Short Open Reading Frames Research Articles

364-OR: Beta-Cell Glucose-Regulated Micropeptide 1 (BGRM1) Controls Cellular Homeostasis in Pancreatic Beta Cells

Accumulating evidence demonstrates that certain types of long-noncoding RNAs (lncRNA) encode very short open reading frames, called micropeptides. By regulating larger protein complexes, these micropeptides can regulate fundamental biological processes such as skeletal muscle development and cell migration. However, the ability of micropeptides to maintain cellular and metabolic homeostasis in islets is unknown. Using a bioinformatics approach, we comprehensively analyzed the protein-coding potential of lncRNAs that are expressed in the human pancreas, and identified more than 30 that likely encode micropeptides. We further characterized one conserved IncRNA that encodes a 65-amino-acid micropeptide, which we named beta cell glucose regulated micropeptide 1 (BGRM1). We found that BGRM1 expression was altered in islets from obese or diabetic humans and mice. Overexpression of BGRM1 elevated glucose-stimulated insulin production in INS-1 cells and in islets from humans and mice. BGRM1 promotes endoplasmic reticulum (ER) calcium efflux and enhances cytosolic calcium levels, which suggests it mediates insulin production is in part by modulating intracellular calcium homeostasis. This notion is further supported by our mass spectra analysis that reveals several BGRM1 binding partners involved in ER calcium homeostasis in beta cells. Notably, BGRM1 is localized in the ER and protects beta cells from ER stress. Taken together, our study provides the first evidence that lncRNA encoded micropeptides play a critical role in pancreatic beta cell function. These results provide a foundation on which to perform future comprehensive analyses of micropeptide function and pathophysiological impact on diabetes. Disclosure M. Li: None. F. Shao: None. M.E. Sweat: None. Q. Qian: None. Y. Guo: None. S.B. Stephens: None. Y. Imai: None. L. Yang: None. H. Cao: None.

Ribosomal Peptides and Small Proteins on the Rise

Genetically encoded and ribosomally synthesised peptides and small proteins act as important regulators in fundamental cellular processes, including gene expression, development, signalling and metabolism. Moreover, they also play a crucial role in eukaryotic and prokaryotic defence against microorganisms. Extremely diverse in size and structure, they are often subject to extensive post-translational modification. Recent technological advances are now allowing the analysis of the whole cellular transcriptome and proteome, revealing the presence of hundreds of long-overlooked alternative and short open reading frames (short ORFs, or sORFs) in mRNA and supposedly noncoding RNAs. However, in many instances the biological roles of their translational products remain to be elucidated. Here we provide an overview on the intriguing structural and functional diversity of ribosomally synthesised peptides and newly discovered peptides and small proteins.

To Code or Not to Code: What Is the Linc?

The annotation of the mammalian protein coding genome is incomplete. Arbitrary open reading frame (ORF) size restriction and the absolute requirement for a methionine codon as the sole initiator of translation, have constrained identification of potentially important transcripts with non‐canonical protein coding potential. Using unbiased transcriptomic approaches in macrophages responding to bacterial infection, we show widespread ribosome association with a large number of RNAs that were previously annotated as “non‐protein coding”. Although the ability of such non‐canonical ORFs to encode functional protein is controversial, we identify a plethora of novel short and non‐ATG initiated ORFs with the ability to generate stable and spatially distinct proteins. Importantly, we show that the translation of a novel ORF ‘hidden’ within the long non‐coding RNA Aw112010 is essential for the orchestration of mucosal immunity during both bacterial infection and colitis. Together this work expands our interpretation of the protein coding genome and demonstrates the critical nature of proteinaceous products generated from non‐canonical ORFs to the immune response in vivo. We therefore propose that the misannotation of non‐canonical ORF‐containing genes as non‐coding RNAs may obscure the essential role of a multitude of previously undiscovered protein coding genes in immunity and disease.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

An Upstream Short Open Reading Frame in the Angiotensin Type 1 Receptor Regulates the β‐Arrestin Signaling Pathway

The angiotensin type 1 receptor (AT1R) plays a critical role in blood pressure control and thus furthering our understanding of its regulation has physiological and clinical significance. We previously showed a short open reading frame (sORF) in exon (E)2 of the 5' leader sequence of the AT1aR mRNA inhibits AT1R mRNA translation and receptor internalization. In this study, we investigated the role of this sORF in AT1R signaling. The sORF start codon in E2 was disrupted by site‐directed mutagenesis [E1,2(−108T),3‐AT1aR] (MT), which along with the wild type unmutated sORF [E1,2,3‐AT1aR] (WT) were cloned into an expression plasmid [EGFPN2]. Human embryonic kidney‐293 cells were then transfected with WT or MT plasmids. No significant differences were found in the time course of Ang II‐induced ERK1/2 activation between 0–80 min at angiotensin doses of 10 nm (n=3) and 100 nM (n=3); however, the peak response to 10 nM Ang II at 5 min was 1.9‐fold higher in cells transfected with MT compared to WT plasmids (p<0.05; n=4). Furthermore, peak ERK1/2 activation was 1.3‐fold higher in cells transfected with MT compared to WT plasmids after stimulation with Sar1,Ile4,Ile8‐Ang II, a biased AT1R agonist which selectively activates the β‐arrestin‐dependent pathway [pERK1/2/total ERK1/2 normalized to AT1R Bmax: WT, 1.8E‐05 ± 9.5E‐07 vs. MT, 2.3E‐05 ± 3.4E‐06, p<0.05; n=3]. A time course of Sar1,Ile4,Ile8‐Ang II‐induced ERK1/2 activation from 0–80 minutes at 30 μM also showed greater ERK1/2 activation in cells transfected with the MT compared to WT plasmids [p<0.05; n=4]. Since β‐arrestins are implicated to play a role in apoptotic signaling, we are also studying the effect of this sORF on Ang II‐induced cell proliferation and both early and late apoptosis by flow cytometry. This study supports our previous findings that a seven amino acid peptide (PEP7) encoded by this sORF is a selective inhibitor of AT1R‐mediated ERK1/2 activation. These findings not only have implications for the regulation of AT1Rs in physiological and pathological conditions but also for the superfamily of G protein coupled receptors, many of which possess sORFs of unknown function in their 5’ leader sequence.Support or Funding InformationNIH/NHLBI R01‐HL121456 to KS NIH TL1 Award TL1TR001431This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Depletion of High-Molecular-Mass Proteins for the Identification of Small Proteins and Short Open Reading Frame Encoded Peptides in Cellular Proteomes.

The identification of small proteins and peptides (below ca. 100-150 amino acids) in complex biological samples is hampered by the dominance of higher-molecular-weight proteins. On the contrary, the increasing knowledge about alternative or short open reading frames creates a need for methods that allow the existence of the corresponding gene products to be proven in proteomics experiments. We present an acetonitrile-based precipitation methodology that depletes the majority of proteins above ca. 15 kDa. Parameters such as depletion mixture composition, pH, and temperature were optimized using a model protein mixture, and the method was evaluated in comparison with the established differential solubility method. The approach was applied to the analysis of the low-molecular-weight proteome of the archaea Methanosarcina mazei by means of LC-MS. The data clearly show a beneficial effect from a reduction of complexity, especially in terms of the quality of MS/MS-based identification of small proteins. This fast, detergent-free method allowed for, with minimal sample manipulation, the successful identification of several not yet identified short open reading frame encoded peptides in M. mazei.

MRNA dynamics and alternative conformations adopted under low and high arginine concentrations control polyamine biosynthesis in Salmonella.

Putrescine belongs to the large group of polyamines, an essential class of metabolites that exists throughout all kingdoms of life. The Salmonella speF gene encodes an inducible ornithine decarboxylase that produces putrescine from ornithine. Putrescine can be also synthesized from arginine in a parallel metabolic pathway. Here, we show that speF expression is controlled at multiple levels through regulatory elements contained in a long leader sequence. At the heart of this regulation is a short open reading frame, orf34, which is required for speF production. Translation of orf34 interferes with Rho-dependent transcription termination and helps to unfold an inhibitory RNA structure sequestering speF ribosome-binding site. Two consecutive arginine codons in the conserved domain of orf34 provide a third level of speF regulation. Uninterrupted translation of orf34 under conditions of high arginine allows the formation of a speF mRNA structure that is degraded by RNase G, whereas ribosome pausing at the consecutive arginine codons in the absence of arginine enables the formation of an alternative structure that is resistant to RNase G. Thus, the rate of ribosome progression during translation of the upstream ORF influences the dynamics of speF mRNA folding and putrescine production. The identification of orf34 and its regulatory functions provides evidence for the evolutionary conservation of ornithine decarboxylase regulatory elements and putrescine production.

A collagen hydrogel loaded with HDAC7-derived peptide promotes the regeneration of infarcted myocardium with functional improvement in a rodent model

Myocardial infarction (MI) leads to the loss of cardiomyocytes, left ventricle (LV) dilation, and cardiac dysfunction, eventually developing into heart failure. Most of the strategies for MI therapy require biomaterials that can support tissue regeneration. In this study, we hypothesized that the extracellular matrix (ECM)-derived collagen I hydrogel loaded with histone deacetylase 7 (HDAC7)-derived-phosphorylated 7-amino-acid peptide (7Ap) could restrain LV remodeling and improve cardiac function after MI. An MI model was established by ligation of the left anterior descending coronary artery (LAD) of C57/B6 mice. The 7Ap-loaded collagen I hydrogel was intramyocardially injected to the infarcted region of the LV wall of the heart. After local delivery, the 7Ap-collagen increased neo-microvessel formation, enhanced stem cell antigen-1 positive (Sca-1+) stem cell recruitment and differentiation, decreased cellular apoptosis, and promoted cardiomyocyte cycle progression. Furthermore, the 7Ap-collagen restricted the fibrosis of the LV wall, reduced the infarct wall thinning, and improved cardiac performance significantly at 2 weeks post-MI. These results highlight the promising implication of 7Ap-collagen as a novel candidate for MI therapy. Statement of SignificanceThe mammalian myocardium has a limited regenerative capability following myocardial infarction (MI). MI leads to extensive loss of cardiomyocytes, thus culminating in adverse cardiac remodeling and congestive heart failure. In situ tissue regeneration through endogenous cell mobilization has great potential for tissue regeneration. A 7-amino-acid-peptide (7A) domain encoded by a short open-reading frame (sORF) of the HDAC7 gene. The phosphorylated from of 7A (7Ap) has been reported to promote in situ tissue repair via the mobilization and recruitment of endogenous stem cell antigen-1 positive (Sca-l+) stem cells. In this study, 7Ap was shown to improve H9C2 cell survival, in vitro. In vivo investigations in a mouse MI model demonstrated that intra-myocardial delivery of 7Ap-loaded collagen hydrogel promoted neovascularization, stimulated Sca-l+ stem cell recruitment and differentiation, reduced cardiomyocyte apoptosis and promoted cell cycle progression. As a result, treated infarcted hearts had increased wall thickness, had improved heart function and exhibited attenuation of adverse cardiac remodeling, observed for up to 2 weeks. Overall, these results highlighted the positive impact of implanting 7Ap-collagen as a novel constituent for MI repair.

Translational Regulation by Upstream Open Reading Frames and Human Diseases.

Short upstream open reading frames (uORFs) are cis-acting elements located within the 5'-leader sequence of transcripts and are defined by an initiation codon in-frame with a termination codon located upstream or downstream of its main ORF (mORF) initiation codon. Recent genome-wide ribosome profiling studies have confirmed the widespread presence of uORFs and have shown that many uORFs can initiate with non-AUG codons. uORFs can impact gene expression of the downstream mORF by triggering mRNA decay or by regulating translation. Thus, disruption or creation of uORFs can elicit the development of several genetic diseases. Here, we review the mechanisms by which AUG- and non-AUG uORFs regulate translation. We also show some examples of uORF deregulation in human genetic diseases, focusing mainly on cancer. The knowledge of how uORF deregulation drives the onset of a disease, points out the need to screen the 5'-leader sequences of the transcripts in search for potential disease-related variants. This information will be relevant for the implementation of new diagnostic and/or therapeutic tools.

The Protein Coded by a Short Open Reading Frame, Not by the Annotated Coding Sequence, Is the Main Gene Product of the Dual-Coding Gene MIEF1

Proteogenomics and ribosome profiling concurrently show that genes may code for both a large and one or more small proteins translated from annotated coding sequences (CDSs) and unannotated alternative open reading frames (named alternative ORFs or altORFs), respectively, but the stoichiometry between large and small proteins translated from a same gene is unknown. MIEF1, a gene recently identified as a dual-coding gene, harbors a CDS and a newly annotated and actively translated altORF located in the 5′UTR. Here, we use absolute quantification with stable isotope-labeled peptides and parallel reaction monitoring to determine levels of both proteins in two human cells lines and in human colon. We report that the main MIEF1 translational product is not the canonical 463 amino acid MiD51 protein but the small 70 amino acid alternative MiD51 protein (altMiD51). These results demonstrate the inadequacy of the single CDS concept and provide a strong argument for incorporating altORFs and small proteins in functional annotations.

The translation of non-canonical open reading frames controls mucosal immunity.

The annotation of the mammalian protein coding genome is incomplete. Arbitrary open reading frame (ORF) size restriction and the absolute requirement for a methionine codon as the sole initiator of translation, have constrained identification of potentially important transcripts with non-canonical protein coding potential1,2. Using unbiased transcriptomic approaches in macrophages responding to bacterial infection, we show widespread ribosome association with a large number of RNAs that were previously annotated as “non-protein coding”. Although the ability of such non-canonical ORFs to encode functional protein is controversial3,4, we identify a plethora of novel short and non-ATG initiated ORFs with the ability to generate stable and spatially distinct proteins. Importantly, we show that the translation of a novel ORF ‘hidden’ within the long non-coding RNA Aw112010 is essential for the orchestration of mucosal immunity during both bacterial infection and colitis. Together this work expands our interpretation of the protein coding genome and demonstrates the critical nature of proteinaceous products generated from non-canonical ORFs to the immune response in vivo. We therefore propose that the misannotation of non-canonical ORF-containing genes as non-coding RNAs may obscure the essential role of a multitude of previously undiscovered protein coding genes in immunity and disease.

Identification of missing proteins in Saccharomyces cerevisiae by small protein-based enrichment

Small proteins (SPs) are defined as peptides of 100 amino acids or less encoded by short open reading frames (sORFs). SPs participate in a wide range of functions in cells, including gene regulating, cell signaling and metabolism. However, most annotated SPs in all living organisms are currently lacking expression evidence at the protein level and regarded as missing proteins (MPs). High efficient SPs identification is the prerequisite for their functional study and contribution to MPs searching. In this study, we identified 72 SPs and successfully validated 9 MPs from Saccharomyces cerevisiae based on SPs enrichment strategy. In-depth analysis showed that the missing factors of MPs were low molecular weight, low abundant, hydrophobicity, lower codon usage bias and unstable. The small protein-based enrichment can be used as MPs searching strategy, which might provide the foundation for their further function research.

A novel uORF-based regulatory mechanism controls translation of the human MDM2 and eIF2D mRNAs during stress

Short upstream open reading frames (uORFs) are the most prevalent cis-acting regulatory elements in the mammalian transcriptome which can orchestrate mRNA translation. Apart from being “passive roadblocks” that decrease expression of the main coding regions, particular uORFs can serve as specific sensors for changing conditions, thus regulating translation in response to cell stress. Here we report a novel uORF-based regulatory mechanism that is employed under conditions of hyperosmotic stress by at least two human mRNAs, coding for translation reinitiation/recycling factor eIF2D and E3 ubiquitin ligase MDM2. This novel mode of translational control selectively downregulates their expression and requires as few as one uORF. Using a set of reporter mRNAs and fleeting mRNA transfection (FLERT) technique, we provide evidence that the phenomenon does not rely on delayed reinitiation, altered AUG recognition, ribosome stalling, mRNA destabilization or other known mechanisms. Instead, it is based on events taking place at uORF stop codon or immediately downstream. Functional aspects and implications of the novel regulatory mechanism to cell physiology are discussed.

Peptid-codierende kleine RNAs in Bakterien

Although short open reading frames (sORFs) are present in all genomes, they have often been missed in annotations. Nevertheless, a small variety of peptides involved in different pathways were identified serendipitously and investigated in more detail. Among them are type I toxins, nucleic acid and metal chaperones, membrane components, stabilizing factors of large protein complexes, and regulatory peptides.

Insights into the Noncoding RNA-encoded Peptides.

Considerable evidence has indicated that most noncoding RNA (ncRNA) transcripts act directly as functional RNAs rather than as encoded peptides. However, a recent study of ribosome occupancy reported that many large intergenic ncRNAs (lincRNAs) are bound by ribosomes, raising the possibility that they are translated into proteins. These lincRNAs contain either Internal Ribosomal Entry Sites (IRES) or short Open Reading Frames (sORFs), and other translation elements, which can be translated into peptides with physiological functions. This review discusses three major types of ncRNA-encoded peptides (ncPEPs). First, microRNA(miRNA)-encoded peptides (miPEPs) are translated from their primary transcripts of miRNA genes and can promote the development of plant roots. Second, a long ncRNA(lncRNA) containing sORF encodes a 90 residues long regulatory peptide. Upon the amino acid response, the lncRNA-encoded peptide reduces the activity of mammalian Target Rapamycin complex 1 (mTORC1) and promotes muscle regeneration. Third, a circular RNA (circRNA) of the Na+/Ca2+ exchanger gene 1 (NCX1) exon 2 transcript encodes a truncated NCX1 protein exhibiting the Na+/Ca2+ exchange activity. It is also of worth noting that the majority of ncRNAs is not translated. This review summarizes the current understanding of the translatability of ncRNAs and the functions of ncPEPs in cellular processes, which may provide novel insights into the roles of ncPEPs.

Lso2 is a conserved ribosome-bound protein required for translational recovery in yeast.

Ribosome-binding proteins function broadly in protein synthesis, gene regulation, and cellular homeostasis, but the complete complement of functional ribosome-bound proteins remains unknown. Using quantitative mass spectrometry, we identified late-annotated short open reading frame 2 (Lso2) as a ribosome-associated protein that is broadly conserved in eukaryotes. Genome-wide crosslinking and immunoprecipitation of Lso2 and its human ortholog coiled-coil domain containing 124 (CCDC124) recovered 25S ribosomal RNA in a region near the A site that overlaps the GTPase activation center. Consistent with this location, Lso2 also crosslinked to most tRNAs. Ribosome profiling of yeast lacking LSO2 (lso2Δ) revealed global translation defects during recovery from stationary phase with translation of most genes reduced more than 4-fold. Ribosomes accumulated at start codons, were depleted from stop codons, and showed codon-specific changes in occupancy in lso2Δ. These defects, and the conservation of the specific ribosome-binding activity of Lso2/CCDC124, indicate broadly important functions in translation and physiology.

Two Crinivirus-Conserved Small Proteins, P5 and P9, Are Indispensable for Efficient Lettuce infectious yellows virus Infectivity in Plants.

Genomic analysis of Lettuce infectious yellows virus (LIYV) has revealed two short open reading frames (ORFs) on LIYV RNA2, that are predicted to encode a 5-kDa (P5) and a 9-kDa (P9) protein. The P5 ORF is part of the conserved quintuple gene block in the family Closteroviridae, while P9 orthologs are found in all Criniviruses. In this study, the expression of LIYV P5 and P9 proteins was confirmed; P5 is further characterized as an endoplasmic reticulum (ER)-localized integral transmembrane protein and P9 is a soluble protein. The knockout LIYV mutants presented reduced symptom severity and virus accumulation in Nicotiana benthamiana or lettuce plants, indicating their importance in efficient virus infection. The P5 mutant was successfully complemented by a dislocated P5 in the LIYV genome. The structural regions of P5 were tested and all were found to be required for the appropriate functions of P5. In addition, P5, as well as its ortholog P6, encoded by Citrus tristeza virus (CTV) and another ER-localized protein encoded by LIYV RNA1, were found to cause cell death when expressed in N. benthamiana plants from a TMV vector, and induce ER stress and the unfolded protein response (UPR).

High-level, constitutive expression of the mgtC gene confers increased thermotolerance on Salmonella enterica serovar Typhimurium.

We found that mutations that increased the transcription of the mgtCBR (Mg2+ transport-related) operon conferred increased thermotolerance on this organism. The 5' leader of the mgtCBR mRNA contains two short open reading frames (ORFs), mgtM and mgtP, whose translation regulates the expression of the mgtCBR operon by a mechanism that is similar to attenuation in amino acid biosynthetic operons. We obtained two types of mutations that resulted in elevated transcription of the operon: defects in the mgtM ribosome-binding site, impairing the translation of this ORF and deletions encompassing the stop codon of mgtM that extend the translation of this ORF across a downstream Rho termination site. These mgtM mutations give further insights into the mechanism of the transcriptional control of the mgtCBR operon that we discuss in this work. We show that the increased thermotolerance requires elevated expression of the mgtC gene, but functional mgtB and mgtR, which respectively encode an Mg2+ transporter and a regulatory protein, are dispensable for this response. MgtC has been shown to have complex functions, including a requirement for virulence, flagella-independent motility and synthesis of cellulose and we now found that it has a role in the regulation of thermotolerance.

Short Open Reading Frames and Their Encoded Peptides.

Proteomics 2018, 18, 1700035 DOI: 10.1002/pmic.201700035 In this article, the following sentences and reference were erroneously published: “In their technical brief, Khitun and Slavoff describe their workflow to detect and validate non-annotated proteins with non-AUG start sites.[11] This proteomic detection method is also exemplified with the characterization of the non-AUG initiated small protein (alt-RPL36) co-encoded with human ribosomal protein L36.” [11] S. A. K. Slavoff, Proteomics 2018 The Editorial Office apologizes for this error.

DENR-MCTS1 heterodimerization and tRNA recruitment are required for translation reinitiation.

The succession of molecular events leading to eukaryotic translation reinitiation—whereby ribosomes terminate translation of a short open reading frame (ORF), resume scanning, and then translate a second ORF on the same mRNA—is not well understood. Density-regulated reinitiation and release factor (DENR) and multiple copies in T-cell lymphoma-1 (MCTS1) are implicated in promoting translation reinitiation both in vitro in translation extracts and in vivo. We present here the crystal structure of MCTS1 bound to a fragment of DENR. Based on this structure, we identify and experimentally validate that DENR residues Glu42, Tyr43, and Tyr46 are important for MCTS1 binding and that MCTS1 residue Phe104 is important for tRNA binding. Mutation of these residues reveals that DENR-MCTS1 dimerization and tRNA binding are both necessary for DENR and MCTS1 to promote translation reinitiation in human cells. These findings thereby link individual residues of DENR and MCTS1 to specific molecular functions of the complex. Since DENR–MCTS1 can bind tRNA in the absence of the ribosome, this suggests the DENR–MCTS1 complex could recruit tRNA to the ribosome during reinitiation analogously to the eukaryotic initiation factor 2 (eIF2) complex in cap-dependent translation.

Reduced expression of C/EBPβ-LIP extends health and lifespan in mice

Ageing is associated with physical decline and the development of age-related diseases such as metabolic disorders and cancer. Few conditions are known that attenuate the adverse effects of ageing, including calorie restriction (CR) and reduced signalling through the mechanistic target of rapamycin complex 1 (mTORC1) pathway. Synthesis of the metabolic transcription factor C/EBPβ-LIP is stimulated by mTORC1, which critically depends on a short upstream open reading frame (uORF) in the Cebpb-mRNA. Here, we describe that reduced C/EBPβ-LIP expression due to genetic ablation of the uORF delays the development of age-associated phenotypes in mice. Moreover, female C/EBPβΔuORF mice display an extended lifespan. Since LIP levels increase upon aging in wild type mice, our data reveal an important role for C/EBPβ in the aging process and suggest that restriction of LIP expression sustains health and fitness. Thus, therapeutic strategies targeting C/EBPβ-LIP may offer new possibilities to treat age-related diseases and to prolong healthspan.