AbstractsORFs.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.

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.

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.

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).

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.