Identification of Novel Bacterial Microproteins Encoded by Small Open Reading Frames Using a Computational Proteogenomics Workflow.

Genome annotation has historically ignored small open reading frames (smORFs), which encode a class of proteins shorter than 100 amino acids, collectively referred to as microproteins. This cutoff was established to avoid thousands of false positives due to limitations of pure genomics pipelines. Proteogenomics, a computational approach that combines genomics, transcriptomics, and proteomics, makes it possible to accurately identify these short sequences by overlaying different levels of omics evidence. In this chapter, we showcase the use of µProteInS, a bioinformatics pipeline developed for the identification of unannotated microproteins encoded by smORFs in bacteria. The workflow covers all the steps from quality control and transcriptome assembly to the scoring and post-processing of mass spectrometry data. Additionally, we provide an example on how to apply the pipeline's machine learning method to identify high-confidence spectra and pinpoint the most reliable identifications from large datasets.

Massively integrated coexpression analysis reveals transcriptional regulation, evolution and cellular implications of the yeast noncanonical translatome.

BACKGROUND: Recent studies uncovered pervasive transcription and translation of thousands of noncanonical open reading frames (nORFs) outside of annotated genes. The contribution of nORFs to cellular phenotypes is difficult to infer using conventional approaches because nORFs tend to be short, of recent de novo origins, and lowly expressed. Here we develop a dedicated coexpression analysis framework that accounts for low expression to investigate the transcriptional regulation, evolution, and potential cellular roles of nORFs in Saccharomyces cerevisiae. RESULTS: Our results reveal that nORFs tend to be preferentially coexpressed with genes involved in cellular transport or homeostasis but rarely with genes involved in RNA processing. Mechanistically, we discover that young de novo nORFs located downstream of conserved genes tend to leverage their neighbors' promoters through transcription readthrough, resulting in high coexpression and high expression levels. Transcriptional piggybacking also influences the coexpression profiles of young de novo nORFs located upstream of genes, but to a lesser extent and without detectable impact on expression levels. Transcriptional piggybacking influences, but does not determine, the transcription profiles of de novo nORFs emerging nearby genes. About 40% of nORFs are not strongly coexpressed with any gene but are transcriptionally regulated nonetheless and tend to form entirely new transcription modules. We offer a web browser interface ( https://carvunislab.csb.pitt.edu/shiny/coexpression/ ) to efficiently query, visualize, and download our coexpression inferences. CONCLUSIONS: Our results suggest that nORF transcription is highly regulated. Our coexpression dataset serves as an unprecedented resource for unraveling how nORFs integrate into cellular networks, contribute to cellular phenotypes, and evolve.

The Cryptic Bacterial Microproteome.

Microproteins encoded by small open reading frames (smORFs) comprise the "dark matter" of proteomes. Although functional microproteins were identified in diverse organisms from all three domains of life, bacterial smORFs remain poorly characterized. In this comprehensive study of intergenic smORFs (ismORFs, 15-70 codons) in 5,668 bacterial genomes of the family Enterobacteriaceae, we identified 67,297 clusters of ismORFs subject to purifying selection. The ismORFs mainly code for hydrophobic, potentially transmembrane, unstructured, or minimally structured microproteins. Using AlphaFold Multimer, we predicted interactions of some of the predicted microproteins encoded by transcribed ismORFs with proteins encoded by neighboring genes, revealing the potential of microproteins to regulate the activity of various proteins, particularly, under stress. We compiled a catalog of predicted microprotein families with different levels of evidence from synteny analysis, structure prediction, and transcription and translation data. This study offers a resource for investigation of biological functions of microproteins.

Microscale marvels: unveiling the macroscopic significance of micropeptides in human health.

Non-coding RNA encodes micropeptides from small open reading frames located within the RNA. Interestingly, these micropeptides are involved in a variety of functions within the body. They are emerging as the resolving piece of the puzzle for complex biomolecular signaling pathways within the body. Recent studies highlight the pivotal role of small peptides in regulating important biological processes like DNA repair, gene expression, muscle regeneration, immune responses, etc. On the contrary, altered expression of micropeptides also plays a pivotal role in the progression of various diseases like cardiovascular diseases, neurological disorders and several types of cancer, including colorectal cancer, hepatocellular cancer, lung cancer, etc. This review delves into the dual impact of micropeptides on health and pathology, exploring their pivotal role in preserving normal physiological homeostasis and probing their involvement in the triggering and progression of diseases.

Microenzymes: Is There Anybody Out There?

Biological macromolecules are found in different shapes and sizes. Among these, enzymes catalyze biochemical reactions and are essential in all organisms, but is there a limit size for them to function properly? Large enzymes such as catalases have hundreds of kDa and are formed by multiple subunits, whereas most enzymes are smaller, with molecular weights of 20-60 kDa. Enzymes smaller than 10 kDa could be called microenzymes and the present literature review brings together evidence of their occurrence in nature. Additionally, bioactive peptides could be a natural source for novel microenzymes hidden in larger peptides and molecular downsizing could be useful to engineer artificial enzymes with low molecular weight improving their stability and heterologous expression. An integrative approach is crucial to discover and determine the amino acid sequences of novel microenzymes, together with their genomic identification and their biochemical biological and evolutionary functions.

Discovering microproteins: making the most of ribosome profiling data.

Building a reference set of protein-coding open reading frames (ORFs) has revolutionized biological process discovery and understanding. Traditionally, gene models have been confirmed using cDNA sequencing and encoded translated regions inferred using sequence-based detection of start and stop combinations longer than 100 amino-acids to prevent false positives. This has led to small ORFs (smORFs) and their encoded proteins left un-annotated. Ribo-seq allows deciphering translated regions from untranslated irrespective of the length. In this review, we describe the power of Ribo-seq data in detection of smORFs while discussing the major challenge posed by data-quality, -depth and -sparseness in identifying the start and end of smORF translation. In particular, we outline smORF cataloguing efforts in humans and the large differences that have arisen due to variation in data, methods and assumptions. Although current versions of smORF reference sets can already be used as a powerful tool for hypothesis generation, we recommend that future editions should consider these data limitations and adopt unified processing for the community to establish a canonical catalogue of translated smORFs.

smAMPsTK: a toolkit to unravel the smORFome encoding AMPs of plant species.

The pervasive repertoire of plant molecules with the potential to serve as a substitute for conventional antibiotics has led to obtaining better insights into plant-derived antimicrobial peptides (AMPs). The massive distribution of Small Open Reading Frames (smORFs) throughout eukaryotic genomes with proven extensive biological functions reflects their practicality as antimicrobials. Here, we have developed a pipeline named smAMPsTK to unveil the underlying hidden smORFs encoding AMPs for plant species. By applying this pipeline, we have elicited AMPs of various functional activity of lengths ranging from 5 to 100 aa by employing publicly available transcriptome data of five different angiosperms. Later, we studied the coding potential of AMPs-smORFs, the inclusion of diverse translation initiation start codons, and amino acid frequency. Codon usage study signifies no such codon usage biases for smORFs encoding AMPs. Majorly three start codons are prominent in generating AMPs. The evolutionary and conservational study proclaimed the widespread distribution of AMPs encoding genes throughout the plant kingdom. Domain analysis revealed that nearly all AMPs have chitin-binding ability, establishing their role as antifungal agents. The current study includes a developed methodology to characterize smORFs encoding AMPs, and their implications as antimicrobial, antibacterial, antifungal, or antiviral provided by SVM score and prediction status calculated by machine learning-based prediction models. The pipeline, complete package, and the results derived for five angiosperms are freely available at https://github.com/skbinfo/smAMPsTK.Communicated by Ramaswamy H. Sarma.

A vast evolutionarily transient translatome contributes to phenotype and fitness.

Translation is the process by which ribosomes synthesize proteins. Ribosome profiling recently revealed that many short sequences previously thought to be noncoding are pervasively translated. To identify protein-coding genes in this noncanonical translatome, we combine an integrative framework for extremely sensitive ribosome profiling analysis, iRibo, with high-powered selection inferences tailored for short sequences. We construct a reference translatome for Saccharomyces cerevisiae comprising 5,400 canonical and almost 19,000 noncanonical translated elements. Only 14 noncanonical elements were evolving under detectable purifying selection. A representative subset of translated elements lacking signatures of selection demonstrated involvement in processes including DNA repair, stress response, and post-transcriptional regulation. Our results suggest that most translated elements are not conserved protein-coding genes and contribute to genotype-phenotype relationships through fast-evolving molecular mechanisms.

Systematic identification of smORFs in domestic silkworm (Bombyx mori).

The silkworm (Bombyx mori) is not only an excellent model species, but also an important agricultural economic insect. Taking it as the research object, its advantages of low maintenance cost and no biohazard risks are considered. Small open reading frames (smORFs) are an important class of genomic elements that can produce bioactive peptides. However, the smORFs in silkworm had been poorly identified and studied. To further study the smORFs in silkworm, systematic genome-wide identification is essential. Here, we identified and analyzed smORFs in the silkworm using comprehensive methods. Our results showed that at least 738 highly reliable smORFs were found in B. mori and that 34,401 possible smORFs were partially supported. We also identified some differentially expressed and tissue-specific-expressed smORFs, which may be closely related to the characteristics and functions of the tissues. This article provides a basis for subsequent research on smORFs in silkworm, and also hopes to provide a reference point for future research methods for smORFs in other species.

The 2022 Report on the Human Proteome from the HUPO Human Proteome Project.

The 2022 Metrics of the Human Proteome from the HUPO Human Proteome Project (HPP) show that protein expression has now been credibly detected (neXtProt PE1 level) for 18 407 (93.2%) of the 19 750 predicted proteins coded in the human genome, a net gain of 50 since 2021 from data sets generated around the world and reanalyzed by the HPP. Conversely, the number of neXtProt PE2, PE3, and PE4 missing proteins has been reduced by 78 from 1421 to 1343. This represents continuing experimental progress on the human proteome parts list across all the chromosomes, as well as significant reclassifications. Meanwhile, applying proteomics in a vast array of biological and clinical studies continues to yield significant findings and growing integration with other omics platforms. We present highlights from the Chromosome-Centric HPP, Biology and Disease-driven HPP, and HPP Resource Pillars, compare features of mass spectrometry and Olink and Somalogic platforms, note the emergence of translation products from ribosome profiling of small open reading frames, and discuss the launch of the initial HPP Grand Challenge Project, "A Function for Each Protein".

Mitochondrial microproteins link metabolic cues to respiratory chain biogenesis.

Electron transport chain (ETC) biogenesis is tightly coupled to energy levels and availability of ETC subunits. Complex III (CIII), controlling ubiquinol:ubiquinone ratio in ETC, is an attractive node for modulating ETC levels during metabolic stress. Here, we report the discovery of mammalian Co-ordinator of mitochondrial CYTB (COM) complexes that regulate the stepwise CIII biogenesis in response to nutrient and nuclear-encoded ETC subunit availability. The COMA complex, consisting of UQCC1/2 and membrane anchor C16ORF91, facilitates translation of CIII enzymatic core subunit CYTB. Subsequently, microproteins SMIM4 and BRAWNIN together with COMA subunits form the COMB complex to stabilize nascent CYTB. Finally, UQCC3-containing COMC facilitates CYTB hemylation and association with downstream CIII subunits. Furthermore, when nuclear CIII subunits are limiting, COMB is required to chaperone nascent CYTB to prevent OXPHOS collapse. Our studies highlight CYTB synthesis as a key regulatory node of ETC biogenesis and uncover the roles of microproteins in maintaining mitochondrial homeostasis.

Origins, evolution, and physiological implications of de novo genes in yeast.

De novo gene birth is the process by which new genes emerge in sequences that were previously noncoding. Over the past decade, researchers have taken advantage of the power of yeast as a model and a tool to study the evolutionary mechanisms and physiological implications of de novo gene birth. We summarize the mechanisms that have been proposed to explicate how noncoding sequences can become protein-coding genes, highlighting the discovery of pervasive translation of the yeast transcriptome and its presumed impact on evolutionary innovation. We summarize current best practices for the identification and characterization of de novo genes. Crucially, we explain that the field is still in its nascency, with the physiological roles of most young yeast de novo genes identified thus far still utterly unknown. We hope this review inspires researchers to investigate the true contribution of de novo gene birth to cellular physiology and phenotypic diversity across yeast strains and species.

A high-resolution map of human RNA translation.

Translated small open reading frames (smORFs) can have important regulatory roles and encode microproteins, yet their genome-wide identification has been challenging. We determined the ribosome locations across six primary human cell types and five tissues and detected 7,767 smORFs with translational profiles matching those of known proteins. The human genome was found to contain highly cell-type- and tissue-specific smORFs and a subset that encodes highly conserved amino acid sequences. Changes in the translational efficiency of upstream-encoded smORFs (uORFs) and the corresponding main ORFs predominantly occur in the same direction. Integration with 456 mass-spectrometry datasets confirms the presence of 603 small peptides at the protein level in humans and provides insights into the subcellular localization of these small proteins. This study provides a comprehensive atlas of high-confidence translated smORFs derived from primary human cells and tissues in order to provide a more complete understanding of the translated human genome.

Small ORFs as New Regulators of Pri-miRNAs and miRNAs Expression in Human and Drosophila.

MicroRNAs (miRNAs) are small regulatory non-coding RNAs, resulting from the cleavage of long primary transcripts (pri-miRNAs) in the nucleus by the Microprocessor complex generating precursors (pre-miRNAs) that are then exported to the cytoplasm and processed into mature miRNAs. Some miRNAs are hosted in pri-miRNAs annotated as long non-coding RNAs (lncRNAs) and defined as MIRHGs (for miRNA Host Genes). However, several lnc pri-miRNAs contain translatable small open reading frames (smORFs). If smORFs present within lncRNAs can encode functional small peptides, they can also constitute cis-regulatory elements involved in lncRNA decay. Here, we investigated the possible involvement of smORFs in the regulation of lnc pri-miRNAs in Human and Drosophila, focusing on pri-miRNAs previously shown to contain translatable smORFs. We show that smORFs regulate the expression levels of human pri-miR-155 and pri-miR-497, and Drosophila pri-miR-8 and pri-miR-14, and also affect the expression and activity of their associated miRNAs. This smORF-dependent regulation is independent of the nucleotidic and amino acidic sequences of the smORFs and is sensitive to the ribosome-stalling drug cycloheximide, suggesting the involvement of translational events. This study identifies smORFs as new cis-acting elements involved in the regulation of pri-miRNAs and miRNAs expression, in both Human and Drosophila melanogaster.

Quantitative high-confidence human mitochondrial proteome and its dynamics in cellular context.

Mitochondria are key organelles for cellular energetics, metabolism, signaling, and quality control and have been linked to various diseases. Different views exist on the composition of the human mitochondrial proteome. We classified >8,000 proteins in mitochondrial preparations of human cells and defined a mitochondrial high-confidence proteome of >1,100 proteins (MitoCoP). We identified interactors of translocases, respiratory chain, and ATP synthase assembly factors. The abundance of MitoCoP proteins covers six orders of magnitude and amounts to 7% of the cellular proteome with the chaperones HSP60-HSP10 being the most abundant mitochondrial proteins. MitoCoP dynamics spans three orders of magnitudes, with half-lives from hours to months, and suggests a rapid regulation of biosynthesis and assembly processes. 460 MitoCoP genes are linked to human diseases with a strong prevalence for the central nervous system and metabolism. MitoCoP will provide a high-confidence resource for placing dynamics, functions, and dysfunctions of mitochondria into the cellular context.

Exploring Evidence of Non-coding RNA Translation With Trips-Viz and GWIPS-Viz Browsers.

Detection of translation in so-called non-coding RNA provides an opportunity for identification of novel bioactive peptides and microproteins. The main methods used for these purposes are ribosome profiling and mass spectrometry. A number of publicly available datasets already exist for a substantial number of different cell types grown under various conditions, and public data mining is an attractive strategy for identification of translation in non-coding RNAs. Since the analysis of publicly available data requires intensive data processing, several data resources have been created recently for exploring processed publicly available data, such as OpenProt, GWIPS-viz, and Trips-Viz. In this work we provide a detailed demonstration of how to use the latter two tools for exploring experimental evidence for translation of RNAs hitherto classified as non-coding. For this purpose, we use a set of transcripts with substantially different patterns of ribosome footprint distributions. We discuss how certain features of these patterns can be used as evidence for or against genuine translation. During our analysis we concluded that the MTLN mRNA, previously misannotated as lncRNA LINC00116, likely encodes only a short proteoform expressed from shorter RNA transcript variants.

LncRNA-encoded microproteins: A new form of cargo in cell culture-derived and circulating extracellular vesicles.

Advancements in omics-based technologies over the past few years have led to the discovery of numerous biologically relevant peptides encoded by small open reading frames (smORFs) embedded in long noncoding RNA (lncRNA) transcripts (referred to as microproteins here) in a variety of species. However, the mechanisms and modes of action that underlie the roles of microproteins have yet to be fully characterized. Herein, we provide the first experimental evidence of abundant microproteins in extracellular vesicles (EVs) derived from glioma cancer cells, indicating that the EV-mediated transfer of microproteins may represent a novel mechanism for intercellular communication. Intriguingly, when examining human plasma, 48, 11 and 3 microproteins were identified from purified EVs, whole plasma and EV-free plasma, respectively, suggesting that circulating microproteins are primarily enriched in EVs. Most importantly, the preliminary data showed that the expression profile of EV microproteins in glioma patient diverged from the health donors, suggesting that the circulating microproteins in EVs might have potential diagnostic application in identifying patients with glioma.

Developmental regulation of canonical and small ORF translation from mRNAs.

BACKGROUND: Ribosomal profiling has revealed the translation of thousands of sequences outside annotated protein-coding genes, including small open reading frames of less than 100 codons, and the translational regulation of many genes. Here we present an improved version of Poly-Ribo-Seq and apply it to Drosophila melanogaster embryos to extend the catalog of in vivo translated small ORFs, and to reveal the translational regulation of both small and canonical ORFs from mRNAs across embryogenesis. RESULTS: We obtain highly correlated samples across five embryonic stages, with nearly 500 million putative ribosomal footprints mapped to mRNAs, and compare them to existing Ribo-Seq and proteomic data. Our analysis reveals, for the first time in Drosophila, footprints mapping to codons in a phased pattern, the hallmark of productive translation. We propose a simple binomial probability metric to ascertain translation probability. Our results also reveal reproducible ribosomal binding apparently not resulting in productive translation. This non-productive ribosomal binding seems to be especially prevalent amongst upstream short ORFs located in the 5' mRNA leaders, and amongst canonical ORFs during the activation of the zygotic translatome at the maternal-to zygotic transition. CONCLUSIONS: We suggest that this non-productive ribosomal binding might be due to cis-regulatory ribosomal binding and to defective ribosomal scanning of ORFs outside periods of productive translation. Our results are compatible with the main function of upstream short ORFs being to buffer the translation of canonical canonical ORFs; and show that, in general, small ORFs in mRNAs display markers compatible with an evolutionary transitory state towards full coding function.

A Proteogenomic Resource Enabling Integrated Analysis of Listeria Genotype-Proteotype-Phenotype Relationships.

Listeria monocytogenes is an opportunistic foodborne pathogen responsible for listeriosis, a potentially fatal foodborne disease. Many different Listeria strains and serotypes exist, but a proteogenomic resource that bridges the gap in our molecular understanding of the relationships between the Listeria genotypes and phenotypes via proteotypes is still missing. Here, we devised a next-generation proteogenomics strategy that enables the community to rapidly proteotype Listeria strains and relate this information back to the genotype. Based on sequencing and de novo assembly of the two most commonly used Listeria model strains, EGD-e and ScottA, we established two comprehensive Listeria proteogenomic databases. A genome comparison established core- and strain-specific genes potentially responsible for virulence differences. Next, we established a DIA/SWATH-based proteotyping strategy, including a new and robust sample preparation workflow, that enables the reproducible, sensitive, and relative quantitative measurement of Listeria proteotypes. This reusable and publicly available DIA/SWATH library covers 70% of open reading frames of Listeria and represents the most extensive spectral library for Listeria proteotype analysis to date. We used these two new resources to investigate the Listeria proteotype in states mimicking the upper gastrointestinal passage. Exposure of Listeria to bile salts at 37 °C, which simulates conditions encountered in the duodenum, showed significant proteotype perturbations including an increase of FlaA, the structural protein of flagella. Given that Listeria is known to lose its flagella above 30 °C, this was an unexpected finding. The formation of flagella, which might have implications on infectivity, was validated by parallel reaction monitoring and light and scanning electron microscopy. flaA transcript levels did not change significantly upon exposure to bile salts at 37 °C, suggesting regulation at the post-transcriptional level. Together, these analyses provide a comprehensive proteogenomic resource and toolbox for the Listeria community enabling the analysis of Listeria genotype-proteotype-phenotype relationships.

The Growth-Arrest-Specific (GAS)-5 Long Non-Coding RNA: A Fascinating lncRNA Widely Expressed in Cancers.

Long non-coding RNA (lncRNA) genes encode non-messenger RNAs that lack open reading frames (ORFs) longer than 300 nucleotides, lack evolutionary conservation in their shorter ORFs, and do not belong to any classical non-coding RNA category. LncRNA genes equal, or exceed in number, protein-coding genes in mammalian genomes. Most mammalian genomes harbor ~20,000 protein-coding genes that give rise to conventional messenger RNA (mRNA) transcripts. These coding genes exhibit sweeping evolutionary conservation in their ORFs. LncRNAs function via different mechanisms, including but not limited to: (1) serving as "enhancer" RNAs regulating nearby coding genes in cis; (2) functioning as scaffolds to create ribonucleoprotein (RNP) complexes; (3) serving as sponges for microRNAs; (4) acting as ribo-mimics of consensus transcription factors binding sites in genomic DNA; (5) hybridizing to other nucleic acids (mRNAs and genomic DNA); and, rarely, (6) as templates encoding small open reading frames (smORFs) that may encode short proteins. Any given lncRNA may have more than one of these functions. This review focuses on one fascinating case-the growth-arrest-specific (GAS)-5 gene, encoding a complicated repertoire of alternatively-spliced lncRNA isoforms. GAS5 is also a host gene of numerous small nucleolar (sno) RNAs, which are processed from its introns. Publications about this lncRNA date back over three decades, covering its role in cell proliferation, cell differentiation, and cancer. The GAS5 story has drawn in contributions from prominent molecular geneticists who attempted to define its tumor suppressor function in mechanistic terms. The evidence suggests that rodent Gas5 and human GAS5 functions may be different, despite the conserved multi-exonic architecture featuring intronic snoRNAs, and positional conservation on syntenic chromosomal regions indicating that the rodent Gas5 gene is the true ortholog of the GAS5 gene in man and other apes. There is no single answer to the molecular mechanism of GAS5 action. Our goal here is to summarize competing, not mutually exclusive, mechanistic explanations of GAS5 function that have compelling experimental support.