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Data availability
Ribo-seq and matched RNA sequencing (RNA-seq) data for adult brain (cortex), liver and testis from the different mammals used to assess potential translation of the loci reported by An et al.3 stem from our previous study7 and can be retrieved from ArrayExpress at www.ebi.ac.uk/arrayexpress/ (accession code E-MTAB-7247). Ribo-seq and matched RNA-seq data for the developing human cortex stem from a previous study21 and were retrieved from the database of Genotypes and Phenotypes (accession code phs002489). The developmental human RNA-seq data stem from our previous study16 and can be retrieved from ArrayExpress (accession code E-MTAB-6814). Analyses of the various gene annotations were based on different versions of Ensembl (http://www.ensembl.org), as detailed in the main text. Protein annotation for ENSG00000205704 was also assessed using UniProt (www.uniprot.org/uniprotkb; accession code A0A590UK83), as described in the main text.
Code availability
To create the plots and analyse the data, we used the R (https://www.r-project.org/) packages ggplot2 (version 3.3.5) and tidyverse (version 1.3.1) throughout the manuscript. We used Integrative Genomics Viewer (version 2.3.60) to visualize the mapped reads. We wrote a custom R script to calculate the translational likelihoods (https://github.com/evgenyleushkin/translationLR). We used codeml from the Phylogenetic Analysis by Maximum Likelihood (version 4.8) package (http://abacus.gene.ucl.ac.uk/software/paml.html) to assess the dN/dS values. The sequence alignments were extracted from existing University of California, Santa Cruz (https://genome.ucsc.edu/) alignments (https://hgdownload.soe.ucsc.edu/downloads.html).
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Acknowledgements
We thank the members of H.K.’s laboratory for discussions.
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E.L. and H.K. conceived of and organized the study, analysed the data and co-wrote the manuscript. E.L. performed all of the large-scale analyses.
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Nature Ecology & Evolution thanks Anne-Ruxandra Carvunis, Sebastiaan van Heesch and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Translation and transcription of the SMIM45 (ENSG00000205704) gene during human cortex development.
a, IGV (Integrative Genomics Viewer24) browser screenshot of ribosome profiling footprints within SMIM45 in fetal (12–23 weeks post-conception) human cortical development, based on ribosome profiling data from Duffy et al. (ref. 21). b, IGV browser screenshot of total RNA sequencing reads during different stages of human cortical development, based on RNA sequencing data from Cardoso-Moreira et al. (ref. 16).
Extended Data Fig. 2 Evolutionary relationships of the SMIM45-201 and SEPTIN3, and developmental expression of SEPTIN3.
a, SMIM45 and SEPTIN-3 are adjacent but independent protein-coding genes in the human genome, and correspond to regions within the single protein-coding gene (annotated as Septin-3) in bony (ray-finned) fish, as illustrated for the fathead minnow. Amino acid sequence similarity was detected with an iterative PSI-BLAST (ref. 25) search (sequence identity, ‘id’, is indicated). b, Expression (transcript abundance) pattern of SEPTIN3 (that is, that of the longest isoform, SEPTIN3-207) across development of different organs in human, based on RNA sequencing data from Cardoso-Moreira et al. (ref. 16). Error bars indicate the range of values across biological replicates.
Extended Data Fig. 3 Translational profile for ENSG00000120742 (SERP1) in the brain.
Transcription/translation signals along the SERP1 sequence. From top to bottom: total RNA (TR) read counts averaged in five trinucleotide windows, ribosome profiling footprint (RP) counts averaged in five trinucleotide windows; translational likelihood ratio (LR) (see legend of Fig. 2 for details); ORFs with LR > 10 in first, second, and third reading frame (relative to position 1 in a spliced transcript; intron positions indicated by gray vertical lines); resulting translated ORF annotation of SERP1 includes a uORF (studied by An et al., to the left) and the main (downstream) canonical ORF/coding region (to the right). See the main text for details.
Extended Data Fig. 4 Translational profile for ENSG00000145063.
Transcription/translation signals along the ENSG00000145063 sequence in three organs (brain, liver, testis). Upper panel: exonic sequence ENSG00000145063, with the translated coding region shaded in turquoise and the ORF studied by An et al. in red (bold). Lower panels, respectively for the three organs, from top to bottom: total RNA (TR) read counts averaged in five trinucleotide windows, ribosome profiling footprint (RP) counts averaged in five trinucleotide windows; translational likelihood ratio (LR) (see legend of Fig. 2 for details); ORFs with LR > 10 in first, second, and third reading frame (relative to position 1 in a spliced transcript; intron positions indicated by gray vertical lines); resulting translated (brain/cortex) ORF annotation of ENSG00000145063 includes the main canonical ORF/coding region (light blue box, bottom figure). The non-translated ORF considered by An et al. is indicated by a red line in lower (left) ORF figure. See the main text for details. Icons reproduced from ref. 16, Springer Nature Limited.
Extended Data Fig. 5 Translational profile for ENSG00000260456 (C16orf95) and its mouse ortholog.
Transcription/translation signals along the ENSG00000260456 sequence in the testis of human and mouse. Panel a (top): human exonic sequence for ENSG00000260456, with the translated coding region shaded in turquoise and the ORF studied by An et al. in red (bold). Panel b (top): mouse exonic sequence for ENSMUSG00000031809, with the translated coding region shaded in turquoise. Below the sequences, respectively for human (a) and mouse (b), from top to bottom: total RNA (TR) read counts averaged in five trinucleotide windows, ribosome profiling footprint (RP) counts averaged in five trinucleotide windows; translational likelihood ratio (LR) (see legend of Fig. 2 for details); ORFs with LR > 10 in first, second, and third reading frame (relative to position 1 in a spliced transcript; intron positions indicated by gray vertical lines); resulting translated (brain/cortex) ORF annotations of ENSG00000260456 and ENSMUSG00000031809, respectively, include the main canonical ORF/coding region (light blue box, bottom figure). The non-translated ORF considered by An et al. in humans is indicated by a red line in the lower (left) ORF figure. See the main text for details. Icons reproduced from ref. 16, Springer Nature Limited.
Extended Data Fig. 6 Translational profile for ENSG00000203930.
Transcription/translation signals along the ENSG00000203930 sequence in the human brain. a, three-exon transcript isoform with the ORF studied by An et al. (ENST00000370535). b, main, efficiently translated two-exon isoform (ENST00000607004). From top to bottom in both panels, respectively: total RNA (TR) read counts averaged in five trinucleotide windows, ribosome profiling footprint (RP) counts averaged in five trinucleotide windows; translational likelihood ratio (LR) (see legend of Fig. 2 for details); ORFs with LR > 10 in first, second, and third reading frame (relative to position 1 in a spliced transcript; intron positions indicated by gray vertical lines); resulting translated (brain/cortex) ORF annotations of the two isoforms, respectively. Only the main isoform ENST00000607004 is efficiently translated (5.1 ribosomal footprints per nucleotide), whereas for the isoform ENST00000370535, the ORF sequence not overlapping with that of the main isoform shows a low translation signal (average footprint coverage across ORF: 0.28 ribosomal footprints per nucleotide). See the main text for details. Icons reproduced from ref. 16, Springer Nature Limited.
Supplementary information
Supplementary Tables
Supplementary Table 1. Reassessment of the 74 loci reported by An et al.3. Supplementary Table 2. Overview of the peptide identifiers from Chen et al.18—where 64 of the loci analysed by An et al.3 had already been reported—for which assignment to a database (PRIDE) and hence identification of the tissue source was possible.
Supplementary Data 1
Annotation of translated ORFs and expressed transcripts for three organs (brain, liver and testis).
Supplementary Data 2
Sequence alignment files for four genes (ENSG00000120742, ENSG00000145063, ENSG00000203930 and ENSG00000260456).
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Leushkin, E., Kaessmann, H. Identification of old coding regions disproves the hominoid de novo status of genes. Nat Ecol Evol 8, 1826–1830 (2024). https://doi.org/10.1038/s41559-024-02513-6
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DOI: https://doi.org/10.1038/s41559-024-02513-6
