PROTEOFORMER: deep proteome coverage through ribosome profiling and MS integration

Crappé, Jeroen; Ndah, Elvis; Koch, Alexander; Steyaert, Sandra; Gawron, Daria; Keulenaer, Sarah De; Meester, Ellen De; Meyer, Tim De; Criekinge, Wim Van; Damme, Petra Van; Menschaert, Gerben

doi:10.1093/nar/gku1283

Cited by 138 publications

(155 citation statements)

References 36 publications

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“…The data may also be useful for characterizing translation of different transcript isoforms. With its high sensitivity for identification of translated ORFs and its quantitative nature, ribosome profiling can also serve as a proxy for the proteome or assist proteomics studies (57)(58)(59). Finally, as the Arabidopsis genome is among the best annotated, we expect ribosome profiling will be an even more powerful approach to uncovering novel ORFs and improving genome annotation when applied to less well-characterized organisms.…”

Section: Discussionmentioning

confidence: 99%

Super-resolution ribosome profiling reveals unannotated translation events inArabidopsis

Hsu

Calviello

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

210

363

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Deep sequencing of ribosome footprints (ribosome profiling) maps and quantifies mRNA translation. Because ribosomes decode mRNA every 3 nt, the periodic property of ribosome footprints could be used to identify novel translated ORFs. However, due to the limited resolution of existing methods, the 3-nt periodicity is observed mostly in a global analysis, but not in individual transcripts. Here, we report a protocol applied to Arabidopsis that maps over 90% of the footprints to the main reading frame and thus offers super-resolution profiles for individual transcripts to precisely define translated regions. The resulting data not only support many annotated and predicted noncanonical translation events but also uncover small ORFs in annotated noncoding RNAs and pseudogenes. A substantial number of these unannotated ORFs are evolutionarily conserved, and some produce stable proteins. Thus, our study provides a valuable resource for plant genomics and an efficient optimization strategy for ribosome profiling in other organisms.translation | ribosome footprint | Ribo-seq | ncRNA | sORF

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Section: Discussionmentioning

confidence: 99%

Super-resolution ribosome profiling reveals unannotated translation events inArabidopsis

Hsu

Calviello

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

210

363

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“…RiboTaper, ORF-RATER and PROTEOFORMER (Crappé et al 2015;Fields et al 2015;Calviello et al 2016;Chun et al 2016), based on integration of ribosome profiling data. PROTEOFORMER and RiboTaper combine ribosome profiling data with an implemented construction of protein sequences from thus detected ORFs.…”

Section: Available Online Resourcesmentioning

confidence: 99%

Recognition of the polycistronic nature of human genes is critical to understanding the genotype-phenotype relationship

et al. 2018

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Technological advances promise unprecedented opportunities for whole exome sequencing and proteomic analyses of populations. Currently, data from genome and exome sequencing or proteomic studies are searched against reference genome annotations. This provides the foundation for research and clinical screening for genetic causes of pathologies. However, current genome annotations substantially underestimate the proteomic information encoded within a gene. Numerous studies have now demonstrated the expression and function of alternative (mainly small, sometimes overlapping) ORFs within mature gene transcripts. This has important consequences for the correlation of phenotypes and genotypes. Most alternative ORFs are not yet annotated because of a lack of evidence, and this absence from databases precludes their detection by standard proteomic methods, such as mass spectrometry. Here, we demonstrate how current approaches tend to overlook alternative ORFs, hindering the discovery of new genetic drivers and fundamental research. We discuss available tools and techniques to improve identification of proteins from alternative ORFs and finally suggest a novel annotation system to permit a more complete representation of the transcriptomic and proteomic information contained within a gene. Given the crucial challenge of distinguishing functional ORFs from random ones, the suggested pipeline emphasizes both experimental data and conservation signatures. The addition of alternative ORFs in databases will render identification less serendipitous and advance the pace of research and genomic knowledge. This review highlights the urgent medical and research need to incorporate alternative ORFs in current genome annotations and thus permit their inclusion in hypotheses and models, which relate phenotypes and genotypes.

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“…Programs that allow non-specialists to easily interpret Ribo-seq data sets have only recently become available (Crappé et al 2015;Legendre et al 2015) and many analyses are not yet supported in published packages. Here we describe an R package riboSeqR (released under Bioconductor, 2014) that provides a set of methods for parsing ribosomal profiling data from multiple samples, aligning to coding sequences, inferring alternative reading frames, and plotting average and transcript-specific behavior of these data.…”

Section: Introductionmentioning

confidence: 99%

The use of duplex-specific nuclease in ribosome profiling and a user-friendly software package for Ribo-seq data analysis

Chung

Hardcastle

Jones

et al. 2015

RNA

120

132

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Ribosome profiling is a technique that permits genome-wide, quantitative analysis of translation and has found broad application in recent years. Here we describe a modified profiling protocol and software package designed to benefit more broadly the translation community in terms of simplicity and utility. The protocol, applicable to diverse organisms, including organelles, is based largely on previously published profiling methodologies, but uses duplex-specific nuclease (DSN) as a convenient, species-independent way to reduce rRNA contamination. We show that DSN-based depletion compares favorably with other commonly used rRNA depletion strategies and introduces little bias. The profiling protocol typically produces high levels of triplet periodicity, facilitating the detection of coding sequences, including upstream, downstream, and overlapping open reading frames (ORFs) and an alternative ribosome conformation evident during termination of protein synthesis. In addition, we provide a software package that presents a set of methods for parsing ribosomal profiling data from multiple samples, aligning reads to coding sequences, inferring alternative ORFs, and plotting average and transcript-specific aspects of the data. Methods are also provided for extracting the data in a form suitable for differential analysis of translation and translational efficiency.

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PROTEOFORMER: deep proteome coverage through ribosome profiling and MS integration

Cited by 138 publications

References 36 publications

Super-resolution ribosome profiling reveals unannotated translation events inArabidopsis

Super-resolution ribosome profiling reveals unannotated translation events inArabidopsis

Recognition of the polycistronic nature of human genes is critical to understanding the genotype-phenotype relationship

The use of duplex-specific nuclease in ribosome profiling and a user-friendly software package for Ribo-seq data analysis

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