Improved Ribo-seq enables identification of cryptic translation events

Erhard, Florian; Halenius, Anne; Zimmermann, Cosima; L’Hernault, Anne; Kowalewski, Daniel J.; Weekes, Michael P.; Stevanović, Stefan; Zimmer, Ralf; Dölken, Lars

doi:10.1038/nmeth.4631

Cited by 158 publications

(183 citation statements)

References 23 publications

(30 reference statements)

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“…Of these, 192 were from smORFs identified in at least two Ribo-Seq experiments (Supplementary Data 3), and 41 lacked an in-frame AUG start codon. A previous study was also able to detect over 100 microprotein peptides in the same proteomics dataset, which is consistent with and expanded by these data 65 . Representative spectra of peptides from smORF-encoded microproteins demonstrate good fragment ion coverage, regardless of the number of times detected and cell lines found in by Ribo-Seq (Fig.…”

Section: Resultssupporting

confidence: 89%

An Improved Human smORF Annnotation Workflow Combining De Novo Transcriptome Assembly and Ribo-Seq

Martínez

Chu

Donaldson

et al. 2019

Preprint

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Protein-coding small open reading frames (smORFs) are emerging as an important class of genes, however, the coding capacity of smORFs in the human genome is unclear. By integrating de novo transcriptome assembly and Ribo-Seq, we confidently annotate thousands of novel translated smORFs in three human cell lines. We find that smORF translation prediction is noisier than for annotated coding sequences, underscoring the importance of analyzing multiple experiments and footprinting conditions. These smORFs are located within non-coding and antisense transcripts, the UTRs of mRNAs, and unannotated transcripts. Analysis of RNA levels and translation efficiency during cellular stress identifies regulated smORFs, providing an approach to select smORFs for further investigation. Sequence conservation and signatures of positive selection indicate that encoded microproteins are likely functional. Additionally, proteomics data from enriched human leukocyte antigen complexes validates the translation of hundreds of smORFs and positions them as a source of novel antigens. Thus, smORFs represent a significant number of important, yet unexplored human genes.

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Section: Resultssupporting

confidence: 89%

An Improved Human smORF Annnotation Workflow Combining De Novo Transcriptome Assembly and Ribo-Seq

Martínez

Chu

Donaldson

et al. 2019

Preprint

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“…The HSV-1 genome is about 152kb in size and known to encode at least 80 open reading frames (ORFs), many of which have been extensively studied. Large-scale RNA-seq and ribosome profiling recently revealed that the coding capacity of three other herpesviruses, namely human cytomegalovirus (HCMV), Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr Virus (EBV) is significantly larger than previously thought [2][3][4][5] . For HCMV and KSHV, in particular, hundreds of new viral gene products were identified.…”

Section: Mainmentioning

confidence: 98%

Integrative functional genomics decodes herpes simplex virus 1

Whisnant

Jürges

Hennig

et al. 2019

Preprint

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SummarySince the genome of herpes simplex virus 1 (HSV-1) was first sequenced more than 30 years ago, its predicted 80 genes have been intensively studied. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identified a total of 201 viral transcripts and 284 open reading frames (ORFs) including all known and 46 novel large ORFs. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of novel viral ORFs as well as N-terminal extensions (NTEs) and truncations thereof. We show that key viral regulators and structural proteins possess NTEs, which initiate from non-canonical start codons and govern subcellular protein localization and packaging. We validated a novel non-canonical large spliced ORF in the ICP0 locus and identified a 93 aa ORF overlapping ICP34.5 that is thus also deleted in the FDA-approved oncolytic virus Imlygic. Finally, we extend the current nomenclature to include all novel viral gene products. Taken together, this work provides a valuable resource for future functional studies, vaccine design and oncolytic therapies.

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“…Often, MS-based immunopeptidomic discoveries are limited to the standard, available protein sequence database, usually containing only annotated protein-coding sequences. Recently, several studies have included protein sequences derived from the translation of transcripts identified from RNA-Seq, or from ribosome profiling, into MS-based searches 9,[23][24][25][26][27][28] . Overall, these studies warrant further development in many key aspects: Importantly, elevated false discovery rates (FDRs) for the non-canonical space can occur when MS reference data are populated with protein sequences derived from all potential three-or six-frame translations of transcribed regions 29 .…”

Section: Introductionmentioning

confidence: 99%

Integrated Proteogenomic Deep Sequencing and Analytics Accurately Identify Non-Canonical Peptides in Tumor Immunopeptidomes

Chong

Müller

Pak

et al. 2019

Preprint

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Efforts to precisely identify tumor human leukocyte antigen (HLA) bound peptides capable of mediating T cell-based tumor rejection still face important challenges. Recent studies suggest that non-canonical tumor-specific HLA peptides that derive from annotated non-coding regions could elicit anti-tumor immune responses. However, sensitive and accurate massspectrometry (MS)-based proteogenomics approaches are required to robustly identify these non-canonical peptides. We present an MS-based analytical approach that characterizes the non-canonical tumor HLA peptide repertoire, by incorporating whole exome sequencing, bulk and single cell transcriptomics, ribosome profiling, and a combination of two MS/MS search tools. This approach results in the accurate identification of hundreds of shared and tumorspecific non-canonical HLA peptides and of an immunogenic peptide from a downstream reading frame in the melanoma stem cell marker gene ABCB5. It holds great promise for the discovery of novel cancer antigens for cancer immunotherapy.

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Improved Ribo-seq enables identification of cryptic translation events

Cited by 158 publications

References 23 publications

An Improved Human smORF Annnotation Workflow Combining De Novo Transcriptome Assembly and Ribo-Seq

An Improved Human smORF Annnotation Workflow Combining De Novo Transcriptome Assembly and Ribo-Seq

Integrative functional genomics decodes herpes simplex virus 1

Integrated Proteogenomic Deep Sequencing and Analytics Accurately Identify Non-Canonical Peptides in Tumor Immunopeptidomes

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