Small Open Reading Frames Research Articles

Refinement of Leishmania donovani Genome Annotations in the Light of Ribosome-Protected mRNAs Fragments (Ribo-Seq Data).

Advances in next-generation sequencing methodologies have facilitated the assembly of an ever-increasing number of genomes. Gene annotations are typically conducted via specialized software, but the most accurate results require additional manual curation that incorporates insights derived from functional and bioinformatic analyses (e.g., transcriptomics, proteomics, and phylogenetics). In this study, we improved the annotation of the Leishmania donovani (strain HU3) genome using publicly available data from the deep sequencing of ribosome-protected mRNA fragments (Ribo-Seq). As a result of this analysis, we uncovered 70 previously non-annotated protein-coding genes and improved the annotation of around 600 genes. Additionally, we present evidence for small upstream open reading frames (uORFs) in a significant number of transcripts, indicating their potential role in the translational regulation of gene expression. The bioinformatics pipelines developed for these analyses can be used to improve the genome annotations of other organisms for which Ribo-Seq data are available. The improvements provided by these studies will bring us closer to the ultimate goal of a complete and accurately annotated L. donovani genome and will enhance future transcriptomics, proteomics, and genetics studies.

LncRNA-encoded peptides: unveiling their significance in cardiovascular physiology and pathology - current research insights.

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 signaling axes, thereby enhancing or suppressing 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 RNO-sORF6/RNO-sORF7/RNO-sORF8, DOWRF, and 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 remodeling. 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.

Regulation of acetate tolerance by small ORF-encoded polypeptides modulating efflux pump specificity in Methylomonas sp. DH-1

BackgroundMethanotrophs have emerged as promising hosts for the biological conversion of methane into value-added chemicals, including various organic acids. Understanding the mechanisms of acid tolerance is essential for improving organic acid production. WatR, a LysR-type transcriptional regulator, was initially identified as involved in lactate tolerance in a methanotrophic bacterium Methylomonas sp. DH-1. In this study, we investigated the role of WatR as a regulator of cellular defense against weak organic acids and identified novel target genes of WatR.ResultsBy conducting an investigation into the genome-wide binding targets of WatR and its role in transcriptional regulation, we identified genes encoding an RND-type efflux pump (WatABO pump) and previously unannotated small open reading frames (smORFs), watS1 to watS5, as WatR target genes activated in response to acetate. The watS1 to watS5 genes encode polypeptides of approximately 50 amino acids, and WatS1 to WatS4 are highly homologous with one predicted transmembrane domain. Deletion of the WatABO pump genes resulted in decreased tolerance against formate, acetate, lactate, and propionate, suggesting its role as an efflux pump for a wide range of weak organic acids. WatR repressed the basal expression of watS genes but activated watS and WatABO pump genes in response to acetate stress. Overexpression of watS1 increased tolerance to acetate but not to other acids, only in the presence of the WatABO pump. Therefore, WatS1 may increase WatABO pump specificity toward acetate, switching the general weak acid efflux pump to an acetate-specific efflux pump for efficient cellular defense against acetate stress.ConclusionsOur study has elucidated the role of WatR as a key transcription factor in the cellular defense against weak organic acids, particularly acetate, in Methylomonas sp. DH-1. We identified the genes encoding WatABO efflux pump and small polypeptides (WatS1 to WatS5), as the target genes regulated by WatR for this specific function. These findings offer valuable insights into the mechanisms underlying weak acid tolerance in methanotrophic bacteria, thereby contributing to the development of bioprocesses aimed at converting methane into value-added chemicals.

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

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

Methods for Interactome Analysis of Microproteins Encoded by Small Open Reading Frames

Recent studies have shown that small open reading frames (sORFs, 100 codons) can encode peptides or microproteins that perform important functions in prokaryotic and eukaryotic cells. It has been established that sORF translation products are involved in the regulation of many processes, for example, they modulate the activity of the mitochondrial respiratory chain or the functions of muscle cells in mammals. However, the identification and subsequent functional analysis of peptides or microproteins encoded by sORFs is a non-trivial task and requires the use of special approaches. One of the critical steps in functional analysis is identification of protein partners of the peptide under study. This review considers the features of the interactome analysis of short protein molecules and describes the approaches currently used for studies in the field.

Small Open Reading Frame-Encoded Micro-Peptides: An Emerging Protein World.

Small open reading frames (sORFs) are often overlooked features in genomes. In the past, they were labeled as noncoding or "transcriptional noise". However, accumulating evidence from recent years suggests that sORFs may be transcribed and translated to produce sORF-encoded polypeptides (SEPs) with less than 100 amino acids. The vigorous development of computational algorithms, ribosome profiling, and peptidome has facilitated the prediction and identification of many new SEPs. These SEPs were revealed to be involved in a wide range of basic biological processes, such as gene expression regulation, embryonic development, cellular metabolism, inflammation, and even carcinogenesis. To effectively understand the potential biological functions of SEPs, we discuss the history and development of the newly emerging research on sORFs and SEPs. In particular, we review a range of recently discovered bioinformatics tools for identifying, predicting, and validating SEPs as well as a variety of biochemical experiments for characterizing SEP functions. Lastly, this review underlines the challenges and future directions in identifying and validating sORFs and their encoded micropeptides, providing a significant reference for upcoming research on sORF-encoded peptides.

Unearth of small open reading frames (sORFs) in drought stress transcriptome of Oryza sativa subsp. indica

Drought is a major abiotic stress that influences rice production. Although the transcriptomic data of rice against drought is widely available, the regulation of small open reading frames (sORFs) in response to drought stress in rice is yet to be investigated. Different levels of drought stress have different regulatory mechanisms in plants. In this study, drought stress was imposed on four-leaf stage rice, divided into two treatments, 40% and 30% soil moisture content (SMC). The RNAs of the samples were extracted, followed by the RNA sequencing analysis on their sORF expression changes under 40%_SMC and 30%_SMC, and lastly, the expression was validated through NanoString. A total of 122 and 143 sORFs were differentially expressed (DE) in 40%_SMC and 30%_SMC, respectively. In 40%_SMC, 69 sORFs out of 696 (9%) DEGs were found to be upregulated. On the other hand, 69 sORFs out of 449 DEGs (11%) were significantly downregulated. The trend seemed to be higher in 30%_SMC, where 112 (12%) sORFs were found to be upregulated from 928 significantly upregulated DEGs. However, only 8% (31 sORFs out of 385 DEGs) sORFs were downregulated in 30%_SMC. Among the identified sORFs, 110 sORFs with high similarity to rice proteome in the PsORF database were detected in 40%_SMC, while 126 were detected in 30%_SMC. The Gene Ontology (GO) enrichment analysis of DE sORFs revealed their involvement in defense-related biological processes, such as defense response, response to biotic stimulus, and cellular homeostasis, whereas enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways indicated that DE sORFs were associated with tryptophan and phenylalanine metabolisms. Several DE sORFs were identified, including the top five sORFs (OsisORF_3394, OsisORF_0050, OsisORF_3007, OsisORF_6407, and OsisORF_7805), which have yet to be characterised. Since these sORFs were responsive to drought stress, they might hold significant potential as targets for future climate-resilient rice development.

Transcriptional and translational landscape fine-tune genome annotation and explores translation control in cotton

IntroductionThe unavailability of intergenic region annotation in whole genome sequencing and pan-genomics hinders efforts to enhance crop improvement. ObjectivesDespite 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. MethodsWe 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. ResultsOur 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. ConclusionReference-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.

Decoding murine cytomegalovirus.

The genomes of both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) were first sequenced over 20 years ago. Similar to HCMV, the MCMV genome had initially been proposed to harbor ≈170 open reading frames (ORFs). More recently, omics approaches revealed HCMV gene expression to be substantially more complex comprising several hundred viral ORFs. Here, we provide a state-of-the art reannotation of lytic MCMV gene expression based on integrative analysis of a large set of omics data. Our data reveal 365 viral transcription start sites (TiSS) that give rise to 380 and 454 viral transcripts and ORFs, respectively. The latter include >200 small ORFs, some of which represented the most highly expressed viral gene products. By combining TiSS profiling with metabolic RNA labelling and chemical nucleotide conversion sequencing (dSLAM-seq), we provide a detailed picture of the expression kinetics of viral transcription. This not only resulted in the identification of a novel MCMV immediate early transcript encoding the m166.5 ORF, which we termed ie4, but also revealed a group of well-expressed viral transcripts that are induced later than canonical true late genes and contain an initiator element (Inr) but no TATA- or TATT-box in their core promoters. We show that viral upstream ORFs (uORFs) tune gene expression of longer viral ORFs expressed in cis at translational level. Finally, we identify a truncated isoform of the viral NK-cell immune evasin m145 arising from a viral TiSS downstream of the canonical m145 mRNA. Despite being ≈5-fold more abundantly expressed than the canonical m145 protein it was not required for downregulating the NK cell ligand, MULT-I. In summary, our work will pave the way for future mechanistic studies on previously unknown cytomegalovirus gene products in an important virus animal model.

A hidden translatome in tumors-the coding lncRNAs.

Long noncoding RNAs (lncRNAs) have been extensively identified in eukaryotic genomes and have been shown to play critical roles in the development of multiple cancers. Through the application and development of ribosome analysis and sequencing technologies, advanced studies have discovered the translation of lncRNAs. Although lncRNAs were originally defined as noncoding RNAs, many lncRNAs actually contain small open reading frames that are translated into peptides. This opens a broad area for the functional investigation of lncRNAs. Here, we introduce prospective methods and databases for screening lncRNAs with functional polypeptides. We also summarize the specific lncRNA-encoded proteins and their molecular mechanisms that promote or inhibit cancerous. Importantly, the role of lncRNA-encoded peptides/proteins holds promise in cancer research, but some potential challenges remain unresolved. This review includes reports on lncRNA-encoded peptides or proteins in cancer, aiming to provide theoretical basis and related references to facilitate the discovery of more functional peptides encoded by lncRNA, and to further develop new anti-cancer therapeutic targets as well as clinical biomarkers of diagnosis and prognosis.

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.

Andes Virus Genome Mutations That Are Likely Associated with Animal Model Attenuation and Human Person-to-Person Transmission.

We performed whole-genome sequencing with bait enrichment techniques to analyze Andes virus (ANDV), a cause of human hantavirus pulmonary syndrome. We used cryopreserved lung tissues from a naturally infected long-tailed colilargo, including early, intermediate, and late cell culture, passages of an ANDV isolate from that animal, and lung tissues from golden hamsters experimentally exposed to that ANDV isolate. The resulting complete genome sequences were subjected to detailed comparative genomic analysis against American orthohantaviruses. We identified four amino acid substitutions related to cell culture adaptation that resulted in attenuation of ANDV in the typically lethal golden hamster animal model of hantavirus pulmonary syndrome. Changes in the ANDV nucleocapsid protein, glycoprotein, and small nonstructural protein open reading frames correlated with mutations typical for ANDV strains associated with increased virulence in the small-animal model. Finally, we identified three amino acid substitutions, two in the small nonstructural protein and one in the glycoprotein, that were only present in the clade of viruses associated with efficient person-to-person transmission. Our results indicate that there are single-nucleotide polymorphisms that could be used to predict strain-specific ANDV virulence and/or transmissibility. IMPORTANCE Several orthohantaviruses cause the zoonotic disease hantavirus pulmonary syndrome (HPS) in the Americas. Among them, HPS caused by Andes virus (ANDV) is of great public health concern because it is associated with the highest case fatality rate (up to 50%). ANDV is also the only orthohantavirus associated with relatively robust evidence of person-to-person transmission. This work reveals nucleotide changes in the ANDV genome that are associated with virulence attenuation in an animal model and increased transmissibility in humans. These findings may pave the way to early severity predictions in future ANDV-caused HPS outbreaks.

Severe acute respiratory syndrome coronavirus-2 accessory proteins ORF3a and ORF7a modulate autophagic flux and Ca2+ homeostasis in yeast.

Virus infection involves the manipulation of key host cell functions by specialized virulence proteins. The Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) small accessory proteins ORF3a and ORF7a have been implicated in favoring virus replication and spreading by inhibiting the autophagic flux within the host cell. Here, we apply yeast models to gain insights into the physiological functions of both SARS-CoV-2 small open reading frames (ORFs). ORF3a and ORF7a can be stably overexpressed in yeast cells, producing a decrease in cellular fitness. Both proteins show a distinguishable intracellular localization. ORF3a localizes to the vacuolar membrane, whereas ORF7a targets the endoplasmic reticulum. Overexpression of ORF3a and ORF7a leads to the accumulation of Atg8 specific autophagosomes. However, the underlying mechanism is different for each viral protein as assessed by the quantification of the autophagic degradation of Atg8-GFP fusion proteins, which is inhibited by ORF3a and stimulated by ORF7a. Overexpression of both SARS-CoV-2 ORFs decreases cellular fitness upon starvation conditions, where autophagic processes become essential. These data confirm previous findings on SARS-CoV-2 ORF3a and ORF7a manipulating autophagic flux in mammalian cell models and are in agreement with a model where both small ORFs have synergistic functions in stimulating intracellular autophagosome accumulation, ORF3a by inhibiting autophagosome processing at the vacuole and ORF7a by promoting autophagosome formation at the ER. ORF3a has an additional function in Ca2+ homeostasis. The overexpression of ORF3a confers calcineurin-dependent Ca2+ tolerance and activates a Ca2+ sensitive FKS2-luciferase reporter, suggesting a possible ORF3a-mediated Ca2+ efflux from the vacuole. Taken together, we show that viral accessory proteins can be functionally investigated in yeast cells and that SARS-CoV-2 ORF3a and ORF7a proteins interfere with autophagosome formation and processing as well as with Ca2+ homeostasis from distinct cellular targets.

Ribosome footprint profiling enables elucidating the systemic regulation of fatty acid accumulation in Acer truncatum

BackgroundThe accumulation of fatty acids in plants covers a wide range of functions in plant physiology and thereby affects adaptations and characteristics of species. As the famous woody oilseed crop, Acer truncatum accumulates unsaturated fatty acids and could serve as the model to understand the regulation and trait formation in oil-accumulation crops. Here, we performed Ribosome footprint profiling combing with a multi-omics strategy towards vital time points during seed development, and finally constructed systematic profiling from transcription to proteomes. Additionally, we characterized the small open reading frames (ORFs) and revealed that the translational efficiencies of focused genes were highly influenced by their sequence features.ResultsThe comprehensive multi-omics analysis of lipid metabolism was conducted in A. truncatum. We applied the Ribo-seq and RNA-seq techniques, and the analyses of transcriptional and translational profiles of seeds collected at 85 and 115 DAF were compared. Key members of biosynthesis-related structural genes (LACS, FAD2, FAD3, and KCS) were characterized fully. More meaningfully, the regulators (MYB, ABI, bZIP, and Dof) were identified and revealed to affect lipid biosynthesis via post-translational regulations. The translational features results showed that translation efficiency tended to be lower for the genes with a translated uORF than for the genes with a non-translated uORF. They provide new insights into the global mechanisms underlying the developmental regulation of lipid metabolism.ConclusionsWe performed Ribosome footprint profiling combing with a multi-omics strategy in A. truncatum seed development, which provides an example of the use of Ribosome footprint profiling in deciphering the complex regulation network and will be useful for elucidating the metabolism of A. truncatum seed oil and the regulatory mechanisms.

Functional PAM sequence for DNA interference by CRISPR-Cas I-B system of Leptospira interrogans and the role of LinCas11b encoded within lincas8b

Pathogenic species of Leptospira are recalcitrant for genetic manipulation using conventional tools, and therefore there is a need to explore techniques of higher efficiency. Application of endogenous CRISPR-Cas tool is emerging and efficient; nevertheless, it is limited by a poor understanding of interference machinery in the bacterial genome and its associated protospacer adjacent motif (PAM). In this study, interference machinery of CRISPR-Cas subtype I-B (Lin_I-B) from L. interrogans was experimentally validated in E. coli using the various identified PAM (TGA, ATG, ATA). The overexpression of the Lin_I-B interference machinery in E. coli demonstrated that LinCas5, LinCas6, LinCas7, and LinCas8b can self-assemble on cognate CRISPR RNA to form an interference complex (LinCascade). Moreover, a robust interference of target plasmids containing a protospacer with a PAM suggested a functional LinCascade. We also recognized a small open reading frame within lincas8b that independently co-translates into LinCas11b. A mutant variant LinCascade-Cas11b that lacks LinCas11b co-expression erred to mount target plasmid interference. At the same time, LinCas11b complementation in LinCascade-Cas11b rescued target plasmid interference. Thus, the present study establishes Leptospira subtype I-B interference machinery to be functional and, soon, may pave the way for scientists to harness it as a programmable endogenous genetic manipulation tool.

Open Reading Frame 4 Is Not Essential in the Replication and Infection of Genotype 1 Hepatitis E Virus

Genotype 1 hepatitis E virus (HEV-1), unlike other genotypes of HEV, has a unique small open reading frame known as ORF4 whose function is not yet known. ORF4 is located in an out-framed manner in the middle of ORF1, which encodes putative 90 to 158 amino acids depending on the strains. To explore the role of ORF4 in HEV-1 replication and infection, we cloned the complete genome of wild-type HEV-1 downstream of a T7 RNA polymerase promoter, and the following ORF4 mutant constructs were prepared: the first construct had TTG instead of the initiation codon ATG (A2836T), introducing an M→L mutation in ORF4 and a D→V mutation in ORF1. The second construct had ACG instead of the ATG codon (T2837C), introducing an M→T mutation in ORF4. The third construct had ACG instead of the second in-frame ATG codon (T2885C), introducing an M→T mutation in ORF4. The fourth construct contained two mutations (T2837C and T2885C) accompanying two M→T mutations in ORF4. For the latter three constructs, the accompanied mutations introduced in ORF1 were all synonymous changes. The capped entire genomic RNAs were generated by in vitro transcription and used to transfect PLC/PRF/5 cells. Three mRNAs containing synonymous mutations in ORF1, i.e., T2837CRNA, T2885CRNA, and T2837C/T2885CRNA, replicated normally in PLC/PRF/5 cells and generated infectious viruses that successfully infected Mongolian gerbils as the wild-type HEV-1 did. In contrast, the mutant RNA, i.e., A2836TRNA, accompanying an amino acid change (D937V) in ORF1 generated infectious viruses upon transfection, but they replicated slower than the wild-type HEV-1 and failed to infect Mongolian gerbils. No putative viral protein(s) derived from ORF4 were detected in the wild-type HEV-1- as well as the mutant virus-infected PLC/PRF/5 cells by Western blot analysis using a high-titer anti-HEV-1 IgG antibody. These results demonstrated that the ORF4-defective HEV-1s had the ability to replicate in the cultured cells, and that these defective viruses had the ability to infect Mongolian gerbils unless the overlapping ORF1 was accompanied by non-synonymous mutation(s), confirming that ORF4 is not essential in the replication and infection of HEV-1.

Long Non-Coding RNA lncWOX11a Suppresses Adventitious Root Formation of Poplar by Regulating the Expression of PeWOX11a.

Long non-coding RNAs (lncRNAs), a class of poorly conserved transcripts without protein-encoding ability, are widely involved in plant organogenesis and stress responses by mediating the transmission and expression of genetic information at the transcriptional, posttranscriptional, and epigenetic levels. Here, we cloned and characterized a novel lncRNA molecule through sequence alignment, Sanger sequencing, transient expression in protoplasts, and genetic transformation in poplar. lncWOX11a is a 215 bp transcript located on poplar chromosome 13, ~50 kbp upstream of PeWOX11a on the reverse strand, and the lncRNA may fold into a series of complex stem-loop structures. Despite the small open reading frame (sORF) of 51 bp within lncWOX11a, bioinformatics analysis and protoplast transfection revealed that lncWOX11a has no protein-coding ability. The overexpression of lncWOX11a led to a decrease in the quantity of adventitious roots on the cuttings of transgenic poplars. Further, cis-regulatory module prediction and CRISPR/Cas9 knockout experiments with poplar protoplasts demonstrated that lncWOX11a acts as a negative regulator of adventitious rooting by downregulating the WUSCHEL-related homeobox gene WOX11, which is supposed to activate adventitious root development in plants. Collectively, our findings imply that lncWOX11a is essential for modulating the formation and development of adventitious roots.

Microprotein Dysregulation in the Serum of Patients with Atrial Fibrillation.

The incidence rate of atrial fibrillation (AF) has stayed at a high level in recent years. Despite the intensive efforts to study the pathologic changes of AF, the molecular mechanism of disease development remains unclarified. Microproteins are ribosomally translated gene products from small open reading frames (sORFs) and are found to play crucial biological functions, while remain rare attention and indistinct in AF study. In this work, we recruited 65 AF patients and 65 healthy subjects for microproteomic profiling. By differential analysis and cross-validation between independent datasets, a total of 4 microproteins were identified as significantly different, including 3 annotated ones and 1 novel one. Additionally, we established a diagnostic model with either microproteins or global proteins by machine learning methods and found the model with microproteins achieved comparable and excellent performance as that with global proteins. Our results confirmed the abnormal expression of microproteins in AF and may provide new perspectives on the mechanism study of AF.

Rescue of Escherichia coli auxotrophy by de novo small proteins.

Increasing numbers of small proteins with diverse physiological roles are being identified and characterized in both prokaryotic and eukaryotic systems, but the origins and evolution of these proteins remain unclear. Recent genomic sequence analyses in several organisms suggest that new functions encoded by small open reading frames (sORFs) may emerge de novo from noncoding sequences. However, experimental data demonstrating if and how randomly generated sORFs can confer beneficial effects to cells are limited. Here, we show that by upregulating hisB expression, de novo small proteins (≤50 amino acids in length) selected from random sequence libraries can rescue Escherichia coli cells that lack the conditionally essential SerB enzyme. The recovered small proteins are hydrophobic and confer their rescue effect by binding to the 5' end regulatory region of the his operon mRNA, suggesting that protein binding promotes structural rearrangements of the RNA that allow increased hisB expression. This study adds RNA regulatory elements as another interacting partner for de novo proteins isolated from random sequence libraries and provides further experimental evidence that small proteins with selective benefits can originate from the expression of nonfunctional sequences.

Identification of small open reading frames in plant lncRNA using class-imbalance learning

Recently, small open reading frames (sORFs) in long noncoding RNA (lncRNA) have been demonstrated to encode small peptides that can help study the mechanisms of growth and development in organisms. Since machine learning-based computational methods are less costly compared with biological experiments, they can be used to identify sORFs and provide a basis for biological experiments. However, few computational methods and data resources have been exploited for identifying sORFs in plant lncRNA. Besides, machine learning models produce underperforming classifiers when faced with a class-imbalance problem. In this study, an alternative method called SMOTE based on weighted cosine distance (WCDSMOTE) which enables interaction with feature selection is put forward to synthesize minority class samples and weighted edited nearest neighbor (WENN) is applied to clean up majority class samples, thus, hybrid sampling WCDSMOTE-ENN is proposed to deal with imbalanced datasets with the multi-angle feature. A heterogeneous classifier ensemble is introduced to complete the classification task. Therefore, a novel computational method that is based on class-imbalance learning to identify the sORFs with coding potential in plant lncRNA (sORFplnc) is presented. Experimental results manifest that sORFplnc outperforms existing computational methods in identifying sORFs with coding potential. We anticipate that the proposed work can be a reference for relevant research and contribute to agriculture and biomedicine.