total number of reads mapping to an ORF and dividing by the length of the ORF. (F) Distribution of mitochondrial codons grouped by the number of mitoribosome P-sites assigned to them.

Inhibition of translation initiation in mitochondria.

(A) (Left) Schematic representation of retapamulin (orange) or tiamulin (yellow) binding to the PTC and disrupting the first round of elongation. (Right) Josamycin (blue) disrupts accommodation of the first aminoacyl-tRNA. All three inhibit initiation in bacteria. (B) MRPF coverage of MT-CYB treated with either retapamulin, tiamulin (note y-axis is segmented), or josamycin. (C) Average fold change increase in the percentage of reads that map to the 5’ of mitochondrial transcripts. (D) Cumulative distribution of ribosome density normalized across all mitochondrial ORFs.

Context-dependent translation arrest by elongation inhibitors.

(A) Schematic representation of chloramphenicol (purple), linezolid (cyan), and telithromycin (green) inducing context-dependent translation arrest. (B) MRPF coverage of MT-CYB from cells treated with either chloramphenicol, linezolid, or telithromycin. (C) Average fold change increase in the percentage of reads that map to the 5’ of mitochondrial transcripts. (D) Cumulative distribution of ribosome density normalized across all mitochondrial ORFs. (E) Volcano plots of changes in MRPF fold change from cells treated with translation inhibitors. Each dot is the mean log2-fold change grouped by the identity of the –1 amino acid (top), P-site amino acid (middle), or A-site amino acid (right). (F) Top 4 strongest stalling sites induced by telithromycin treatment. Red amino acids indicate a classical R/K-X-R/K stalling motif. (G) Fold change in ribosome density, calculated as 2mean(log2(Telithromycin/Control)). Bars differ in the identity of the P-site amino acid and contain either a lysine in the A-site (top) or arginine (bottom).

MRPF sizes indicate use of alternative initiation sites.

(A) Schematic representation of changing MRPF sizes as a function of the mitoribosome’s position near the 3’ end of mRNAs. (B) Read length measurement of reads mapping to the 5’ ends of mitochondrial transcripts (red) compared to the entire ORF (black). (C) Read lengths of MRPFs mapping to the 5’ ends of mitochondrial transcripts. Each row contains the nucleotide sequence of each transcript starting from the 5’ end. The coloring of the box is proportional abundance of MRPFs with read lengths from 20-34 nts. Nucleotide sequence and read abundance are overlayed to compare the abundance of reads to potential ribosome start sites. Transcripts are grouped by being leaderless, having a leader, or having a potential alternative start site. Black boxes indicate annotated start codons and green boxes indicate alternative initiation sites. (D) Visual representation of the peptide coverage (green bars or highlights) from mass spectrometry data covering the N-terminus of MT-ND1 or (E) MT-ND5.

MRPFs on ncRNAs indicate potential novel ORFs

(A) Read lengths of MRPFs mapping to the 5’ end of ncRNAs. Each row contains the nucleotide sequence beginning from the 5’ end of listed ncRNAs. The coloring of the box is proportional the abundance of MRPFs with read lengths 20-34 nts. The nucleotide sequence and read MRPF abundance are overlayed to identify potential new start sites. Putative start sites are indicated by green boxes. (B) Read phasing analysis of all canonical ORFs (grey), a mutative ORF located on MT-RNR1, the non-coding region of MT-RNR1, and Humanin. (C) A correlation plot comparing the frequence of amino acid usage in canonical ORFs compared to the putative ORF on MT-RNR1 or (D) the non-coding region of MT-RNR1.

MRPF lengths after treatment with initiation inhibitors correlate to alternative initiation sites.

(A) Read lengths of MNase-generated MRPFs from cells treated with josamycin or (B) retapamulin.