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.

FuncPEP v2.0: An Updated Database of Functional Short Peptides Translated from Non-Coding RNAs.

Over the past decade, there have been reports of short novel functional peptides (less than 100 aa in length) translated from so-called non-coding RNAs (ncRNAs) that have been characterized using mass spectrometry (MS) and large-scale proteomics studies. Therefore, understanding the bivalent functions of some ncRNAs as transcripts that encode both functional RNAs and short peptides, which we named ncPEPs, will deepen our understanding of biology and disease. In 2020, we published the first database of functional peptides translated from non-coding RNAs-FuncPEP. Herein, we have performed an update including the newly published ncPEPs from the last 3 years along with the categorization of host ncRNAs. FuncPEP v2.0 contains 152 functional ncPEPs, out of which 40 are novel entries. A PubMed search from August 2020 to July 2023 incorporating specific keywords was performed and screened for publications reporting validated functional peptides derived from ncRNAs. We did not observe a significant increase in newly discovered functional ncPEPs, but a steady increase. The novel identified ncPEPs included in the database were characterized by a wide array of molecular and physiological parameters (i.e., types of host ncRNA, species distribution, chromosomal density, distribution of ncRNA length, identification methods, molecular weight, and functional distribution across humans and other species). We consider that, despite the fact that MS can now easily identify ncPEPs, there still are important limitations in proving their functionality.

Novel insights into small open reading frame-encoded micropeptides in hepatocellular carcinoma: A potential breakthrough.

Traditionally, non-coding RNAs (ncRNAs) are regarded as a class of RNA transcripts that lack encoding capability; however, advancements in technology have revealed that some ncRNAs contain small open reading frames (sORFs) that are capable of encoding micropeptides of approximately 150 amino acids in length. sORF-encoded micropeptides (SEPs) have emerged as intriguing entities in hepatocellular carcinoma (HCC) research, shedding light on this previously unexplored realm. Recent studies have highlighted the regulatory functions of SEPs in the occurrence and progression of HCC. Some SEPs exhibit inhibitory effects on HCC, but others facilitate its development. This discovery has revolutionized the landscape of HCC research and clinical management. Here, we introduce the concept and characteristics of SEPs, summarize their associations with HCC, and elucidate their carcinogenic mechanisms in HCC metabolism, signaling pathways, cell proliferation, and metastasis. In addition, we propose a step-by-step workflow for the investigation of HCC-associated SEPs. Lastly, we discuss the challenges and prospects of applying SEPs in the diagnosis and treatment of HCC. This review aims to facilitate the discovery, optimization, and clinical application of HCC-related SEPs, inspiring the development of early diagnostic, individualized, and precision therapeutic strategies for HCC.

Non-Mature miRNA-Encoded Micropeptide miPEP166c Stimulates Anthocyanin and Proanthocyanidin Synthesis in Grape Berry Cells.

The phenylpropanoid and flavonoid pathways exhibit intricate regulation, not only influenced by environmental factors and a complex network of transcription factors but also by post-transcriptional regulation, such as silencing by microRNAs and miRNA-encoded micropeptides (miPEPs). VviMYBC2-L1 serves as a transcriptional repressor for flavonoids, playing a crucial role in coordinating the synthesis of anthocyanin and proanthocyanidin. It works in tandem with their respective transcriptional activators, VviMYBA1/2 and VviMYBPA1, to maintain an equilibrium of flavonoids. We have discovered a miPEP encoded by miR166c that appears to target VviMYBC2-L1. We conducted experiments to test the hypothesis that silencing this transcriptional repressor through miPEP166c would stimulate the synthesis of anthocyanins and proanthocyanidins. Our transcriptional analyses by qPCR revealed that the application of exogenous miPEP166c to Gamay Fréaux grape berry cells resulted in a significant upregulation in flavonoid transcriptional activators (VviMYBA1/2 and VviMYBPA1) and structural flavonoid genes (VviLDOX and VviDFR), as well as genes involved in the synthesis of proanthocyanidins (VviLAR1 and VviANR) and anthocyanins (VviUFGT1). These findings were supported by the increased enzyme activities of the key enzymes UFGT, LAR, and ANR, which were 2-fold, 14-fold, and 3-fold higher, respectively, in the miPEP166c-treated cells. Ultimately, these changes led to an elevated total content of anthocyanins and proanthocyanidins.

Emphasizing the Role of Long Non-Coding RNAs (lncRNA), Circular RNA (circRNA), and Micropeptides (miPs) in Plant Biotic Stress Tolerance.

Biotic stress tolerance in plants is complex as it relies solely on specific innate immune responses from different plant species combating diverse pathogens. Each component of the plant immune system is crucial to comprehend the molecular basis underlying sustainable resistance response. Among many other regulatory components, long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) have recently emerged as novel regulatory control switches in plant development and stress biology. Besides, miPs, the small peptides (100-150 amino acids long) encoded by some of the non-coding portions of the genome also turned out to be paramount regulators of plant stress. Although some studies have been performed in deciphering the role of miPs in abiotic stress tolerance, their function in regulating biotic stress tolerance is still largely elusive. Hence, the present review focuses on the roles of long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) in combating biotic stress in plants. The probable role of miPs in plant-microbe interaction is also comprehensively highlighted. This review enhances our current understanding of plant lncRNAs, circRNAs, and miPs in biotic stress tolerance and raises intriguing questions worth following up.

Micropeptides: origins, identification, and potential role in metabolism-related diseases. / å¾®è‚½ï¼šèµ·æºã€é‰´å®šåŠå ¶åœ¨ä»£è°¢ç›¸å ³ç–¾ç— ä¸­çš„ä½œç”¨.

With the development of modern sequencing techniques and bioinformatics, genomes that were once thought to be noncoding have been found to encode abundant functional micropeptides (miPs), a kind of small polypeptides. Although miPs are difficult to analyze and identify, a number of studies have begun to focus on them. More and more miPs have been revealed as essential for energy metabolism homeostasis, immune regulation, and tumor growth and development. Many reports have shown that miPs are especially essential for regulating glucose and lipid metabolism and regulating mitochondrial function. MiPs are also involved in the progression of related diseases. This paper reviews the sources and identification of miPs, as well as the functional significance of miPs for metabolism-related diseases, with the aim of revealing their potential clinical applications.

Microproteins transitioning into a new Phase: Defining the undefined.

Recent advancements in omics technologies have unveiled a hitherto unknown group of short polypeptides called microproteins (miPs). Despite their size, accumulating evidence has demonstrated that miPs exert varied and potent biological functions. They act in paracrine, juxtracrine, and endocrine fashion, maintaining cellular physiology and driving diseases. The present study focuses on biochemical and biophysical analysis and characterization of twenty-four human miPs using distinct computational methods, including RIDAO, AlphaFold2, D2P2, FuzDrop, STRING, and Emboss Pep wheel. miPs often lack well-defined tertiary structures and may harbor intrinsically disordered regions (IDRs) that play pivotal roles in cellular functions. Our analyses define the physicochemical properties of an essential subset of miPs, elucidating their structural characteristics and demonstrating their propensity for driving or participating in liquid-liquid phase separation (LLPS) and intracellular condensate formation. Notably, miPs such as NoBody and pTUNAR revealed a high propensity for LLPS, implicating their potential involvement in forming membrane-less organelles (MLOs) during intracellular LLPS and condensate formation. The results of our study indicate that miPs have functionally profound implications in cellular compartmentalization and signaling processes essential for regulating normal cellular functions. Taken together, our methodological approach explains and highlights the biological importance of these miPs, providing a deeper understanding of the unusual structural landscape and functionality of these newly defined small proteins. Understanding their functions and biological behavior will aid in developing targeted therapies for diseases that involve miPs.

Small open reading frames: a comparative genetics approach to validation.

Open reading frames (ORFs) with fewer than 100 codons are generally not annotated in genomes, although bona fide genes of that size are known. Newer biochemical studies have suggested that thousands of small protein-coding ORFs (smORFs) may exist in the human genome, but the true number and the biological significance of the micropeptides they encode remain uncertain. Here, we used a comparative genomics approach to identify high-confidence smORFs that are likely protein-coding. We identified 3,326 high-confidence smORFs using constraint within human populations and evolutionary conservation as additional lines of evidence. Next, we validated that, as a group, our high-confidence smORFs are conserved at the amino-acid level rather than merely residing in highly conserved non-coding regions. Finally, we found that high-confidence smORFs are enriched among disease-associated variants from GWAS. Overall, our results highlight that smORF-encoded peptides likely have important functional roles in human disease.

linc-mipep and linc-wrb encode micropeptides that regulate chromatin accessibility in vertebrate-specific neural cells.

Thousands of long intergenic non-coding RNAs (lincRNAs) are transcribed throughout the vertebrate genome. A subset of lincRNAs enriched in developing brains have recently been found to contain cryptic open-reading frames and are speculated to encode micropeptides. However, systematic identification and functional assessment of these transcripts have been hindered by technical challenges caused by their small size. Here, we show that two putative lincRNAs (linc-mipep, also called lnc-rps25, and linc-wrb) encode micropeptides with homology to the vertebrate-specific chromatin architectural protein, Hmgn1, and demonstrate that they are required for development of vertebrate-specific brain cell types. Specifically, we show that NMDA receptor-mediated pathways are dysregulated in zebrafish lacking these micropeptides and that their loss preferentially alters the gene regulatory networks that establish cerebellar cells and oligodendrocytes - evolutionarily newer cell types that develop postnatally in humans. These findings reveal a key missing link in the evolution of vertebrate brain cell development and illustrate a genetic basis for how some neural cell types are more susceptible to chromatin disruptions, with implications for neurodevelopmental disorders and disease.

The Meeting of Micropeptides with Major Ca2+ Pumps in Inner Membranes-Consideration of a New Player, SERCA1b.

Calcium is a major signalling bivalent cation within the cell. Compartmentalization is essential for regulation of calcium mediated processes. A number of players contribute to intracellular handling of calcium, among them are the sarco/endoplasmic reticulum calcium ATP-ases (SERCAs). These molecules function in the membrane of ER/SR pumping Ca2+ from cytoplasm into the lumen of the internal store. Removal of calcium from the cytoplasm is essential for signalling and for relaxation of skeletal muscle and heart. There are three genes and over a dozen isoforms of SERCA in mammals. These can be potentially influenced by small membrane peptides, also called regulins. The discovery of micropeptides has increased in recent years, mostly because of the small ORFs found in long RNAs, annotated formerly as noncoding (lncRNAs). Several excellent works have analysed the mechanism of interaction of micropeptides with each other and with the best known SERCA1a (fast muscle) and SERCA2a (heart, slow muscle) isoforms. However, the array of tissue and developmental expressions of these potential regulators raises the question of interaction with other SERCAs. For example, the most abundant calcium pump in neonatal and regenerating skeletal muscle, SERCA1b has never been looked at with scrutiny to determine whether it is influenced by micropeptides. Further details might be interesting on the interaction of these peptides with the less studied SERCA1b isoform.

Micropeptides: origins, identification, and potential role in metabolism-related diseases / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

With the development of modern sequencing techniques and bioinformatics, genomes that were once thought to be noncoding have been found to encode abundant functional micropeptides (miPs), a kind of small polypeptides. Although miPs are difficult to analyze and identify, a number of studies have begun to focus on them. More and more miPs have been revealed as essential for energy metabolism homeostasis, immune regulation, and tumor growth and development. Many reports have shown that miPs are especially essential for regulating glucose and lipid metabolism and regulating mitochondrial function. MiPs are also involved in the progression of related diseases. This paper reviews the sources and identification of miPs, as well as the functional significance of miPs for metabolism-related diseases, with the aim of revealing their potential clinical applications.

De novo birth of functional microproteins in the human lineage.

Small open reading frames (sORFs) can encode functional "microproteins" that perform crucial biological tasks. However, their size makes them less amenable to genomic analysis, and their origins and conservation are poorly understood. Given their short length, it is plausible that some of these functional microproteins have recently originated entirely de novo from noncoding sequences. Here we sought to identify such cases in the human lineage by reconstructing the evolutionary origins of human microproteins previously found to have measurable, statistically significant fitness effects. By tracing the formation of each ORF and its transcriptional activation, we show that novel microproteins with significant phenotypic effects have emerged de novo throughout animal evolution, including two after the human-chimpanzee split. Notably, traditional methods for assessing coding potential would miss most of these cases. This evidence demonstrates that the functional potential intrinsic to sORFs can be relatively rapidly and frequently realized through de novo gene emergence.

Regulation of the regulator: Endopeptidase cleavage controls the expression of a micropeptide that regulates SERCA.

Micropeptides regulate cellular calcium handling by modulating the function of the calcium transporter SERCA. In a recent Nature Communications paper [4] authors Schiemann et al. describe regulation of an invertebrate SERCA-active micropeptide, sarcolamban, by an endopeptidase called neprilysin 4 (NEP4). NEP4 activity limits sarcolamban expression by cleavage of luminal residues near the micropeptide's c-terminus. This cleavage event liberates sarcolamban from the membrane, reduces its oligomerization, and prevents it from inhibiting SERCA. The study reveals a novel mechanism for "regulation of the regulator" that may be a general feature of micropeptide/SERCA physiology.

Micropeptide MIAC inhibits the tumor progression by interacting with AQP2 and inhibiting EREG/EGFR signaling in renal cell carcinoma.

BACKGROUND: Although, micropeptides encoded by non-coding RNA have been shown to have an important role in a variety of tumors processes, there have been no reports on micropeptide in renal cell carcinoma (RCC). Based on the micropeptide MIAC (micropeptide inhibiting actin cytoskeleton) discovered and named in the previous work, this study screened its tumor spectrum, and explored its mechanism of action and potential diagnosis and treatment value in the occurrence and development of renal carcinoma. METHODS: The clinical significance of MIAC in RCC was explored by bioinformatics analysis through high-throughput RNA-seq data from 530 patients with kidney renal clear cell carcinoma (KIRC) in the TCGA database, and the detection of clinical samples of 70 cases of kidney cancer. In vitro and in vivo experiments to determine the role of MIAC in renal carcinoma cell growth and metastasis; High-throughput transcriptomics, western blotting, immunoprecipitation, molecular docking, affinity experiments, and Streptavidin pulldown experiments identify MIAC direct binding protein and key regulatory pathways. RESULTS: The analysis of 600 renal carcinoma samples from different sources revealed that the expression level of MIAC is significantly decreased, and corelated with the prognosis and clinical stage of tumors in patients with renal carcinoma. Overexpression of MIAC in renal carcinoma cells can significantly inhibit the proliferation and migration ability, promote apoptosis of renal carcinoma cells, and affect the distribution of cells at various stages. After knocking down MIAC, the trend is reversed. In vivo experiments have found that MIAC overexpression inhibit the growth and metastasis of RCC, while the synthetized MIAC peptides can significantly inhibit the occurrence and development of RCC in vitro and in vivo. Further mechanistic studies have demonstrated that MIAC directly bind to AQP2 protein, inhibit EREG/EGFR expression and activate downstream pathways PI3K/AKT and MAPK to achieve anti-tumor effects. CONCLUSIONS: This study revealed for the first time the tumor suppressor potential of the lncRNA-encoded micropeptide MIAC in RCC, which inhibits the activation of the EREG/EGFR signaling pathway by direct binding to AQP2 protein, thereby inhibiting renal carcinoma progression and metastasis. This result emphasizes that the micropeptide MIAC can provide a new strategy for the diagnosis and treatment of RCC.

Research progress on high throughput discovery and functional verification of functional micropeptides.

With the rapid development of computational biology and deep sequencing technology, more and more studies have shown that a large number of non-classical open reading frames that have not been annotated and hidden in non-coding RNA can encode functional micropeptide. This article reviewed the current research status and technology strategy of gene sources, biological properties, predicted methods and functional verification of micropeptide, providing theoretical and reference basis for the subsequent discovery of micropeptides, research on regulatory mechanisms and development of novel targets and biomarkers.

Mitochondrion-located peptides and their pleiotropic physiological functions.

With the development of advanced technologies, many small open reading frames (sORFs) have been found to be translated into micropeptides. Interestingly, a considerable proportion of micropeptides are located in mitochondria, which are designated here as mitochondrion-located peptides (MLPs). These MLPs often contain a transmembrane domain and show a high degree of conservation across species. They usually act as co-factors of large proteins and play regulatory roles in mitochondria such as electron transport in the respiratory chain, reactive oxygen species (ROS) production, metabolic homeostasis, and so on. Deficiency of MLPs disturbs diverse physiological processes including immunity, differentiation, and metabolism both in vivo and in vitro. These findings reveal crucial functions for MLPs and provide fresh insights into diverse mitochondrion-associated biological processes and diseases.

Cancer-related micropeptides encoded by ncRNAs: Promising drug targets and prognostic biomarkers.

An exciting emerging topic in the noncoding RNA (ncRNA) field is the discovery of short peptides called micropeptides (≤100 amino acids), whose novel therapeutic opportunities remain under-explored. Micropeptides have been suggested to play essential regulatory roles in diverse species of physiological and pathological processes. Genomics studies have revealed that these micropeptides are encoded by small open reading frames (sORFs) concealed in misannotated ncRNAs, generally lncRNAs (long noncoding RNAs) and circRNAs (circular RNAs). These ncRNA-encoded micropeptides have been shown to contribute to tumorigenesis but little is known about their pathological mechanism because of challenges in translated sORF identification techniques. Here, we review the best-validated micropeptides involved in the progression of human tumors and discuss their therapeutic and/or prognostic potential, at the same time, we also give our own suggestions on the concept of potential-coding RNA and micropeptides.

Inhibitory and stimulatory micropeptides preferentially bind to different conformations of the cardiac calcium pump.

The ATP-dependent ion pump sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) sequesters Ca2+ in the endoplasmic reticulum to establish a reservoir for cell signaling. Because of its central importance in physiology, the activity of this transporter is tightly controlled via direct interactions with tissue-specific regulatory micropeptides that tune SERCA function to match changing physiological conditions. In the heart, the micropeptide phospholamban (PLB) inhibits SERCA, while dwarf open reading frame (DWORF) stimulates SERCA. These competing interactions determine cardiac performance by modulating the amplitude of Ca2+ signals that drive the contraction/relaxation cycle. We hypothesized that the functions of these peptides may relate to their reciprocal preferences for SERCA binding; SERCA binds PLB more avidly at low cytoplasmic [Ca2+] but binds DWORF better when [Ca2+] is high. In the present study, we demonstrated this opposing Ca2+ sensitivity is due to preferential binding of DWORF and PLB to different intermediate states that SERCA samples during the Ca2+ transport cycle. We show PLB binds best to the SERCA E1-ATP state, which prevails at low [Ca2+]. In contrast, DWORF binds most avidly to E1P and E2P states that are more populated when Ca2+ is elevated. Moreover, FRET microscopy revealed dynamic shifts in SERCA-micropeptide binding equilibria during cellular Ca2+ elevations. A computational model showed that DWORF exaggerates changes in PLB-SERCA binding during the cardiac cycle. These results suggest a mechanistic basis for inhibitory versus stimulatory micropeptide function, as well as a new role for DWORF as a modulator of dynamic oscillations of PLB-SERCA regulatory interactions.