Functional protein-coding small open reading frames (smORFs) are emerging as an important class of genes. However, the number of translated smORFs in the human genome is unclear because proteogenomic methods are not sensitive enough, and, as we show, Ribo-seq strategies require additional measures to ensure comprehensive and accurate smORF annotation. Here, we integrate de novo transcriptome assembly and Ribo-seq into an improved workflow that overcomes obstacles with previous methods, to more confidently annotate thousands of smORFs. Evolutionary conservation analyses suggest that hundreds of smORF-encoded microproteins are likely functional. Additionally, many smORFs are regulated during fundamental biological processes, such as cell stress. Peptides derived from smORFs are also detectable on human leukocyte antigen complexes, revealing smORFs as a source of antigens. Thus, by including additional validation into our smORF annotation workflow, we accurately identify thousands of unannotated translated smORFs that will provide a rich pool of unexplored, functional human genes.
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All sequencing datasets generated in this study are available through GEO (GSE125218).
A custom java script used for three-frame in silico translation of assembled transcripts is included as Supplementary Data 4.
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We thank the Saghatelian laboratory for helpful comments and suggestions throughout the study, and N. Ingolia for advice on RNase I digestion conditions. We also thank M. Ku, N. Hah and the Salk Institute Next Generation Sequencing Core for preparation of RNA-seq libraries and high-throughput sequencing of Ribo-seq and RNA-seq libraries. This research was supported by NIH/NIGMS (R01 GM102491, A.S.), Leona M. and Harry B. Helmsley Charitable Trust grant (A.S.), Dr Frederick Paulsen Chair/Ferring Pharmaceuticals (A.S.), NIH/NIGMS postdoctoral fellowship (F32 GM123685, T.F.M.), George E. Hewitt Foundation for medical research (Q.C.) and a Pioneer Fellowship (D.T.). This work was also supported by the Razavi Newman Integrative Genomics and Bioinformatics Core and the Next Generation Sequencing Core Facilities of the Salk Institute with funding from the NIH-NCICCSG (P30 014195) and the Chapman Foundation.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Figs. 1–14.
List of protein-coding smORFs identified in this study and their properties.
List of significantly regulated ER stress smORFs and annotated genes.
List of smORFs containing conserved protein domains and predicted transmembrane helices, as well as smORFs encoding peptides identified in HLA-I proteomics datasets.
Custom java script used to generate a three-frame ORF database from transcriptome assembly in gtf format.
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Martinez, T.F., Chu, Q., Donaldson, C. et al. Accurate annotation of human protein-coding small open reading frames. Nat Chem Biol 16, 458–468 (2020). https://doi.org/10.1038/s41589-019-0425-0
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