Journal: Genome Biology

Article Title: Balanced mitochondrial and cytosolic translatomes underlie the biogenesis of human respiratory complexes

doi: 10.1186/s13059-022-02732-9

Figure Lengend Snippet: Evidence for the translation of a novel mitochondrial noncanonical open reading frame. A Translation initiation occurs on an internal AUG in the MT-ND5 3′ UTR across cell lines. A-site density on the MT-ND5 stop codon (shaded in red) and P-site density (shaded in green, A-site density one codon downstream) of initiating ribosomes on the internal AUG are highlighted. Codons in the three frames are shown below with stop (UAA, UAG), start (AUG), and mitochondrial alternative start (AUU, AUA) highlighted (red, green, yellow, respectively). B Stacked frame plot highlighting number of A-site transformed reads in each subcodon position on heavy strand. The example analysis (left panel) shows how raw A site read counts are transformed to line plots, and how the line plots highlight which frame is being read. The color of the arrowheads indicates which frame each ATG is in. The blue ATG starts an open reading frame. Mitoribosome profiling data are from two fibroblast replicates, summed. Amino acid sequence of putative MT-ND5-dORF peptide is shown with putative (put.) start and stop codons highlighted with green and red lines, respectively. Bar plots below show the average percentage of reads in each subcodon position for regions indicated, with error bars showing range between the replicates summed above. C Distribution of initiation scores after applying ad hoc scoring method to fibroblasts and HeLa S3 cells. Gene names in black: known genes with no leader; gray: known genes with 1 to 3 nt leader; cyan: known genes with long 5′ UTR. D Estimated relative synthesis of MT-ND5-dORF compared to other mtDNA-encoded genes. A-site transformed reads were summed across the first four codons with signal for each gene and normalized by the total number of such reads. For genes with 5′ UTRs ( MT-ATP6 , MT-ND4 , MT-CO3 , MT-ND5-dORF ), this includes codons 2–5, where codon 5 is a stop codon for MT-ND5-dORF . For genes with a very short or no leader (all others), this includes codons 6–9. For MT-ATP6 , the signal is confounded by reads from MT-ATP8 , which overlaps the first 15 codons. Samples are plotted in the order listed and error bars show range across replicates. E Variants at MT-ND5-dORF ATG (in green), which is antisense to MT-ND6 (in grey) in the orientation indicated by the schematic. ATA: canonical alternative mitochondrial start codon. GTG: variant start codon shown to efficiently initiate translation. ACG: capable of initiation in reconstituted mammalian mitochondria translation (Lee et al, 2021). AGG: ambiguous (stop or frame-shift inducing). F Genome-wide analysis of SNVs at ATGs. Only equivalently oriented ATGs opposite to codons in known ORFs were included to equalize the constraints (see “ ”). ATGs were required to have at least 2 SNVs to be included

Article Snippet: Mitoribosome profiling datasets for BJ fibroblasts (“Fibro-Rooijers”) [ ], additional HEK293T data (“HEK_Pearce”) [ , ], HCT116 cells (HCT116_Li-1, -2) [ ], and RNase footprinting for HeLa (Hela_Jilab-1, -2) [ ] were downloaded from GEO (GSE48933, GSE133315, and GSE151987), ArrayExpress (E-MTAB-5519), and the SRA (SRR10491343 and SRR10491342).

Techniques: Transformation Assay, Sequencing, Variant Assay, Genome Wide

Journal: Genome Biology

Article Title: Balanced mitochondrial and cytosolic translatomes underlie the biogenesis of human respiratory complexes

doi: 10.1186/s13059-022-02732-9

Figure Lengend Snippet: Balanced translatomes exist in five human cell lines. A Flow of genetic information to encode dual-origin OXPHOS complexes in human cells. B Relative synthesis values of mtDNA- (left graph) and nDNA-encoded (right graph) OXPHOS subunits. Cytoribosome profiling data are from Wu et al. . Each replicate is shown as an individual bar, values given in Additional file : Table S2. Dotted lines show averages across all subunits and samples. Numbers below bars show stoichiometry of each subunit. Synthesis units are tpm/100 for mitochondrial synthesis and tpm for cytosolic synthesis (note that mitochondrial and cytosolic synthesis absolute values cannot be compared across compartments). C Cumulative distribution of relative synthesis rates for nDNA-encoded subunits (top, Additional file : Table S2 and “Methods” for details) and mtDNA-encoded subunits (bottom) of the dual-origin OXPHOS complexes. Log -fold differences in synthesis levels compared to the median value for each complex are shown. Shaded region indicates 2-fold spread. D Synthesis of mtDNA-encoded subunits (tpm/100) compared to synthesis of nDNA-encoded core subunits (tpm) for each complex. Small dots show individual subunits, large dots show average synthesis of subunits within each corresponding complex, following the color code displayed. Fibroblast and HeLa S3 cytoribosome profiling data are from Tirosh et al. and Wu et al. , respectively. Myocytes are post-differentiation-induction day 2 myoblasts. Individual subunit synthesis for each replicate is shown in Fig. 3B (HeLa S3), and Additional file : Figure S3A. Error bars show range in averages across replicates. E RNA abundance of mtDNA-encoded subunits (tpm/100) compared to RNA abundance of nDNA-encoded subunits (tpm) for each complex. Subunits included are identical to those in D . Small dots show individual subunit RNA abundance, large dots show average RNA abundance of subunits within each corresponding complex, following the color code displayed. Error bars show range in averages across replicates. All panels show Pearson correlation, r . F Stoichiometry of average synthesis values for each complex relative to Complex III, which is set to 3, following published convention. Dark gray and light gray bars show empirically determined complex stoichiometry as determined in [ , ], respectively

Article Snippet: Mitoribosome profiling datasets for BJ fibroblasts (“Fibro-Rooijers”) [ ], additional HEK293T data (“HEK_Pearce”) [ , ], HCT116 cells (HCT116_Li-1, -2) [ ], and RNase footprinting for HeLa (Hela_Jilab-1, -2) [ ] were downloaded from GEO (GSE48933, GSE133315, and GSE151987), ArrayExpress (E-MTAB-5519), and the SRA (SRR10491343 and SRR10491342).

Techniques: