An update on sORFs.org: a repository of small ORFs identified by ribosome profiling.

sORFs.org (http://www.sorfs.org) is a public repository of small open reading frames (sORFs) identified by ribosome profiling (RIBO-seq). This update elaborates on the major improvements implemented since its initial release. sORFs.org now additionally supports three more species (zebrafish, rat and Caenorhabditis elegans) and currently includes 78 RIBO-seq datasets, a vast increase compared to the three that were processed in the initial release. Therefore, a novel pipeline was constructed that also enables sORF detection in RIBO-seq datasets comprising solely elongating RIBO-seq data while previously, matching initiating RIBO-seq data was necessary to delineate the sORFs. Furthermore, a novel noise filtering algorithm was designed, able to distinguish sORFs with true ribosomal activity from simulated noise, consequently reducing the false positive identification rate. The inclusion of other species also led to the development of an inner BLAST pipeline, assessing sequence similarity between sORFs in the repository. Building on the proof of concept model in the initial release of sORFs.org, a full PRIDE-ReSpin pipeline was now released, reprocessing publicly available MS-based proteomics PRIDE datasets, reporting on true translation events. Next to reporting those identified peptides, sORFs.org allows visual inspection of the annotated spectra within the Lorikeet MS/MS viewer, thus enabling detailed manual inspection and interpretation.

Comprehensive Peptide Analysis of Mouse Brain Striatum Identifies Novel sORF-Encoded Polypeptides.

Bio-active peptides are involved in the regulation of most physiological processes in the body. Classical bio-active peptides (CBAPs) are cleaved from a larger precursor protein and stored in secretion vesicles from which they are released in the extracellular space. Recently, another non-classical type of bio-active peptides (NCBAPs) has gained interest. These typically are not secreted but instead appear to be translated from short open reading frames (sORF) and released directly into the cytoplasm. In contrast to CBAPs, these peptides are involved in the regulation of intra-cellular processes such as transcriptional control, calcium handling and DNA repair. However, bio-chemical evidence for the translation of sORFs remains elusive. Comprehensive analysis of sORF-encoded polypeptides (SEPs) is hampered by a number of methodological and biological challenges: the low molecular mass (many 4-10 kDa), the low abundance, transient expression and complications in data analysis. We developed a strategy to address a number of these issues. Our strategy is to exclude false positive identifications. In total sample, we identified 926 peptides originated from 37 known (neuro)peptide precursors in mouse striatum. In addition, four SEPs were identified including NoBody, a SEP that was previously discovered in humans and three novel SEPS from 5' untranslated transcript regions (UTRs).

sORFs.org: a repository of small ORFs identified by ribosome profiling.

With the advent of ribosome profiling, a next generation sequencing technique providing a "snap-shot'' of translated mRNA in a cell, many short open reading frames (sORFs) with ribosomal activity were identified. Follow-up studies revealed the existence of functional peptides, so-called micropeptides, translated from these 'sORFs', indicating a new class of bio-active peptides. Over the last few years, several micropeptides exhibiting important cellular functions were discovered. However, ribosome occupancy does not necessarily imply an actual function of the translated peptide, leading to the development of various tools assessing the coding potential of sORFs. Here, we introduce sORFs.org (http://www.sorfs.org), a novel database for sORFs identified using ribosome profiling. Starting from ribosome profiling, sORFs.org identifies sORFs, incorporates state-of-the-art tools and metrics and stores results in a public database. Two query interfaces are provided, a default one enabling quick lookup of sORFs and a BioMart interface providing advanced query and export possibilities. At present, sORFs.org harbors 263 354 sORFs that demonstrate ribosome occupancy, originating from three different cell lines: HCT116 (human), E14_mESC (mouse) and S2 (fruit fly). sORFs.org aims to provide an extensive sORFs database accessible to researchers with limited bioinformatics knowledge, thus enabling easy integration into personal projects.

proBAMconvert: A Conversion Tool for proBAM/proBed.

The introduction of new standard formats, proBAM and proBed, improves the integration of genomics and proteomics information, thus aiding proteogenomics applications. These novel formats enable peptide spectrum matches (PSM) to be stored, inspected, and analyzed within the context of the genome. However, an easy-to-use and transparent tool to convert mass spectrometry identification files to these new formats is indispensable. proBAMconvert enables the conversion of common identification file formats (mzIdentML, mzTab, and pepXML) to proBAM/proBed using an intuitive interface. Furthermore, ProBAMconvert enables information to be output both at the PSM and peptide levels and has a command line interface next to the graphical user interface. Detailed documentation and a completely worked-out tutorial is available at http://probam.biobix.be .

Identification of Small Novel Coding Sequences, a Proteogenomics Endeavor.

The identification of small proteins and peptides has consistently proven to be challenging. However, technological advances as well as multi-omics endeavors facilitate the identification of novel small coding sequences, leading to new insights. Specifically, the application of next generation sequencing technologies (NGS), providing accurate and sample specific transcriptome / translatome information, into the proteomics field led to more comprehensive results and new discoveries. This book chapter focuses on the inclusion of RNA-Seq and RIBO-Seq also known as ribosome profiling, an RNA-Seq based technique sequencing the +/- 30 bp long fragments captured by translating ribosomes. We emphasize the identification of micropeptides and neo-antigens, two distinct classes of small translation products, triggering our current understanding of biology. RNA-Seq is capable of capturing sample specific genomic variations, enabling focused neo-antigen identification. RIBO-Seq can identify translation events in small open reading frames which are considered to be non-coding, leading to the discovery of micropeptides. The identification of small translation products requires the integration of multi-omics data, stressing the importance of proteogenomics in this novel research area.

MYCN-induced nucleolar stress drives an early senescence-like transcriptional program in hTERT-immortalized RPE cells.

MYCN is an oncogenic driver in neural crest-derived neuroblastoma and medulloblastoma. To better understand the early effects of MYCN activation in a neural-crest lineage context, we profiled the transcriptome of immortalized human retina pigment epithelial cells with inducible MYCN activation. Gene signatures associated with elevated MYC/MYCN activity were induced after 24 h of MYCN activation, which attenuated but sustained at later time points. Unexpectedly, MYCN activation was accompanied by reduced cell growth. Gene set enrichment analysis revealed a senescence-like signature with strong induction of p53 and p21 but in the absence of canonical hallmarks of senescence such as ß-galactosidase positivity, suggesting incomplete cell fate commitment. When scrutinizing the putative drivers of this growth attenuation, differential gene expression analysis identified several regulators of nucleolar stress. This process was also reflected by phenotypic correlates such as cytoplasmic granule accrual and nucleolar coalescence. Hence, we propose that the induction of MYCN congests the translational machinery, causing nucleolar stress and driving cells into a transient pre-senescent state. Our findings shed new light on the early events induced by MYCN activation and may help unravelling which factors are required for cells to tolerate unscheduled MYCN overexpression during early malignant transformation.

A methodology for discovering novel brain-relevant peptides: Combination of ribosome profiling and peptidomics.

Brain derived peptides function as signaling molecules in the brain and regulate various physiological and behavioral processes. The low abundance and atypical fragmentation of these brain derived peptides makes detection using traditional proteomic methods challenging. In this study, we introduce and validate a new methodology for the discovery of novel peptides derived from mammalian brain. This methodology combines ribosome profiling and mass spectrometry-based peptidomics. Using this framework, we have identified a novel peptide in mouse whole brain whose expression is highest in the basal ganglia, hypothalamus and amygdala. Although its functional role is unknown, it has been previously detected in peripheral tissue as a component of the mRNA decapping complex. Continued discovery and studies of novel regulating peptides in mammalian brain may also provide insight into brain disorders.

Mitochondrial peptide BRAWNIN is essential for vertebrate respiratory complex III assembly.

The emergence of small open reading frame (sORF)-encoded peptides (SEPs) is rapidly expanding the known proteome at the lower end of the size distribution. Here, we show that the mitochondrial proteome, particularly the respiratory chain, is enriched for small proteins. Using a prediction and validation pipeline for SEPs, we report the discovery of 16 endogenous nuclear encoded, mitochondrial-localized SEPs (mito-SEPs). Through functional prediction, proteomics, metabolomics and metabolic flux modeling, we demonstrate that BRAWNIN, a 71 a.a. peptide encoded by C12orf73, is essential for respiratory chain complex III (CIII) assembly. In human cells, BRAWNIN is induced by the energy-sensing AMPK pathway, and its depletion impairs mitochondrial ATP production. In zebrafish, Brawnin deletion causes complete CIII loss, resulting in severe growth retardation, lactic acidosis and early death. Our findings demonstrate that BRAWNIN is essential for vertebrate oxidative phosphorylation. We propose that mito-SEPs are an untapped resource for essential regulators of oxidative metabolism.

Using the sORFs.Org Database.

Ribosome profiling involves sequencing of approximately 30-base-long stretches of ribosome-protected mRNA. The technique enables genome-wide mapping of RNA undergoing active translation. Numerous small open reading frames have been identified by using ribosome profiling, leading researchers to question the assumed non-functional character of sORFs and to the identification of various important sORF translation products. sORFs.org (https://www.sorfs.org) is a public repository of small open reading frames identified by ribosome profiling in a database of over 3 million sORFs across 78 datasets from six species. sORFs.org is a multi-omics endeavor providing tools and metrics to assess the coding potential of the delineated sORFs. A pipeline is also in place to systematically rescan public mass spectrometry datasets to acquire new experimental evidence for sORF-encoded polypeptides. sORFs.org provides two distinct query interfaces, export functionality, and various visualization tools to enable inspection of the available information. © 2018 by John Wiley & Sons, Inc.

The proBAM and proBed standard formats: enabling a seamless integration of genomics and proteomics data.

On behalf of The Human Proteome Organization (HUPO) Proteomics Standards Initiative, we introduce here two novel standard data formats, proBAM and proBed, that have been developed to address the current challenges of integrating mass spectrometry-based proteomics data with genomics and transcriptomics information in proteogenomics studies. proBAM and proBed are adaptations of the well-defined, widely used file formats SAM/BAM and BED, respectively, and both have been extended to meet the specific requirements entailed by proteomics data. Therefore, existing popular genomics tools such as SAMtools and Bedtools, and several widely used genome browsers, can already be used to manipulate and visualize these formats "out-of-the-box." We also highlight that a number of specific additional software tools, properly supporting the proteomics information available in these formats, are now available providing functionalities such as file generation, file conversion, and data analysis. All the related documentation, including the detailed file format specifications and example files, are accessible at http://www.psidev.info/probam and at http://www.psidev.info/probed .