Auswahl der wissenschaftlichen Literatur zum Thema „SmORF peptide“

The last decade witnesses the emergence of the abundant family of smORF peptides, encoded by small ORF (<100 codons), whose biological functions remain largely unexplored. Bioinformatic analyses here identify hundreds of putative smORF peptides expressed in Drosophila imaginal leg discs. Thanks to a functional screen in leg, we found smORF peptides involved in morphogenesis, including the pioneer smORF peptides Pri. Since we identified its target Ubr3 in the epidermis and pri was known to control leg development through poorly understood mechanisms, we investigated the role of Ubr3 in mediating pri function in leg. We found that pri plays several roles during leg development both in patterning and in cell survival. During larval stage, pri activates independently of Ubr3 tarsal transcriptional programs and Notch and EGFR signaling pathways, whereas at larval pupal transition, Pri peptides cooperate with Ubr3 to insure cell survival and leg morphogenesis. Our results highlight Ubr3 dependent and independent functions of Pri peptides and their pleiotropy. Moreover, we reveal that the smORF peptide family is a reservoir of overlooked developmental regulators, displaying distinct molecular functions and orchestrating leg development.

Thousands of naturally occurring peptides differing in their origin, abundance and possible functions have been identified in the tissue and biological fluids of vertebrates, insects, fungi, plants and bacteria. These peptide pools are referred to as intracellular or extracellular peptidomes, and besides a small proportion of well-characterized peptide hormones and defense peptides, are poorly characterized. However, a growing body of evidence suggests that unknown bioactive peptides are hidden in the peptidomes of different organisms. In this review, we present a comprehensive overview of the mechanisms of generation and properties of peptidomes across different organisms. Based on their origin, we propose three large peptide groups—functional protein “degradome”, small open reading frame (smORF)-encoded peptides (smORFome) and specific precursor-derived peptides. The composition of peptide pools identified by mass-spectrometry analysis in human cells, plants, yeast and bacteria is compared and discussed. The functions of different peptide groups, for example the role of the “degradome” in promoting defense signaling, are also considered.

The expression of long noncoding RNAs is highly enriched in the human nervous system. However, the function of neuronal lncRNAs in the cytoplasm and their potential translation remains poorly understood. Here we performed Poly-Ribo-Seq to understand the interaction of lncRNAs with the translation machinery and the functional consequences during neuronal differentiation of human SH-SY5Y cells. We discovered 237 cytoplasmic lncRNAs up-regulated during early neuronal differentiation, 58%–70% of which are associated with polysome translation complexes. Among these polysome-associated lncRNAs, we find 45 small ORFs to be actively translated, 17 specifically upon differentiation. Fifteen of 45 of the translated lncRNA-smORFs exhibit sequence conservation within Hominidea, suggesting they are under strong selective constraint in this clade. The profiling of publicly available data sets revealed that 8/45 of the translated lncRNAs are dynamically expressed during human brain development, and 22/45 are associated with cancers of the central nervous system. One translated lncRNA we discovered is LINC01116, which is induced upon differentiation and contains an 87 codon smORF exhibiting increased ribosome profiling signal upon differentiation. The resulting LINC01116 peptide localizes to neurites. Knockdown of LINC01116 results in a significant reduction of neurite length in differentiated cells, indicating it contributes to neuronal differentiation. Our findings indicate cytoplasmic lncRNAs interact with translation complexes, are a noncanonical source of novel peptides, and contribute to neuronal function and disease. Specifically, we demonstrate a novel functional role for LINC01116 during human neuronal differentiation.

Ribosome profiling reveals the translational dynamics of mRNAs by capturing a ribosomal footprint snapshot. Growing evidence shows that several long non-coding RNAs (lncRNAs) contain small open reading frames (smORFs) that are translated into functional peptides. The difficulty in identifying bona-fide translated smORFs is a constant challenge in experimental and bioinformatics fields due to their unconventional characteristics. This motivated us to isolate human adipose-derived stem cells (hASC) from adipose tissue and perform a ribosome profiling followed by bioinformatics analysis of transcriptome, translatome, and ribosome-protected fragments of lncRNAs. Here, we demonstrated that 222 lncRNAs were associated with the translational machinery in hASC, including the already demonstrated lncRNAs coding microproteins. The ribosomal occupancy of some transcripts was consistent with the translation of smORFs. In conclusion, we were able to identify a subset of 15 lncRNAs containing 35 smORFs that likely encode functional microproteins, including four previously demonstrated smORF-derived microproteins, suggesting a possible dual role of these lncRNAs in hASC self-renewal.

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.

In 2004, when the protein estimate from the finished human genome was only 24,000, the surprise was compounded as reviewed estimates fell to 19,000 by 2014. However, variability in the total canonical protein counts (i.e. excluding alternative splice forms) of open reading frames (ORFs) in different annotation portals persists. This work assesses these differences and possible causes. A 16-year analysis of Ensembl and UniProtKB/Swiss-Prot shows convergence to a protein number of ~20,000. The former had shown some yo-yoing, but both have now plateaued. Nine major annotation portals, reviewed at the beginning of 2017, gave a spread of counts from 21,819 down to 18,891. The 4-way cross-reference concordance (within UniProt) between Ensembl, Swiss-Prot, Entrez Gene and the Human Gene Nomenclature Committee (HGNC) drops to 18,690, indicating methodological differences in protein definitions and experimental existence support between sources. The Swiss-Prot and neXtProt evidence criteria include mass spectrometry peptide verification and also cross-references for antibody detection from the Human Protein Atlas. Notwithstanding, hundreds of Swiss-Prot entries are classified as non-coding biotypes by HGNC. The only inference that protein numbers might still rise comes from numerous reports of small ORF (smORF) discovery. However, while there have been recent cases of protein verifications from previous miss-annotation of non-coding RNA, very few have passed the Swiss-Prot curation and genome annotation thresholds. The post-genomic era has seen both advances in data generation and improvements in the human reference assembly. Notwithstanding, current numbers, while persistently discordant, show that the earlier yo-yoing has largely ceased. Given the importance to biology and biomedicine of defining the canonical human proteome, the task will need more collaborative inter-source curation combined with broader and deeper experimental confirmation in vivo and in vitro of proteins predicted in silico. The eventual closure could be well be below ~19,000.

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.

Glioma is a lethal malignant brain cancer, and many reports have shown that abnormalities in the behavior of water and ion channels play an important role in regulating tumor proliferation, migration, apoptosis, and differentiation. Recently, new studies have suggested that some long noncoding RNAs containing small open reading frames can encode small peptides and form oligomers for water or ion regulation. However, because the peptides are difficult to identify, their functional mechanisms are far from being clearly understood. In this study, we used bioinformatics methods to identify and evaluate lncRNAs, which may encode small transmembrane peptides in gliomas. Combining ab initio homology modeling, molecular dynamics simulations, and free energy calculations, we constructed a predictive model and predicted the oligomer channel activity of peptides by identifying the lncRNA ORFs. We found that one key hub lncRNA, namely, DLEU1, which contains two smORFs (ORF1 and ORF8), encodes small peptides that form pentameric channels. The mechanics of water and ion (Na+ and Cl-) transport through this pentameric channel were simulated. The potential mean force of the H2O molecules along the two ORF-encoded peptide channels indicated that the energy barrier was different between ORF1 and ORF8. The ORF1-encoded peptide pentamer acted as a self-assembled water channel but not as an ion channel, and the ORF8 permeated neither ions nor water. This work provides new methods and theoretical support for further elucidation of the function of lncRNA-encoded small peptides and their role in cancer. Additionally, this study provides a theoretical basis for drug development.

Small open reading frames (smORFs) are short DNA sequences that are able to encode small peptides of less than 100 amino acids. Study of these elements has been neglected despite thousands existing in our genomes. We and others previously showed that peptides as short as 11 amino acids are translated and provide essential functions during insect development. Here, we describe two peptides of less than 30 amino acids regulating calcium transport, and hence influencing regular muscle contraction, in the Drosophila heart. These peptides seem conserved for more than 550 million years in a range of species from flies to humans, in which they have been implicated in cardiac pathologies. Such conservation suggests that the mechanisms for heart regulation are ancient and that smORFs may be a fundamental genome component that should be studied systematically.

Non-coding RNAs (ncRNAs) are essential players in many cellular processes, from normal development to oncogenic transformation. Initially, ncRNAs were defined as transcripts that lacked an open reading frame (ORF). However, multiple lines of evidence suggest that certain ncRNAs encode small peptides of less than 100 amino acids. The sequences encoding these peptides are known as small open reading frames (smORFs), many initiating with the traditional AUG start codon but terminating with atypical stop codons, suggesting a different biogenesis. The ncRNA-encoded peptides (ncPEPs) are gradually becoming appreciated as a new class of functional molecules that contribute to diverse cellular processes, and are deregulated in different diseases contributing to pathogenesis. As multiple publications have identified unique ncPEPs, we appreciated the need for assembling a new web resource that could gather information about these functional ncPEPs. We developed FuncPEP, a new database of functional ncRNA encoded peptides, containing all experimentally validated and functionally characterized ncPEPs. Currently, FuncPEP includes a comprehensive annotation of 112 functional ncPEPs and specific details regarding the ncRNA transcripts that encode these peptides. We believe that FuncPEP will serve as a platform for further deciphering the biologic significance and medical use of ncPEPs. The link for FuncPEP database can be found at the end of the Introduction Section.

De récentes avancées technologiques ont révélé que des petits ORF (Open Reading Frame), dont la taille est inférieure à 100 codons et qui étaient jusqu'à présent considérés comme non codants à cause de leur petite taille, peuvent être traduits en des milliers de petits peptides smORF (small ORF) dans tous les organismes. Plusieurs études ont montré que des peptides smORF peuvent interagir avec des protéines canoniques et réguler leur activité. La famille des peptides smORF représente ainsi un réservoir largement inexploré de potentiels régulateurs. Un crible fonctionnel mené chez la Drosophile nous a permis d'identifier de nouveaux peptides smORF impliqués dans le développement, dont plusieurs sont issus de la traduction d'ARN polycistroniques. Mon projet de recherche de thèse a été de caractériser la fonction chez la Drosophile de 1) Tic et Tac, deux peptides smORF inconnus codés par un ARN bicistronique , et de 2) Sem1/DSS1, un peptide très conservé. La première partie de mes travaux de thèse a été d'analyser la fonction biologique de deux peptides smORF inconnus codés par un ARN bicistronique que nous avons nommé tictac. J'ai démontré que Tic et Tac sont deux peptides traduits à partir du même ARN bicistronique tictac, dont la séquence en AA et la localisation cellulaire sont différentes. In vivo, tictac est fortement exprimé dans les organes reproducteurs mâles. A la suite d'un accouplement, les changements comportementaux et physiologiques caractérisant la réponse post-accouplement de la femelle, induits par le fluide séminal, sont affectés en absence de tictac chez les mâles. Grâce à l'analyse du transcriptome sur les organes reproducteurs mâles, je montre que l'absence de tictac conduit à une dérégulation transcriptionnelle de 150 gènes, dont 10% codent pour des composants du liquide séminal, montrant que tictac régule sa composition. La délétion spécifique de tic ou de tac, montre qu'ils ont des fonctions in vivo différentes mais que tous deux sont impliqués dans la synthèse du liquide séminal. L'analyse des glandes accessoires, l'organe fonctionnel équivalent de la prostate humaine fabriquant le fluide séminal, révèle que leur maturation et que leur fonction sécrétrice sont régulées par tictac. La recherche d'homologues de Tic et de Tac indique que Tic possède un domaine peptidique présentant une homologie avec un sous-domaine de RQC1 de levure, impliquée dans le contrôle qualité des ribosomes, alors que Tac possède un domaine serglycine, connu chez l'homme pour avoir des fonctions importantes dans la sécrétion. Ainsi, les peptides smORF Tic et Tac contribueraient à la synthèse et à la sécrétion des protéines du fluide séminal. Dans un second temps, j'ai mené l'analyse moléculaire de Sem1, une protéine intrinsèquement désordonnée connue pour interagir et réguler plusieurs complexes macromoléculaires. Comme ces données ont été générées dans des modèles in cellulo et in vitro, j'ai mené l'analyse moléculaire de Sem1 pour la première fois in vivo. J'ai montré que Sem1 est essentielle car sa délétion induit une létalité au stade larvaire. De plus, son absence entraine une apoptose massive se généralisant à l'ensemble du tissu de manière non-cellulaire autonome. Pour découpler les fonctions moléculaires de Sem1 et identifier le(s) domaine(s) protéique(s) essentiels à la survie cellulaire, j'ai généré pour chaque domaine fonctionnel une forme mutée, dont j'ai induit l'expression dans un contexte déplété de Sem1. Cette analyse structure-fonction met en évidence que les deux domaines interagissant avec l'ubiquitine sont essentiels au développement. En conclusion, mes travaux de thèse mettent en lumière de nouvelles fonctions des peptides smORF dans la reproduction et la survie cellulaire, illustrant la capacité des peptides smORF à agir sur de multiples complexes macro-moléculaires et contrôler ainsi de nombreux processus physiologiques
Recent technological advances have revealed that small ORFs (Open Reading Frame), which are less than 100 codons in size and were previously considered non-coding due to their small size, can be translated into thousands of smORF (small ORF) peptides in all organisms. Several studies have shown that smORF peptides can interact with canonical proteins and regulate their activity. Thus, the smORF peptide family represents a vast unexplored reservoir of potential regulators. A functional screen carried out in Drosophila identified new smORF peptides involved in development, several of which are derived from polycistronic RNA translation. My thesis research project was to characterize the function in Drosophila of 1) Tic and Tac, two unknown smORF peptides encoded by a bicistronic RNA, and 2) Sem1/DSS1, a highly conserved peptide. The first part of my thesis was to analyze the biological function of two unknown smORF peptides encoded by a bicistronic RNA that we named tictac. I demonstrated that Tic and Tac are two peptides translated from the same bicistronic RNA tictac, but with different AA sequences and cellular localization. In vivo, tictac is highly expressed in male reproductive organs. Following mating, behavioral and physiological changes characterizing the post-mating response in female, which are mainly induced by the seminal fluid, are affected in the absence of tictac in males. Using transcriptome analysis on male reproductive organs, I show that the absence of tictac leads to transcriptional deregulation of 150 genes. Among them, 10% code for seminal fluid components, proving that tictac regulates its composition. Specific deletion of either tic or tac highlights that they have different in vivo functions, but that both are involved in seminal fluid synthesis. The analysis of accessory glands, the functional seminal fluid-producing organ equivalent to the human prostate, reveals that their maturation and secretory function are regulated by tictac. The search for homologs of Tic and Tac indicates that Tic has a peptide domain with strong homology to a subdomain of RQC1, a yeast protein involved in ribosome quality control, and Tac has a serglycin domain, known in human to have important functions in secretion. My data therefore suggests that Tic and Tac smORF peptides may contribute to the synthesis and secretion of seminal fluid proteins. In a second step, I carried out the molecular analysis of Sem1, an intrinsically disordered protein known to interact with and regulate several macromolecular complexes. As these data were only generated in cellulo and in vitro models, I carried out the molecular analysis of Sem1 for the first time in vivo. I showed that Sem1 is essential, as its deletion induces lethality at the larval stage. Moreover, its absence leads to massive apoptosis that generalizes to the whole tissue in a non-cell-autonomous manner. To uncouple the molecular functions of Sem1 and identify the protein domains essential for cell survival, I generated a mutated form of each functional domain, then induced its expression in the absence of endogenous Sem1. This structure-function analysis revealed that two domains known to interact with ubiquitin are essential for development. In conclusion, my thesis work highlights new functions of smORF peptides in reproduction and cell survival, illustrating the ability of smORF peptides to act on multiple macro-molecular complexes, thus controlling numerous physiological processes