Multiple myeloma (MM) is a common yet incurable hematological malignancy characterized by bone marrow infiltration. A major clinical challenge is the resistance to chemotherapy, highlighting the urgent need to better understand the molecular mechanisms underlying chemotherapeutic resistance to available drugs. Recent studies have emphasized the role of micropeptides in solid tumors and leukemia, but their functions in MM remain unclear. In this study, we identified a novel micropeptide, altKLF4, derived from the transcription factor KLF4, which is highly expressed in newly diagnosed myeloma patient samples. We found that ectopic expression of altKLF4 interfered with chemotherapy sensitivity induced by proteasome inhibitors in myeloma cells. Additionally, confocal microscopy and transcriptome sequencing revealed that altKLF4 co-localizes with the mitochondrial inner marker TOMM20 and participates in mitochondria-related biological processes, suggesting that altKLF4 partially localizes to the mitochondria. Mitochondria may also play a role in regulating ferroptosis. Our results further demonstrated that altKLF4 inhibited drug sensitivity and ferroptosis induced by the GPX4 inhibitor RSL3 in multiple myeloma cells through a direct interaction with GPX4. In vivo experiments showed that RSL3 significantly suppressed primary myeloma growth, which could be rescued by the micropeptide altKLF4. Taken together, our study identifies altKLF4 as a novel micropeptide that serves as a potential biomarker for chemotherapeutic resistance in multiple myeloma, offering insights for diagnosis and management of drug-resistant MM.

In this study, we analyze the long noncoding RNA, lncMN3, that is predominantly expressed in motor neurons and shows potential coding capabilities. Utilizing custom antibodies, we demonstrate the production of a lncMN3-derived type I transmembrane micropeptide, SERTM2. Patch-clamp experiments performed on both wild-type and SERTM2 knockout motor neurons, differentiated in vitro from mouse embryonic stem cells, show a difference in the resting membrane potential and overall decreased excitability upon SERTM2 depletion. In vivo studies indicate that the absence of the peptide impairs treadmill test performance. At the mechanistic level, we identify a two-pore domain potassium channel, TASK1, known to be a major determinant of the resting membrane potential in motor neurons, as a SERTM2 interactor. Our study characterizes one of the first lncRNA-derived micropeptides involved in neuronal physiology.

Multidrug-resistant (MDR) bacteria pose a major threat to public health, and additional sources of antibacterial candidates are urgently needed. Noncanonical peptides (NCPs), derived from noncanonical small open reading frames, represent small biological molecules with important roles in biology. However, the antibacterial activity of NCPs remains largely unknown. Here, we discovered a plant-derived noncanonical antibacterial peptide (NCBP1) against both Gram-positive and Gram-negative bacteria. NCBP1 is composed of 11 amino acid residues with cationic surface potential and favorable safety and stability. Mechanistic studies revealed that NCBP1 displayed antibacterial activity by targeting phosphatidylglycerol and cardiolipin in bacterial membrane, resulting in membrane damage and dysfunction. Notably, NCBP1 showed promising efficacy in mice. Furthermore, NCBP1 effectively inhibited the growth of plant fungal pathogens and enhanced disease resistance in maize. Our results demonstrate the unexplored antimicrobial potential of plant-derived NCPs and provide an accessible source for the discovery of antimicrobial substances against MDR bacterial and fungal pathogens.

Microproteins encoded by small open reading frames comprise the "dark matter" of proteomes. Although microproteins have been detected in diverse organisms from all three domains of life, many more remain to be identified, and only a few have been functionally characterized. In this comprehensive study of intergenic small open reading frames (ismORFs, 15-70 codons) in 5,668 bacterial genomes of the family Enterobacteriaceae, we identify 67,297 clusters of ismORFs subject to purifying selection. Expression of tagged Escherichia coli microproteins is detected for 11 of the 16 tested, validating the predictions. Although the ismORFs mainly code for hydrophobic, potentially transmembrane, unstructured, or minimally structured microproteins, some globular folds, oligomeric structures, and possible interactions with proteins encoded by neighboring genes are predicted. Complete information on the predicted microprotein families, including evidence of transcription and translation, and structure predictions are available as an easily searchable resource for investigation of microprotein functions.

Ribosome profiling is a high-throughput sequencing technique that captures the positions of translating ribosomes on RNAs. Recent advancements in ribosome profiling include achieving highly phased ribosome footprints for plant translatomes and more recently for bacterial translatomes. This substantially increases the specificity of detecting open reading frames (ORFs) that can be translated, such as small ORFs located upstream and downstream of the annotated ORFs. However, most genomes (e.g. bacterial genomes) lack the annotations for the transcription start and termination sites. This hinders the systematic discovery of novel ORFs in the 'untranslated' regions in ribosome profiling data. Here, we develop a new computational pipeline called RIBOSS to discover noncanonical ORFs and assess their translational potential against annotated ORFs. The RIBOSS Python modules are versatile, and we use them to analyse both prokaryotic and eukaryotic data. We present a resulting list of noncanonical ORFs with high translational potential in Homo sapiens, Arabidopsis thaliana, and Salmonella enterica. We further illustrate RIBOSS utility when studying organisms with incomplete transcriptome annotations. We leverage long-read and short-read data for reference-guided transcriptome assembly and highly phased ribosome profiling data for detecting novel translational events in the assembled transcriptome for S. enterica. In sum, RIBOSS is the first integrated computational pipeline for noncanonical ORF detection and translational potential assessment that incorporates long- and short-read sequencing technologies to investigate translation. RIBOSS is freely available at https://github.com/lcscs12345/riboss.

Glioblastoma (GB), the most common and aggressive brain tumor, demonstrates intrinsic resistance to current therapies, resulting in poor clinical outcomes. Cancer progression can be partially attributed to the deregulation of protein translation mechanisms that drive cancer cell growth. In this study, we present the translatome landscape of GB as a valuable data resource. Eight patient-derived GB sphere cultures (GSCs) were analyzed using ribosome profiling and messenger RNA (mRNA) sequencing. We investigated inter-cell-line differences through differential expression analysis at both the translatome and transcriptome levels. Translational changes post-radiotherapy were assessed at 30 and 60 min. The translation of non-coding RNAs (ncRNAs) was validated using in-house and public mass spectrometry (MS) data, whereas RNA expression was confirmed by quantitative PCR (qPCR). Our findings demonstrate that ribosome sequencing provides more detailed information than MS or transcriptional analyses. Transcriptional similarities among GSCs correlate with translational similarities, aligning with previously defined subtypes such as proneural and mesenchymal. Additionally, we identified a broad spectrum of open reading frame types in both coding and non-coding mRNA regions, including long non-coding RNAs (lncRNAs) and pseudogenes undergoing active translation. Translation of ncRNAs into peptides was independently confirmed by in-house data and external MS data. We also observed that translational regulation of histones (downregulated) and splicing factors (upregulated) occurs in response to radiotherapy. These data offer new insights into genome-wide protein synthesis, identifying translationally regulated genes and alternative translation initiation sites in GB under normal and radiotherapeutic conditions, providing a rich resource for GB research. Further functional validation of differentially expressed genes after radiotherapy is needed. Understanding translational control in GB can reveal mechanistic insights and identify currently unknown biomarkers, ultimately enhancing the diagnosis and treatment of this aggressive brain cancer.

In the present era, noncoding RNAs (ncRNAs) have become a subject of considerable scientific interest, with peptides encoded by ncRNAs representing a particularly promising avenue of investigation. The identification of ncRNA-encoded peptides in human cancers is increasing. These peptides regulate cancer progression through multiple molecular mechanisms. Here, we delineate the patterns of diverse ncRNA-encoded peptides and provide a synopsis of the methodologies employed for the identification of ncRNAs that possess the capacity to encode these peptides. Furthermore, we discuss the impacts of ncRNA-encoded peptides on the biological behavior of cancer cells and the underlying molecular mechanisms. In conclusion, we describe the prospects of ncRNA-encoded peptides in cancer and the challenges that need to be overcome.

OVO is required for female germ cell viability but has no known function in the male germline in Drosophila. ovo is autoregulated by 2 antagonistic isoforms, OVO-A and OVO-B. All ovo- alleles were created as partial revertants of the antimorphic ovoD1 allele. Creation of new targeted alleles in an ovo+ background indicated that disrupting the germline-specific exon extension of ovo-B leads to an arrested egg chamber phenotype, rather than germ cell death. RNA sequencing analysis, including >1 K full-length cDNAs, indicates that ovo has several unannotated splice variations in the extended exon and a minor population of ovo-B transcripts has an alternative splice. This indicates that classical ovo alleles, such as ovoD1rv23, are not truly null for ovo and are likely to be weak antimorphs. To generate bonafide nulls, we deleted the ovo-A and ovo-B promoters showing that only ovo-B is required for female germ cell viability, and there is an early and continual developmental requirement for ovo-B in the female germline. To visualize OVO expression and localization, we endogenously tagged ovo and found nuclear OVO in all differentiating female germ cells throughout oogenesis in adults. We also found that OVO is maternally deposited into the embryo, where it showed nuclear localization in newly formed pole cells. Maternal OVO persisted in embryonic germ cells until zygotic OVO expression was detectable, suggesting that there is continuous nuclear OVO expression in the female germline in the transition from one generation to the next.

Hundreds of thousands of small open reading frames (smORFs) of less than 100 codons exist in every genome, especially in long noncoding RNAs (lncRNAs) and in the 5' leaders of mRNAs. smORFs are often discarded as nonfunctional, but ribosomal profiling (RiboSeq) reveals that thousands are translated, while characterised smORF functions have risen from anecdotal to identifiable trends: smORFs can either have a cis-noncoding regulatory function (involving low translation of nonfunctional peptides) or full coding function mediated by robustly translated peptides, often having cellular and physiological roles as membrane-associated regulators of canonical proteins. The evolutionary context reveals that many smORFs represent new genes emerging de novo from noncoding sequences. We suggest a mechanism for this process, where cis-noncoding smORF functions provide niches for the subsequent evolution of full peptide functions.

Long non-coding RNAs (lncRNAs), once thought to be mere transcriptional noise, are now revealing a hidden code. Recent advancements like ribosome sequencing have unveiled that many lncRNAs harbor small open reading frames and can potentially encode functional micropeptides. Emerging research suggests these micropeptides, not the lncRNAs themselves, play crucial roles in regulating homeostasis, inflammation, metabolism, and especially in breast cancer progression. This review delves into the rapidly evolving computational tools used to predict and validate lncRNA-encoded micropeptides. We then explore the diverse functions and mechanisms of action of these micropeptides in breast cancer pathogenesis, with a focus on their roles in various species. Ultimately, this review aims to illuminate the functional landscape of lncRNA-encoded micropeptides and their potential as therapeutic targets in cancer.

Small Open Reading Frames (smORFs) of less than 100 codons remain mostly uncharacterised. About a thousand smORFs per genome encode peptides and microproteins about 70-80 aa long, often containing recognisable protein structures and markers of translation, and these are referred to as short Coding Sequences (sCDSs). The characterisation of individual sCDSs has provided examples of smORFs' function and conservation, but we cannot infer the functionality of all other metazoan smORFs from these. sCDS function has been characterised at a genome-wide scale in yeast and bacteria, showing that hundreds can produce a phenotype, but attempts in metazoans have been less successful. Either most sCDSs are not functional, or classic experimental techniques do not work with smORFs due to their shortness. Here, we combine extensive proteomics with bioinformatics and genetics in order to detect and corroborate sCDS function in Drosophila. Our studies nearly double the number of sCDSs with detected peptides and microproteins and an experimentally corroborated function. Finally, we observe a correlation between proven sCDS protein function and bioinformatic markers such as conservation and GC content. Our results support that sCDSs peptides and microproteins act as membrane-related regulators of canonical proteins, regulators whose functions are best understood at the cellular level, and whose mutants produce little, if any, overt morphological phenotypes.

Bacterial proteins of ≤50 amino acids, denoted small proteins or microproteins, have been traditionally understudied and overlooked, as standard computational, biochemical, and genetic approaches often do not detect proteins of this size. However, with the realization that small proteins are stably expressed and have important cellular roles, there has been increased identification of small proteins in bacteria and eukaryotes. Gradually, the functions of a few of these small proteins are being elucidated. Many interact with larger protein products to modulate their subcellular localization, stabilities, or activities. Here, we provide an overview of these diverse functions in bacteria, highlighting generalities among bacterial small proteins and similarly sized proteins in eukaryotic organisms and discussing questions for future research.

sORF-encoded peptides (SEPs) refer to proteins encoded by small open reading frames (sORFs) with a length of less than 100 amino acids, which play an important role in various life activities. Analysis of known SEPs showed that using non-canonical initiation codons of SEPs was more common. However, the current analysis of SEP sequences mainly relies on bioinformatics prediction, and most of them use AUG as the start site, which may not be completely correct for SEPs. Chemical labeling was used to systematically analyze the N-terminal sequences of SEPs to accurately define the start sites of SEPs. By comparison, we found that dimethylation and guanidinylation are more efficient than acetylation. The ACN precipitation and heating precipitation performed better in SEP enrichment. As an N-terminal peptide enrichment material, Hexadhexaldehyde was superior to CNBr-activated agarose and NHS-activated agarose. Combining these methods, we identified 128 SEPs with 131 N-terminal sequences. Among them, two-thirds are novel N-terminal sequences, and most of them start from the 11-31st amino acids of the original sequence. Partial novel N-termini were produced by proteolysis or signal peptide removal. Some SEPs' transcription start sites were corrected to be non-AUG start codons. One novel start codon was validated using GFP-tag vectors. These results demonstrated that the chemical labeling approaches would be beneficial for identifying the start codons of sORFs and the real N-terminal of their encoded peptides, which helps better understand the characterization of SEPs.

Recently developed experimental and computational approaches to identify putative coding small ORFs (smORFs) in genomes have revealed thousands of smORFs localized within coding and non-coding RNAs. They can be translated into smORF peptides or microproteins, which are defined as less than 100 amino acids in length. The identification of such a large number of potential biological regulators represents a major challenge, notably for elucidating the in vivo functions of these microproteins. Since the emergence of this field, Drosophila has proved to be a valuable model for studying the biological functions of microproteins in vivo. In this review, we outline how the smORF field emerged and the nomenclature used in this domain. We summarize the technical challenges associated with identifying putative coding smORFs in the genome and the relevant translated microproteins. Finally, recent findings on one of the best studied smORF peptides, Pri, and other microproteins studied so far in Drosophila are described. These studies highlight the diverse roles that microproteins can fulfil in the regulation of various molecular targets involved in distinct cellular processes during animal development and physiology. Given the recent emergence of the microprotein field and the associated discoveries, the microproteome represents an exquisite source of potentially bioactive molecules, whose in vivo biological functions can be explored in the Drosophila model.

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.

Noncanonical peptides (NCPs) are a class of peptides generated from regions previously thought of as noncoding, such as introns, 5' UTRs, 3' UTRs, and intergenic regions. In recent years, the significance and diverse functions of NCPs have come to light, yet a systematic and comprehensive NCP database remains absent. Here, we developed NCPbook (https://ncp.wiki/ncpbook/), a database of evidence-supported NCPs, which aims to provide a resource for efficient exploration, analysis, and manipulation of NCPs. NCPbook incorporates data from diverse public databases and scientific literature. The current version of NCPbook includes 180,676 NCPs across 29 different species, evidenced by MS, ribosome profiling, or molecular experiments. These NCPs are distributed across kingdoms, comprising 123,408 from 14 plant species, 56,999 from 7 animal species, and 269 from 8 microbial species. Furthermore, NCPbook encompasses 9,166 functionally characterized NCPs playing important roles in immunity, stress resistance, growth, and development. Equipped with a user-friendly interface, NCPbook allows users to search, browse, visualize, and retrieve data, making it an indispensable platform for researching NCPs in various plant, animal, and microbial species.

Chagas disease affects around 8 million people globally, with Latin America bearing approximately 10,000 deaths each year. Combatting the disease relies heavily on vector control methods, necessitating the identification of new targets. Within insect genomes, genes harboring small open reading frames (smORFs - < 100 amino acids) present numerous potential candidates. In our investigation, we elucidate the pivotal role of the archetypal smORF-containing gene, mille-pattes/polished-rice/tarsalless (mlpt/pri/tal), in the post-embryonic development of the kissing bug Rhodnius prolixus. Injection of double-stranded RNA targeting mlpt (dsmlpt) during nymphal stages yields a spectrum of phenotypes hindering post-embryonic growth. Notably, fourth or fifth stage nymphs subjected to dsmlpt do not undergo molting. These dsmlpt nymphs display heightened mRNA levels of JHAMT-like and EPOX-like, enzymes putatively involved in the juvenile hormone (JH) pathway, alongside increased expression of the transcription factor Kr-h1, indicating changes in the hormonal control. Histological examination reveals structural alterations in the hindgut and external cuticle of dsmlpt nymphs compared to control (dsGFP) counterparts. Furthermore, significant changes in the vector's digestive physiology were observed, with elevated hemozoin and glucose levels in the posterior midgut of dsmlpt nymphs. Importantly, dsmlpt nymphs exhibit impaired metacyclogenesis of Trypanosoma cruzi, the causative agent of Chagas disease, underscoring the crucial role of proper gut organization in parasite differentiation. Thus, our findings constitute the first evidence of a smORF-containing gene's regulatory influence on vector physiology, parasitic cycle, and disease transmission.

Perioperative neurocognitive disorders (PND) refer to neurocognitive abnormalities during perioperative period, which are a great challenge for elderly patients and associated with increased morbidity and mortality. Our studies showed that long non-coding RNAs (lncRNAs) regulate mitochondrial function and aging-related pathologies in the aged hippocampus after anesthesia, and lncRNAs are associated with multiple neurodegenerations. However, the regulatory role of lncRNAs in PND-related pathological processes remains unclear.

A total of 18-month mice were assigned to control and surgery (PND) groups, mice in PND group received sevoflurane anesthesia and laparotomy. Cognitive function was assessed with fear conditioning test. Hippocampal RNAs were isolated for sequencing, lncRNA and microRNA libraries were constructed, mRNAs were identified, Gene Ontology (GO) analysis were performed, and lncRNA-microRNA-mRNA networks were established. qPCR was performed for gene expression verification.

A total of 312 differentially expressed (DE) lncRNAs, 340 DE-Transcripts of Uncertain Coding Potential (TUCPs), and 2,003 DEmRNAs were identified in the hippocampus between groups. The lncRNA-microRNA-mRNA competing endogenous RNA (ceRNA) network was constructed with 29 DElncRNAs, 90 microRNAs, 493 DEmRNAs, 148 lncRNA-microRNA interaction pairs, 794 microRNA-mRNA interaction pairs, and 110 lncRNA-mRNA co-expression pairs. 795 GO terms were obtained. Based on the frequencies of involved pathological processes, BP terms were divided into eight categories: neurological system alternation, neuronal development, metabolism alternation, immunity and neuroinflammation, apoptosis and autophagy, cellular communication, molecular modification, and behavior changes. LncRNA-microRNA-mRNA ceRNA networks in these pathological categories were constructed, and involved pathways and targeted genes were revealed. The top relevant lncRNAs in these ceRNA networks included RP23-65G6.4, RP24-396L14.1, RP23-251I16.2, XLOC_113622, RP24-496E14.1, etc., and the top relevant mRNAs in these ceRNA networks included Dlg4 (synaptic function), Avp (lipophagy), Islr2 (synaptic function), Hcrt (regulation of awake behavior), Tnc (neurotransmitter uptake).

In summary, we have constructed the lncRNA-associated ceRNA network during PND development in mice, explored the role of lncRNAs in multiple pathological processes in the mouse hippocampus, and provided insights into the potential mechanisms and therapeutic gene targets for PND.

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.

We determined RNA spectrum of the human RSK4 (hRSK4) gene (also called RPS6KA6) and identified 29 novel mRNA variants derived from alternative splicing, which, plus the NCBI-documented ones and the five we reported previously, totaled 50 hRSK4 RNAs that, by our bioinformatics analyses, encode 35 hRSK4 protein isoforms of 35-762 amino acids. Many of the mRNAs are bicistronic or tricistronic for hRSK4. The NCBI-normalized NM_014496.5 and the protein it encodes are designated herein as the Wt-1 mRNA and protein, respectively, whereas the NM_001330512.1 and the long protein it encodes are designated as the Wt-2 mRNA and protein, respectively. Many of the mRNA variants responded differently to different situations of stress, including serum starvation, a febrile temperature, treatment with ethanol or ethanol-extracted clove buds (an herbal medicine), whereas the same stressed situation often caused quite different alterations among different mRNA variants in different cell lines. Mosifloxacin, an antibiotics and also a functional inhibitor of hRSK4, could inhibit the expression of certain hRSK4 mRNA variants. The hRSK4 gene likely uses alternative splicing as a handy tool to adapt to different stressed situations, and the mRNA and protein multiplicities may partly explain the incongruous literature on its expression and comports.

The unavailability of intergenic region annotation in whole genome sequencing and pan-genomics hinders efforts to enhance crop improvement.

Despite advances in research, the impact of post-transcriptional regulation on fiber development and translatome profiling at different stages of fiber growth in cotton (G. hirsutum) remains unexplored.

We utilized a combination of reference-guided de novo transcriptome assembly and ribosome profiling techniques to uncover the hidden mechanisms of translational control in eight distinct tissues of upland cotton.

Our study identified P-site distribution at three-nucleotide periodicity and dominant ribosome footprint at 27 nucleotides. Specifically, we have detected 1,589 small open reading frames (sORFs), including 1,376 upstream ORFs (uORFs) and 213 downstream ORFs (dORFs), as well as 552 long non-coding RNAs (lncRNAs) with potential coding functions, which fine-tune the annotation of the cotton genome. Further, we have identified novel genes and lncRNAs with strong translation efficiency (TE), while sORFs were found to affect mRNA transcription levels during fiber elongation. The reliability of these findings was confirmed by the high consistency in correlation and synergetic fold change between RNA-sequencing (RNA-seq) and Ribosome-sequencing (Ribo-seq) analyses. Additionally, integrated omics analysis of the normal fiber ZM24 and short fiber pag1 cotton mutant revealed several differentially expressed genes (DEGs), and fiber-specific expressed (high/low) genes associated with sORFs (uORFs and dORFs). These findings were further supported by the overexpression and knockdown of GhKCS6, a gene associated with sORFs in cotton, and demonstrated the potential regulation of the mechanism governing fiber elongation on both the transcriptional and post-transcriptional levels.

Reference-guided transcriptome assembly and the identification of novel transcripts fine-tune the annotation of the cotton genome and predicted the landscape of fiber development. Our approach provided a high-throughput method, based on multi-omics, for discovering unannotated ORFs, hidden translational control, and complex regulatory mechanisms in crop plants.

The heart, once considered a mere blood pump, is now recognized as a multifunctional metabolic and endocrine organ. Its function is tightly regulated by various metabolic processes, at the same time it serves as an endocrine organ, secreting bioactive molecules that impact systemic metabolism. In recent years, research has shed light on the intricate interplay between the heart and other metabolic organs, such as adipose tissue, liver, and skeletal muscle. The metabolic flexibility of the heart and its ability to switch between different energy substrates play a crucial role in maintaining cardiac function and overall metabolic homeostasis. Gaining a comprehensive understanding of how metabolic disorders disrupt cardiac metabolism is crucial, as it plays a pivotal role in the development and progression of cardiac diseases. The emerging understanding of the heart as a metabolic and endocrine organ highlights its essential contribution to whole body metabolic regulation and offers new insights into the pathogenesis of metabolic diseases, such as obesity, diabetes, and cardiovascular disorders. In this review, we provide an in-depth exploration of the heart's metabolic and endocrine functions, emphasizing its role in systemic metabolism and the interplay between the heart and other metabolic organs. Furthermore, emerging evidence suggests a correlation between heart disease and other conditions such as aging and cancer, indicating that the metabolic dysfunction observed in these conditions may share common underlying mechanisms. By unraveling the complex mechanisms underlying cardiac metabolism, we aim to contribute to the development of novel therapeutic strategies for metabolic diseases and improve overall cardiovascular health.

To achieve a highly differentiated state, cells undergo multiple transcriptional processes whose coordination and timing are not well understood. In Drosophila embryonic epidermal cells, polished-rice (Pri) smORF peptides act as temporal mediators of ecdysone to activate a transcriptional program leading to cell shape remodeling. Here, we show that the ecdysone/Pri axis concomitantly represses the transcription of a large subset of cuticle genes to ensure proper differentiation of the insect exoskeleton. The repression relies on the transcription factor Ken and persists for several days throughout early larval stages, during which a soft cuticle allows larval crawling. The onset of these cuticle genes normally awaits the end of larval stages when the rigid pupal case assembles, and their premature expression triggers abnormal sclerotization of the larval cuticle. These results uncovered a temporal switch to set up distinct structures of cuticles adapted to the animal lifestyle and which might be involved in the evolutionary history of insects.

Evolutionary analyses have estimated that ∼60% of nucleotides in intergenic regions of the Drosophila melanogaster genome are functionally relevant, suggesting that regulatory information may be encoded more densely in intergenic regions than has been revealed by most functional dissections of regulatory DNA. Here, we approached this issue through a functional dissection of the regulatory region of the gene shavenbaby (svb). Most of the ∼90 kb of this large regulatory region is highly conserved in the genus Drosophila, though characterized enhancers occupy a small fraction of this region. By analyzing the regulation of svb in different contexts of Drosophila development, we found that the regulatory information that drives svb expression in the abdominal pupal epidermis is organized in a different way than the elements that drive svb expression in the embryonic epidermis. While in the embryonic epidermis svb is activated by compact enhancers separated by large inactive DNA regions, svb expression in the pupal epidermis is driven by regulatory information distributed over broader regions of svb cis-regulatory DNA. In the same vein, we observed that other developmental genes also display a dense distribution of putative regulatory elements in their regulatory regions. Furthermore, we found that a large percentage of conserved noncoding DNA of the Drosophila genome is contained within regions of open chromatin. These results suggest that part of the evolutionary constraint on noncoding DNA of Drosophila is explained by the density of regulatory information, which may be greater than previously appreciated.

The transcription factor Shavenbaby (Svb), the only member of the OvoL family in Drosophila, controls the fate of various epithelial embryonic cells and adult stem cells. Post-translational modification of Svb produces two protein isoforms, Svb-ACT and Svb-REP, which promote adult intestinal stem cell renewal or differentiation, respectively. To define Svb mode of action, we used engineered cell lines and develop an unbiased method to identify Svb target genes across different contexts. Within a given cell type, Svb-ACT and Svb-REP antagonistically regulate the expression of a set of target genes, binding specific enhancers whose accessibility is constrained by chromatin landscape. Reciprocally, Svb-REP can influence local chromatin marks of active enhancers to help repressing target genes. Along the intestinal lineage, the set of Svb target genes progressively changes, together with chromatin accessibility. We propose that Svb-ACT-to-REP transition promotes enterocyte differentiation of intestinal stem cells through direct gene regulation and chromatin remodeling.

The low-molecular-weight proteins (LMWP) in serum and plasma are related to various human diseases and can be valuable biomarkers. A small open reading frame-encoded peptide (SEP) is one kind of LMWP, which has been found to function in many bioprocesses and has also been found in human blood, making it a potential biomarker. The detection of LMWP by a mass spectrometry (MS)-based proteomic assay is often inhibited by the wide dynamic range of serum/plasma protein abundance. Nanoparticle protein coronas are a newly emerging protein enrichment method. To analyze SEPs in human serum, we have developed a protocol integrated with nanoparticle protein coronas and liquid chromatography (LC)/MS/MS. With three nanoparticles, TiO2, Fe3O4@SiO2, and Fe3O4@SiO2@TiO2, we identified 164 new SEPs in the human serum sample. Fe3O4@SiO2 and a nanoparticle mixture obtained the maximum number and the largest proportion of identified SEPs, respectively. Compared with acetonitrile-based extraction, nanoparticle protein coronas can cover more small proteins and SEPs. The magnetic nanoparticle is also fit for high-throughput parallel protein separation before LC/MS. This method is fast, efficient, reproducible, and easy to operate in 96-well plates and centrifuge tubes, which will benefit the research on SEPs and biomarkers.

The genomic integrity of every organism is endangered by various intrinsic and extrinsic stresses. To maintain genomic integrity, a sophisticated DNA damage response (DDR) network is activated rapidly after DNA damage. Notably, the fundamental DDR mechanisms are conserved in eukaryotes. However, knowledge about many regulatory aspects of the plant DDR is still limited. Important, yet little understood, regulatory factors of the DDR are the long non-coding RNAs (lncRNAs). In humans, 13 lncRNAs functioning in DDR have been characterized to date, whereas no such lncRNAs have been characterized in plants yet. By meta-analysis, we identified the putative long intergenic non-coding RNA induced by DNA damage (LINDA) that responds strongly to various DNA double-strand break-inducing treatments, but not to replication stress induced by mitomycin C. After DNA damage, LINDA is rapidly induced in an ATM- and SOG1-dependent manner. Intriguingly, the transcriptional response of LINDA to DNA damage is similar to that of its flanking hypothetical protein-encoding gene. Phylogenetic analysis of putative Brassicales and Malvales LINDA homologs indicates that LINDA lncRNAs originate from duplication of a flanking small protein-encoding gene followed by pseudogenization. We demonstrate that LINDA is not only needed for the regulation of this flanking gene but also fine-tuning of the DDR after the occurrence of DNA double-strand breaks. Moreover, Δlinda mutant root stem cells are unable to recover from DNA damage, most likely due to hyper-induced cell death.

As conserved regulatory agents, noncoding RNAs (ncRNAs) have an important impact on many aspects of plant life, including growth, development, and environmental response. Noncoding RNAs can travel through not only plasmodesma and phloem but also intercellular barriers to regulate distinct processes. Increasing evidence shows that the intercellular trans-kingdom transmission of ncRNAs is able to modulate many important interactions between plants and other organisms, such as plant response to pathogen attack, the symbiosis between legume plants and rhizobia and the interactions with parasitic plants. In these interactions, plant ncRNAs are believed to be sorted into extracellular vesicles (EVs) or other nonvesicular vehicles to pass through cell barriers and trigger trans-kingdom RNA interference (RNAi) in recipient cells from different species. There is evidence that the features of extracellular RNAs and associated RNA-binding proteins (RBPs) play a role in defining the RNAs to retain in cell or secrete outside cells. Despite the few reports about RNA secretion pathway in plants, the export of extracellular ncRNAs is orchestrated by a series of pathways in plants. The identification and functional analysis of mobile small RNAs (sRNAs) are attracting increasing attention in recent years. In this review, we discuss recent advances in our understanding of the function, sorting, transport, and regulation of plant extracellular ncRNAs.

Advances in proteogenomic technologies have revealed hundreds to thousands of translated small open reading frames (sORFs) that encode microproteins in genomes across evolutionary space. While many microproteins have now been shown to play critical roles in biology and human disease, a majority of recently identified microproteins have little or no experimental evidence regarding their functionality. Computational tools have some limitations for analysis of short, poorly conserved microprotein sequences, so additional approaches are needed to determine the role of each member of this recently discovered polypeptide class. A currently underexplored avenue in the study of microproteins is structure prediction and determination, which delivers a depth of functional information. In this review, we provide a brief overview of microprotein discovery methods, then examine examples of microprotein structures (and, conversely, intrinsic disorder) that have been experimentally determined using crystallography, cryo-electron microscopy, and NMR, which provide insight into their molecular functions and mechanisms. Additionally, we discuss examples of predicted microprotein structures that have provided insight or context regarding their function. Analysis of microprotein structure at the angstrom level, and confirmation of predicted structures, therefore, has potential to identify translated microproteins that are of biological importance and to provide molecular mechanism for their in vivo roles.

Short polypeptides encoded by small open reading frames (smORFs) are ubiquitously found in eukaryotic genomes and are important regulators of physiology, development, and mitochondrial processes. Here, we focus on a subset of 298 smORFs that are evolutionarily conserved between Drosophila melanogaster and humans. Many of these smORFs are conserved broadly in the bilaterian lineage, and ∼182 are conserved in plants. We observe remarkably heterogeneous spatial and temporal expression patterns of smORF transcripts-indicating wide-spread tissue-specific and stage-specific mitochondrial architectures. In addition, an analysis of annotated functional domains reveals a predicted enrichment of smORF polypeptides localizing to mitochondria. We conduct an embryonic ribosome profiling experiment and find support for translation of 137 of these smORFs during embryogenesis. We further embark on functional characterization using CRISPR knockout/activation, RNAi knockdown, and cDNA overexpression, revealing diverse phenotypes. This study underscores the importance of identifying smORF function in disease and phenotypic diversity.

Long noncoding RNAs (lncRNAs) have been confirmed to play a crucial role in various biological processes across several species. Though many efforts have been devoted to the expansion of the lncRNAs landscape, much about lncRNAs is still unknown due to their great complexity. The development of high-throughput technologies and the constantly improved bioinformatic methods have resulted in a rapid expansion of lncRNA research and relevant databases. In this review, we introduced genome-wide research of lncRNAs in three parts: (i) novel lncRNA identification by high-throughput sequencing and computational pipelines; (ii) functional characterization of lncRNAs by expression atlas profiling, genome-scale screening, and the research of cancer-related lncRNAs; (iii) mechanism research by large-scale experimental technologies and computational analysis. Besides, primary experimental methods and bioinformatic pipelines related to these three parts are summarized. This review aimed to provide a comprehensive and systemic overview of lncRNA genome-wide research strategies and indicate a genome-wide lncRNA research system.

Long non-coding RNAs (lncRNAs), which are RNA transcripts exceeding 200 nucleotides were believed to lack any protein-coding capacity. But advancements in -omics technology have revealed that some lncRNAs have small open reading frames (sORFs) that can be translated by ribosomes to encode peptides, some of which have important biological functions. These encoded peptides subserve important biological functions by interacting with their targets to modulate transcriptional or signalling axes, thereby enhancing or suppressing cardiovascular disease (CVD) occurrence and progression. In this review, we summarize what is known about the research strategy of lncRNA-encoded peptides, mainly comprising predictive websites/tools and experimental methods that have been widely used for prediction, identification, and validation. More importantly, we have compiled a list of lncRNA- encoded peptides, with a focus on those that play significant roles in cardiovascular physiology and pathology, including ENSRNOT (RNO)-sORF6/RNO-sORF7/RNO-sORF8, dwarf open reading frame (DOWRF), myoregulin (NLN), etc. Additionally, we have outlined the functions and mechanisms of these peptides in cardiovascular physiology and pathology, such as cardiomyocyte hypertrophy, myocardial contraction, myocardial infarction, and vascular remodelling. Finally, an overview of the existing challenges and potential future developments in the realm of lncRNA-encoded peptides was provided, with consideration given to prospective avenues for further research. Given that many lncRNA-encoded peptides have not been functionally annotated yet, their application in CVD diagnosis and treatment still requires further research.

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.

Pattern recognition receptor-mediated innate immunity is critical for host defense against viruses. A growing number of coding and noncoding genes are found to encode microproteins. However, the landscape and functions of microproteins in responsive to virus infection remain uncharacterized. Here, we systematically identified microproteins that are responsive to vesicular stomatitis virus infection. A conserved and endoplasmic reticulum-localized membrane microprotein, MAVI1 (microprotein in antiviral immunity 1), was found to interact with mitochondrion-localized MAVS protein and inhibit MAVS aggregation and type I interferon signaling activation. The importance of MAVI1 was highlighted that viral infection was attenuated and survival rate was increased in Mavi1-knockout mice. A peptide inhibitor targeting the interaction between MAVI1 and MAVS activated the type I interferon signaling to defend viral infection. Our findings uncovered that microproteins play critical roles in regulating antiviral innate immune responses, and targeting microproteins might represent a therapeutic avenue for treating viral infection.

OVO is required for karyotypically female germ cell viability but has no known function in the male germline in Drosophila. ovo is autoregulated by two antagonistic isoforms, OVO-A and OVO-B. All ovo- alleles were created as partial revertants of the antimorphic ovoD1 allele. Creation of new targeted alleles in an ovo+ background indicated that disrupting the germline-specific exon extension of ovo-B leads to an arrested egg chamber phenotype, rather than germ cell death. RNA-seq analysis, including >1K full length cDNAs, indicates that ovo utilizes a number of unannotated splice variations in the extended exon and a minor population of ovo-B transcripts utilizes an alternative splice. This indicates that classical ovo alleles such as ovoD1rv23, are not truly null for ovo, and are likely to be weak antimorphs. To generate bonafide nulls, we deleted the ovo-A and ovo-B promoters showing that only ovo-B is required for female germ cell viability and there is an early and polyphasic developmental requirement for ovo-B in the female germline. To visualize OVO expression and localization, we endogenously tagged ovo and found nuclear OVO in all differentiating female germ cells throughout oogenesis in adults. We also found that OVO is maternally deposited into the embryo, where it showed nuclear localization in newly formed pole cells. Maternal OVO persisted in embryonic germ cells until zygotic OVO expression was detectable, suggesting that there is continuous nuclear OVO expression in the female germline in the transition from one generation to the next.

Non-coding RNAs (ncRNAs) are not conventionally involved in protein encoding. However, recent findings indicate that ncRNAs possess the capacity to code for proteins or peptides. These ncRNA-encoded peptides (ncPEPs) are vital for diverse plant life processes and exhibit significant potential value. Despite their importance, research on plant ncPEPs is limited, with only a few studies conducted and less information on the underlying mechanisms, and the field remains in its nascent stage. This manuscript provides a comprehensive overview of ncPEPs mining methods in plants, focusing on prediction, identification, and functional analysis. We discuss the strengths and weaknesses of various techniques, identify future research directions in the ncPEPs domain, and elucidate the biological functions and agricultural application prospects of plant ncPEPs. By highlighting the immense potential and research value of ncPEPs, we aim to lay a solid foundation for more in-depth studies in plant science.

Long noncoding RNAs (lncRNAs) in eukaryotic transcripts have long been believed to regulate various aspects of cellular processes, including carcinogenesis. Herein, it is found that lncRNA AFAP1-AS1 encodes a conserved 90-amino acid peptide located on mitochondria, named lncRNA AFAP1-AS1 translated mitochondrial-localized peptide (ATMLP), and it is not the lncRNA but the peptide that promotes the malignancy of nonsmall cell lung cancer (NSCLC). As the tumor progresses, the serum level of ATMLP increases. NSCLC patients with high levels of ATMLP display poorer prognosis. Translation of ATMLP is controlled by m6 A methylation at the 1313 adenine locus of AFAP1-AS1. Mechanistically, ATMLP binds to the 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) and inhibits its transport from the inner to the outer mitochondrial membrane, which antagonizes the NIPSNAP1-mediated regulation of cell autolysosome formation. The findings uncover a complex regulatory mechanism of NSCLC malignancy orchestrated by a peptide encoded by a lncRNA. A comprehensive judgment of the application prospects of ATMLP as an early diagnostic biomarker for NSCLC is also made.

Hepatocellular carcinoma (HCC) was the sixth most prevalent cancer worldwide. Long non-coding RNA TGFB2-OT1 has been proven to mediate inflammation and autophagy in vascular endothelial cells. However, its function in HCC is still unknown.

We analyzed the relationship between TGFB2-OT1 expression and the clinicopathological features of 202 HCC patients. RT-qPCR was used to analyze the TGFB2-OT1 expression in HCC cell lines and tissues. In vitro and in vivo assays were conducted to verify the effect of TGFB2-OT1 on the phenotype of HCC. RNA pull-down assays were applied to reveal the proteins binding to the TGFB2-OT1. Western-blot assays were conducted to analyze the protein expression in HCC cell lines.

TGFB2-OT1 was found to be highly expressed in HCC samples and hepatoma cells. TGFB2-OT1 expression was significantly associated with age (P = 0.001), cirrhosis (P = 0.003), tumor size (P < 0.001), tumor encapsulation (P = 0.029), tumor protruding from the liver surface (P = 0.040), and alpha fetoprotein (AFP, P < 0.001) levels. TGFB2-OT1 promoted proliferation, migration, invasion, and angiogenesis in HCC cells, both in vitro and in vivo. TGFB2-OT1 binds to β-catenin and competitively impaired the binding of β-catenin to GSK3β, thus suppressing the phosphorylation of β-catenin at Ser33, Ser37, and Thr41.

TGFB2-OT1 is overexpressed in HCC and predicts the poor prognosis of HCC patients. TGFB2-OT1 impedes the phosphorylation of β-catenin and acts as an alternative activator of the Wnt/β-catenin pathway to promote the progression and angiogenesis of HCC.