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MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally. In animals, this regulation is achieved via base-pairing with partially complementary sequences on mainly 3' UTR region of messenger RNAs (mRNAs). Computational approaches that predict miRNA target interactions (MTIs) facilitate the process of narrowing down potential targets for experimental validation. The availability of new datasets of high-throughput, direct MTIs has led to the development of machine learning (ML) based methods for MTI prediction. To train an ML algorithm, it is beneficial to provide entries from all class labels (i.e., positive and negative). Currently, no high-throughput assays exist for capturing negative examples. Therefore, current ML approaches must rely on either artificially generated or inferred negative examples deduced from experimentally identified positive miRNA-target datasets. Moreover, the lack of uniform standards for generating such data leads to biased results and hampers comparisons between studies. In this comprehensive study, we collected methods for generating negative data for animal miRNAs–target interactions and investigated their impact on the classification of true human MTIs. Our study relies on training ML models on a fixed positive dataset in combination with different negative datasets and evaluating their intra- and cross-dataset performance. As a result, we were able to examine each method independently and evaluate ML models' sensitivity to the methodologies utilized in negative data generation. To achieve a deep understanding of the performance results, we analyzed unique features that distinguish between datasets. In addition, we examined whether one-class classification models that utilize solely positive interactions for training are suitable for the task of MTI classification. We demonstrate the importance of negative data in MTI classification, analyze specific methodological characteristics that differentiate negative datasets, and highlight the challenge of ML models generalizing interaction rules from training to testing sets derived from different approaches. This study provides valuable insights into the computational prediction of MTIs that can be further used to establish standards in the field. Author summary: Gene expression regulation is fundamental for all organisms' development, homeostasis, and environmental adaptation. microRNAs (miRNAs) play a central role in post-transcriptional gene regulation by binding to target mRNAs and repressing their translation or mediating their degradation. Technical challenges in experimental miRNA target identification led to growing interest in computational target prediction. While machine learning (ML) models have shown success in this area, they rely heavily on artificially generated negative examples due to limited experimental data. The diversity of methods for generating negative interactions and the lack of a uniform standardized approach introduce bias and hinder the comparison of results across different studies. In this study, we collected methods for generating negative data for animal miRNAs–target interactions and analyzed their impact on classifying true interactions in humans. Using an ML approach, we evaluated miRNA–target prediction performance within and across negative datasets. We also analyzed unique features distinguishing between the negative datasets to understand the performance results better. Our study shows that negative data is essential for accurately classifying miRNA–target interactions and that ML models struggle to apply what they have learned from the training set when faced with new data derived from different approaches. This study particularly appeals to researchers interested in miRNA–target classification. It emphasizes the need for standardized methods to enhance comparability between studies. [ABSTRACT FROM AUTHOR]

Sessile organisms, such as plants, developed various ways to sense and respond to external and internal stimuli to maximize their fitness through evolutionary time. Transcripts and protein regulation are, among many, the main mechanisms that plants use to respond to environmental changes. SKIP protein is one such, presenting an SNKW interacting domain, which is highly conserved among eukaryotes, where SKI interacting protein acts in regulating key processes.In the present work, many bioinformatics tools, such as phylogenetic relationships, gene structure, physical–chemical properties, conserved motifs, prediction of regulatory cis‐elements, chromosomal localization, and protein–protein interaction network, were used to better understand the genome‐wide SNW/SKIP domain‐containing proteins.In total, 28 proteins containing the SNW/SKIP domain were identified in different plant species, including plants of agronomic interest. Two main protein clusters were formed in phylogenetic analysis, and gene structure analysis revealed that, in general, the coding region had no introns. Also, expression of these genes is possibly induced by abiotic stress stimuli. Primary structure analysis of the proteins revealed the existence of an evolutionarily conserved functional unit. But physicochemical properties show that proteins containing the SNW/SKIP domain are commonly unstable under in vivo conditions.In addition, the protein network, demonstrated that SKIP homologues could act by modulating plant fitness through gene expression regulation at the transcriptional and post‐transcriptional levels. This could be corroborated by the expression number of gene copies of SKIP proteins in many species, highlighting it's crucial role in plant development and tolerance through the course of evolution. [ABSTRACT FROM AUTHOR]

N6-methyladenosine (m6A) is the most abundant mRNA modification and plays diverse roles in eukaryotes, including plants. It regulates various processes, including plant growth, development, and responses to external or internal stress responses. However, the mechanisms underlying how m6A is related to environmental stresses in both mammals and plants remain elusive. Here, we identified EVOLUTIONARILY CONSERVED C-TERMINAL REGION 8 (ECT8) as an m6A reader protein and showed that its m6A-binding capability is required for salt stress responses in Arabidopsis (Arabidopsis thaliana). ECT8 accelerates the degradation of its target transcripts through direct interaction with the decapping protein DECAPPING 5 within processing bodies. We observed a significant increase in the ECT8 expression level under various environmental stresses. Using salt stress as a representative stressor, we found that the transcript and protein levels of ECT8 rise in response to salt stress. The increased abundance of ECT8 protein results in the enhanced binding capability to m6A-modified mRNAs, thereby accelerating their degradation, especially those of negative regulators of salt stress responses. Our results demonstrated that ECT8 acts as an abiotic stress sensor, facilitating mRNA decay, which is vital for maintaining transcriptome homeostasis and enhancing stress tolerance in plants. Our findings not only advance the understanding of epitranscriptomic gene regulation but also offer potential applications for breeding more resilient crops in the face of rapidly changing environmental conditions. An m6A reader accelerates target mRNA degradation by directly interacting with a decapping protein within processing bodies. [ABSTRACT FROM AUTHOR]

Gene regulation is crucial for cellular function and homeostasis. It involves diverse mechanisms controlling the production of specific gene products and contributing to tissue-specific variations in gene expression. The dysregulation of genes leads to disease, emphasizing the need to understand these mechanisms. Computational methods have jointly studied transcription factors (TFs), microRNA (miRNA), and messenger RNA (mRNA) to investigate gene regulatory networks. However, there remains a knowledge gap in comprehending gene regulatory networks. On the other hand, super-enhancers (SEs) have been implicated in miRNA biogenesis and function in recent experimental studies, in addition to their pivotal roles in cell identity and disease progression. However, statistical/computational methodologies harnessing the potential of SEs in deciphering gene regulation networks remain notably absent. However, to understand the effect of miRNA on mRNA, existing statistical/computational methods could be updated, or novel methods could be developed by accounting for SEs in the model. In this review, we categorize existing computational methods that utilize TF and miRNA data to understand gene regulatory networks into three broad areas and explore the challenges of integrating enhancers/SEs. The three areas include unraveling indirect regulatory networks, identifying network motifs, and enriching pathway identification by dissecting gene regulators. We hypothesize that addressing these challenges will enhance our understanding of gene regulation, aiding in the identification of therapeutic targets and disease biomarkers. We believe that constructing statistical/computational models that dissect the role of SEs in predicting the effect of miRNA on gene regulation is crucial for tackling these challenges. [ABSTRACT FROM AUTHOR]

Summary N6‐methyladenosine (m6A) RNA modification is the most prevalent messenger RNA (mRNA) modification in eukaryotes and plays critical roles in the regulation of gene expression. m6A is a reversible RNA modification that is deposited by methyltransferases (writers) and removed by demethylases (erasers). The function of m6A erasers in plants is highly diversified and their roles in cereal crops, especially in reproductive development essential for crop yield, are largely unknown. Here, we demonstrate that rice OsALKBH5 acts as an m6A demethylase required for the normal progression of male meiosis. OsALKBH5 is a nucleo‐cytoplasmic protein, highly enriched in rice anthers during meiosis, that associates with P‐bodies and exon junction complexes, suggesting that it is involved in regulating mRNA processing and abundance. Mutations of OsALKBH5 cause reduced double‐strand break (DSB) formation, severe defects in DSB repair, and delayed meiotic progression, leading to complete male sterility. Transcriptome analysis and m6A profiling indicate that OsALKBH5‐mediated m6A demethylation stabilizes the mRNA level of multiple meiotic genes directly or indirectly, including several genes that regulate DSB formation and repair. Our study reveals the indispensable role of m6A metabolism in post‐transcriptional regulation of meiotic progression in rice. [ABSTRACT FROM AUTHOR]

The subcellular localization of messenger RNAs (mRNAs) is a pivotal aspect of biomolecules, tightly linked to gene regulation and protein synthesis, and offers innovative insights into disease diagnosis and drug development in the field of biomedicine. Several computational methods have been proposed to predict the subcellular localization of mRNAs within cells. However, there remains a deficiency in the accuracy of these predictions. In this study, we propose an mRCat predictor based on the gradient boosting tree algorithm specifically to predict whether mRNAs are localized in the nucleus or in the cytoplasm. This predictor firstly uses large language models to thoroughly explore hidden information within sequences and then integrates traditional sequence features to collectively characterize mRNA gene sequences. Finally, it employs CatBoost as the base classifier for predicting the subcellular localization of mRNAs. The experimental validation on an independent test set demonstrates that mRCat obtained accuracy of 0.761, F1 score of 0.710, MCC of 0.511, and AUROC of 0.751. The results indicate that our method has higher accuracy and robustness compared to other state-of-the-art methods. It is anticipated to offer deep insights for biomolecular research. [ABSTRACT FROM AUTHOR]

Messenger RNA (mRNA) is vital for post-transcriptional gene regulation, acting as the direct template for protein synthesis. However, the methods available for predicting mRNA subcellular localization need to be improved and enhanced. Notably, few existing algorithms can annotate mRNA sequences with multiple localizations. In this work, we propose the mRNA-CLA, an innovative multi-label subcellular localization prediction framework for mRNA, leveraging a deep learning approach with a multi-head self-attention mechanism. The framework employs a multi-scale convolutional layer to extract sequence features across different regions and uses a self-attention mechanism explicitly designed for each sequence. Paired with Position Weight Matrices (PWMs) derived from the convolutional neural network layers, our model offers interpretability in the analysis. In particular, we perform a base-level analysis of mRNA sequences from diverse subcellular localizations to determine the nucleotide specificity corresponding to each site. Our evaluations demonstrate that the mRNA-CLA model substantially outperforms existing methods and tools. • Visualization of CNN and attention layers used for mRNA localization prediction. • Multiscale modeling and Bayesian optimization enhance model performance. • Integrated analyses reveal base content and sequence patterns in mRNA compartments. [ABSTRACT FROM AUTHOR]

Sepsis, a life-threatening condition caused by the body's dysregulated response to infection, presents a significant challenge in clinical management. Timely and accurate diagnosis is paramount for initiating appropriate interventions and improving patient outcomes. In recent years, there has been growing interest in identifying biomarkers that can aid in the early detection and prognostication of sepsis. MicroRNAs (miRNAs) have emerged as potential biomarkers for sepsis due to their involvement in the regulation of gene expression and their stability in various biological fluids, including blood. MiRNAs are small non-coding RNA molecules that play crucial roles in post-transcriptional gene regulation by binding to target messenger RNAs (mRNAs), leading to mRNA degradation or translational repression. The diagnostic and prognostic potential of miRNAs in sepsis stems from their ability to serve as sensitive and specific biomarkers reflective of the underlying pathophysiological processes. Compared to traditional biomarkers such as C-reactive protein (CRP) and procalcitonin (PCT), miRNAs offer several advantages, including their early and sustained elevation during sepsis, as well as their stability in stored samples, making them attractive candidates for clinical use. However, despite their promise, the clinical translation of miRNAs as sepsis biomarkers faces several challenges. These include the need for standardized sample collection and processing methods, the identification of optimal miRNA panels or signatures for differentiating sepsis from other inflammatory conditions, and the validation of findings across diverse patient populations and clinical settings. In conclusion, miRNAs hold great promise as diagnostic and prognostic biomarkers for sepsis, offering insights into the underlying molecular mechanisms and potential therapeutic targets. However, further research is needed to overcome existing challenges and realize the full clinical utility of miRNAs in improving sepsis outcomes. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small non-coding RNAs which contribute to the regulation of many physiological and pathological processes. Conventionally, miRNAs perform their activity in the cytoplasm where they regulate gene expression by interacting in a sequence-specific manner with mature messenger RNAs. Recent studies point to the presence of mature miRNAs in the nucleus. This review summarizes current findings regarding the molecular activities of nuclear miRNAs. These molecules can regulate gene expression at the transcriptional level by directly binding DNA on the promoter or the enhancer of regulated genes. miRNAs recruit different protein complexes to these regions, resulting in activation or repression of transcription, through a number of molecular mechanisms. Hematopoiesis is presented as a paradigmatic biological process whereby nuclear miRNAs possess a relevant regulatory role. Nuclear miRNAs can influence gene expression by affecting nuclear mRNA processing and by regulating pri-miRNA maturation, thus impacting the biogenesis of miRNAs themselves. Overall, nuclear miRNAs are biologically active molecules that can be critical for the fine tuning of gene expression and deserve further studies in a number of physiological and pathological conditions. [ABSTRACT FROM AUTHOR]

Ataxin-2 (ATXN2) is a gene implicated in spinocerebellar ataxia type II (SCA2), amyotrophic lateral sclerosis (ALS) and Parkinsonism. The encoded protein is a therapeutic target for ALS and related conditions. ATXN2 (or Atx2 in insects) can function in translational activation, translational repression, mRNA stability and in the assembly of mRNP-granules, a process mediated by intrinsically disordered regions (IDRs). Previous work has shown that the LSm (Like-Sm) domain of Atx2, which can help stimulate mRNA translation, antagonizes mRNP-granule assembly. Here we advance these findings through a series of experiments on Drosophila and human Ataxin-2 proteins. Results of Targets of RNA Binding Proteins Identified by Editing (TRIBE), co-localization and immunoprecipitation experiments indicate that a polyA-binding protein (PABP) interacting, PAM2 motif of Ataxin-2 may be a major determinant of the mRNA and protein content of Ataxin-2 mRNP granules. Transgenic Experiments with transgenic Drosophila indicate that while the Atx2-LSm domain may protect against neurodegeneration, structured PAM2- and unstructured IDR- interactions both support Atx2-induced cytotoxicity. Taken together, the data lead to a proposal for how Ataxin-2 interactions are remodelled during translational control and how structured and non-structured interactions contribute differently to the specificity and efficiency of RNP granule condensation as well as to neurodegeneration. Author summary: Mutations in genes that promote protein aggregation are primarily associated with neurodegenerative diseases. RNA-binding proteins, crucial for gene regulation and cellular stress response, are also major constituents of protein aggregate-rich mRNP granules. Recent studies aim to understand the molecular mechanisms governing the formation of stress granules identified Intrinsically Disordered Regions (IDRs) within mRNP granule proteins as a key factor that facilitates the formation of amyloid-like structures. A deeper understanding of stress granule dynamics holds promise for elucidating neurodegenerative disease mechanisms. Ataxin-2, is an RNA-binding protein implicated in neurodegenerative disorders like spinocerebellar ataxia type II, amyotrophic lateral sclerosis, and Parkinsonism. Reducing the levels of Ataxin-2 levels or deleting its IDR prevents RNP aggregation and offers neuroprotection in animal models of neurodegenerative diseases. We show that deleting the structured LSm domain of Ataxin-2, which impedes granule assembly, enhances cytotoxicity. Moreover, specific molecular interactions involving Ataxin-2 PAM2 motif binding to polyA-binding protein is crucial for recruiting mRNAs and other proteins into RNP granules. These interactions not only shape granule composition but also influence disease progression. Our findings propose a model illustrating the dynamic remodeling of Ataxin-2 interactions, emphasizing the distinct roles of structured and unstructured domains in governing mRNP-granule condensation and neurodegeneration. [ABSTRACT FROM AUTHOR]

The eukaryotic mRNA life cycle includes transcription, nuclear mRNA export and degradation. To quantify all these processes simultaneously, we perform thiol-linked alkylation after metabolic labeling of RNA with 4-thiouridine (4sU), followed by sequencing of RNA (SLAM-seq) in the nuclear and cytosolic compartments of human cancer cells. We develop a model that reliably quantifies mRNA-specific synthesis, nuclear export, and nuclear and cytosolic degradation rates on a genome-wide scale. We find that nuclear degradation of polyadenylated mRNA is negligible and nuclear mRNA export is slow, while cytosolic mRNA degradation is comparatively fast. Consequently, an mRNA molecule generally spends most of its life in the nucleus. We also observe large differences in the nuclear export rates of different 3'UTR transcript isoforms. Furthermore, we identify genes whose expression is abruptly induced upon metabolic labeling. These transcripts are exported substantially faster than average mRNAs, suggesting the existence of alternative export pathways. Our results highlight nuclear mRNA export as a limiting factor in mRNA metabolism and gene regulation. Author summary: In our work, we aim to quantify two fundamental processes in the life cycle of a eukaryotic messenger RNA (mRNA), namely export from the nuclear compartment to the cytosol, and degradation. To enable a genome-wide evaluation, we have implemented an experimental-bioinformatics approach. We use a chemical moiety, 4-thiouridine (4sU), which is incorporated into RNA molecules when added to the solution, to mark RNA which is recently synthesized and distinguish it from already existing RNA. We take a time series of recent and already existing RNA within both the nuclear and cytoplasmic cellular compartments. We have devised a computational model that can reliably quantify mRNA nuclear export and cytosolic degradation rates from this data. We revealed that the export of mRNA from the nucleus to the cytoplasm emerges as a comparatively tardy event. Subsequently, mRNA molecules within the cytoplasm experience swift degradation. This identifies the nucleus as the predominant residence for the greater portion of an mRNA's lifecycle. Yet, we have also found notable exceptions from this scheme which are immediately exported after transcription without delay, suggesting the plausible existence of alternative mRNA export pathways. In sum, our research underscores the pivotal role of nuclear mRNA export as a determining factor in the orchestration of mRNA metabolism and the regulation of gene expression within eukaryotic cells. [ABSTRACT FROM AUTHOR]

Increasing technological development results in more sources of the extremely low-frequency electromagnetic field (ELF-EMF), which is recognized as an environmental risk factor. The results of the past study indicate that the ELF-EMF can affect the level of DNA methylation. The study aimed to determine whether the ELF-EMF induces changes in epigenetic regulation of gene expression in the endometrium of pigs during the peri-implantation period. Endometrial slices (100 ± 5 mg) collected on days 15–16 of pregnancy were exposed in vitro to the ELF-EMF at a frequency of 50 Hz for 2 h of treatment duration. To determine the impact of the ELF-EMF on elements of epigenetic regulations involved in DNA methylation, histone modification, and microRNA biogenesis in the endometrium, the DNMT1 and DNMT3a; EZH2, UHRF1, and MBD1; DICER1 and DGCR8 mRNA transcript and protein abundance were analyzed using Real-Time PCR and Western blot, respectively. Moreover, EED and SUZ1 2 mRNA transcript, global DNA methylation, and the activity of histone deacetylase (HDAC) were analyzed. The changes in the abundance of DNMT1 and DNMT3a, EZH2 mRNA transcript and protein, EED and SUZ1 2 mRNA transcript, global DNA methylation level, HDAC activity, and the abundance of proteins involved in microRNA biogenesis evoked by the ELF-EMF in the endometrium were observed. The ELF-EMF possesses the potential to alter epigenetic regulation of gene expression in the porcine endometrium. Observed alterations may be the reason for changes in the transcriptomic profile of the endometrium exposed to the ELF-EMF which in turn may disrupt biological processes in the uterus during peri-implantation. • The ELF-EMF impacts epigenetic regulation of gene expression in endometrium. • The ELF-EMF alters DNMTs abundance and global DNA methylation in endometrium. • The ELF-EMF affects the potential for microRNA biogenesis in endometrium. • Histone deacetylases activity is increased in endometrium treated by ELF-EMF. [ABSTRACT FROM AUTHOR]

Chondrosarcoma, the second most common primary malignant bone tumor, originates from cartilaginous tissue and accounts for almost 20% of all primary bone tumors. The management of chondrosarcoma remains challenging due to its diverse clinical course and prognosis, which can range from benign to highly aggressive with a huge risk of metastasis. Emerging research has demonstrated the importance of microRNA (miRNA) dysregulation in the pathogenesis of chondrosarcoma. MiRNAs are small, noncoding RNA molecules that play an essential role in gene expression regulation by targeting specific messenger RNAs (mRNAs) for degradation or translational repression. This article provides an extensive review of current miRNA research in chondrosarcoma, focusing on diagnostic strategies, cell cycle regulation, drug resistance, biomarkers of progression, and stem cell phenotype. We will examine recent studies identifying differentially expressed miRNAs in chondrosarcoma compared to normal cartilage tissue, exploring their potential as diagnostic and prognostic biomarkers. Furthermore, we will discuss the role of miRNAs in regulating cell cycle progression and their potential as therapeutic targets to overcome drug resistance. We will also investigate the prospective utility of miRNAs as biomarkers of progression and their role in modulating the stem cell phenotype of chondrosarcoma cells. This article offers a comprehensive analysis of current miRNA research in chondrosarcoma, focusing on its potential as diagnostic and prognostic biomarkers, therapeutic targets, and regulators of disease progression. By integrating the latest discoveries in this field, we aim to contribute to the development of novel approaches to the prevention, diagnosis, and treatment of chondrosarcoma, ultimately enhancing patient outcomes. [ABSTRACT FROM AUTHOR]

The road from transcription to protein synthesis is paved with many obstacles, allowing for several modes of post-transcriptional regulation of gene expression. A fundamental player in mRNA biology is DDX3X, an RNA binding protein that canonically regulates mRNA translation. By monitoring dynamics of mRNA abundance and translation following DDX3X depletion, we observe stabilization of translationally suppressed mRNAs. We use interpretable statistical learning models to uncover GC content in the coding sequence as the major feature underlying RNA stabilization. This result corroborates GC content-related mRNA regulation detectable in other studies, including hundreds of ENCODE datasets and recent work focusing on mRNA dynamics in the cell cycle. We provide further evidence for mRNA stabilization by detailed analysis of RNA-seq profiles in hundreds of samples, including a Ddx3x conditional knockout mouse model exhibiting cell cycle and neurogenesis defects. Our study identifies a ubiquitous feature underlying mRNA regulation and highlights the importance of quantifying multiple steps of the gene expression cascade, where RNA abundance and protein production are often uncoupled. Synopsis: Monitoring the dynamics of mRNA changes after DDX3X depletion indicates translation suppression followed by mRNA stabilization. GC content in the CDS is a strong predictor of mRNA stabilization and it is detectable in multiple transcriptomics datasets, with a potential link to cell-cycle regulation. RNA-seq and Ribo-seq on a time course of DDX3X depletion show regulation of translation and mRNA levels. Intron-exon count modeling and SLAM-seq demonstrate post-transcriptional mRNA stabilization. Random Forest and Lasso show GC content in the CDS (GCcds) as a main predictor of mRNA stabilization. ENCODE RBP knockdowns and in vivo datasets show widespread GCcds-dependent mRNA stabilization. Monitoring the dynamics of mRNA changes after DDX3X depletion indicates translation suppression followed by mRNA stabilization. GC content in the CDS is a strong predictor of mRNA stabilization and it is detectable in multiple transcriptomics datasets, with a potential link to cell-cycle regulation. [ABSTRACT FROM AUTHOR]

3′ untranslated regions (3′ UTRs) of mRNAs have many functions, including mRNA processing and transport, translational regulation, and mRNA degradation and stability. These different functions require cis‐elements in 3′ UTRs that can be either sequence motifs or RNA structures. Here we review the role of secondary structures in the functioning of 3′ UTRs and discuss some of the trans‐acting factors that interact with these secondary structures in eukaryotic organisms. We propose potential participation of 3′‐UTR secondary structures in cytoplasmic polyadenylation in the model organism Drosophila melanogaster. Because the secondary structures of 3′ UTRs are essential for post‐transcriptional regulation of gene expression, their disruption leads to a wide range of disorders, including cancer and cardiovascular diseases. Trans‐acting factors, such as STAU1 and nucleolin, which interact with 3′‐UTR secondary structures of target transcripts, influence the pathogenesis of neurodegenerative diseases and tumor metastasis, suggesting that they are possible therapeutic targets. [ABSTRACT FROM AUTHOR]

Alternative polyadenylation (APA) is a widespread mechanism of gene regulation that generates mRNA isoforms with alternative 3' untranslated regions (3' UTRs). Our previous study has revealed the global 3' UTR shortening of host mRNAs through APA upon viral infection. However, how the dynamic changes in the APA landscape occur upon viral infection remains largely unknown. Here we further found that, the reduced protein abundance of CPSF6, one of the core 3' processing factors, promotes the usage of proximal poly(A) sites (pPASs) of many immune related genes in macrophages and fibroblasts upon viral infection. Shortening of the 3' UTR of these transcripts may improve their mRNA stability and translation efficiency, leading to the promotion of type I IFN (IFN-I) signalling-based antiviral immune responses. In addition, dysregulated expression of CPSF6 is also observed in many immune related physiological and pathological conditions, especially in various infections and cancers. Thus, the global APA dynamics of immune genes regulated by CPSF6, can fine-tune the antiviral response as well as the responses to other cellular stresses to maintain the tissue homeostasis, which may represent a novel regulatory mechanism for antiviral immunity. Author summary: Innate antiviral immunity endows the host with a first line of defense against viral infection. Efficient regulatory manners are needed to modulate the strength and duration of the response and recover initial state. APA, a widespread gene regulatory mechanism in eukaryotes that generates messenger RNA isoforms with different 3' UTR lengths, has emerged as a key mechanistic step in the regulation of the cellular response to environmental stimuli or perturbation, including viral infection. The global 3' UTR shortening of mRNAs through APA that occurs during viral infection, as described in our previous study, may represent an important yet poorly understood regulatory mechanism of antiviral immune responses. Here, we further find that the reduced protein expression of CPSF6, a core 3' processing factor, effectively contributes to the global 3' UTR shortening of many immune related genes after viral infection, which improves their mRNA stability and translation efficiency and promote the establishment of antiviral defense. Hence, the global 3' UTR shortening caused by reduced CPSF6 during viral infection orchestrates the cellular antiviral capacity to inhibit viral infection. These findings suggest that CPSF6-mediated APA is a smart regulator of antiviral immune responses, opening up new research ideas for the treatment of viral infections and related diseases. [ABSTRACT FROM AUTHOR]

Transcriptional and post-transcriptional regulations control gene expression in most cells. However, critical transitions during the development of the female gamete relies exclusively on regulation of mRNA translation in the absence of de novo mRNA synthesis. Specific temporal patterns of maternal mRNA translation are essential for the oocyte progression through meiosis, for generation of a haploid gamete ready for fertilization and for embryo development. In this review, we will discuss how mRNAs are translated during oocyte growth and maturation using mostly a genome-wide perspective. This broad view on how translation is regulated reveals multiple divergent translational control mechanisms required to coordinate protein synthesis with progression through the meiotic cell cycle and with development of a totipotent zygote. [ABSTRACT FROM AUTHOR]

Background: The mRNA subcellular localization bears substantial impact in the regulation of gene expression, cellular migration, and adaptation. However, the methods employed for experimental determination of this localization are arduous, time-intensive, and come with a high cost. Methods: In this research article, we tackle the essential challenge of predicting the subcellular location of messenger RNAs (mRNAs) through Unified mRNA Subcellular Localization Predictor (UMSLP), a machine learning (ML) based approach. We embrace an in silico strategy that incorporate four distinct feature sets: kmer, pseudo k-tuple nucleotide composition, nucleotide physicochemical attributes, and the 3D sequence depiction achieved via Z-curve transformation for predicting subcellular localization in benchmark dataset across five distinct subcellular locales, encompassing nucleus, cytoplasm, extracellular region (ExR), mitochondria, and endoplasmic reticulum (ER). Results: The proposed ML model UMSLP attains cutting-edge outcomes in predicting mRNA subcellular localization. On independent testing dataset, UMSLP ahcieved over 87% precision, 94% specificity, and 94% accuracy. Compared to other existing tools, UMSLP outperformed mRNALocator, mRNALoc, and SubLocEP by 11%, 21%, and 32%, respectively on average prediction accuracy for all five locales. SHapley Additive exPlanations analysis highlights the dominance of k-mer features in predicting cytoplasm, nucleus, ER, and ExR localizations, while Z-curve based features play pivotal roles in mitochondria subcellular localization detection. Availability: We have shared datasets, code, Docker API for users in GitHub at: https://github.com/smusleh/UMSLP. [ABSTRACT FROM AUTHOR]

Central to the development and survival of all organisms is the regulation of gene expression, which begins with the process of transcription catalyzed by RNA polymerases. During transcription of protein-coding genes, the general transcription factors (GTFs) work alongside RNA polymerase II (Pol II) to assemble the preinitiation complex at the transcription start site, open the promoter DNA, initiate synthesis of the nascent messenger RNA, transition to productive elongation, and ultimately terminate transcription. Through these different stages of transcription, Pol II is dynamically phosphorylated at the C-terminal tail of its largest subunit, serving as a control mechanism for Pol II elongation and a signaling/binding platform for co-transcriptional factors. The large number of core protein factors participating in the fundamental steps of transcription add dense layers of regulation that contribute to the complexity of temporal and spatial control of gene expression within any given cell type. The Pol II transcription system is highly conserved across different levels of eukaryotes; however, most of the information here will focus on the human Pol II system. This review walks through various stages of transcription, from preinitiation complex assembly to termination, highlighting the functions and mechanisms of the core machinery that participates in each stage. [ABSTRACT FROM AUTHOR]

Background: In recent years, microRNAs (miRNAs) have emerged as key players in the pathophysiology of multiple diseases including Alzheimer's disease (AD). Messenger RNA (mRNA) targeting for regulation of gene expression by miRNAs has been implicated in the annotation of disease pathophysiology as well as in the explication of their starring role in contemporary therapeutic interventions. One such miRNA is miR-153 which mediates the survival of cortical neurons and inhibits plaque formation. However, the core mRNA targets of miR-153 have not been fully illustrated. Objective: The present study aimed to elucidate the potential involvement of miR-153 in AD pathogenesis and to reveal its downstream targets. Methods: miRanda was used to identify AD-associated targets of miR-153. TargetScan, PicTar, miRmap, and miRDB were further used to validate these targets. STRING 12 was employed to assess the protein-protein interaction network while Gene ontology (GO) analysis was carried out to identify the molecular functions exhibited by these gene targets. Results: In silico analysis using miRanda predicted five important AD-related targets of miR-153, including APP, SORL1, PICALM, USF1, and PSEN1. All five target genes are negatively regulated by miR-153 and are substantially involved in AD pathogenesis. A protein interaction network using STRING 12 uncovered 30 potential interacting partners for SORL1, PICALM, and USF1. GO analysis revealed that miR-153 target genes play a critical role in neuronal survival, differentiation, exon guidance, amyloid precursor protein processing, and synapse formation. Conclusion: These findings unravel the potential role of miR-153 in the pathogenesis of AD and provide the basis for forthcoming experimental studies. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra, leading to various motor and non‐motor symptoms. Several cellular and molecular mechanisms such as alpha‐synuclein (α‐syn) accumulation, mitochondrial dysfunction, oxidative stress and neuroinflammation are involved in the pathogenesis of this disease. MicroRNAs (miRNAs) play important roles in post‐transcriptional gene regulation. They are typically about 21–25 nucleotides in length and are involved in the regulation of gene expression by binding to the messenger RNA (mRNA) molecules. miRNAs like miR‐221 play important roles in various biological processes, including development, cell proliferation, differentiation and apoptosis. miR‐221 promotes neuronal survival against oxidative stress and neurite outgrowth and neuronal differentiation. Additionally, the role of miR‐221 in PD has been investigated in several studies. According to the results of these studies, (1) miR‐221 protects PC12 cells against oxidative stress induced by 6‐hydroxydopamine; (2) miR‐221 prevents Bax/caspase‐3 signalling activation by stopping Bim; (3) miR‐221 has moderate predictive power for PD; (4) miR‐221 directly targets PTEN, and PTEN over‐expression eliminates the protective action of miR‐221 on p‐AKT expression in PC12 cells; and (5) miRNA‐221 controls cell viability and apoptosis by manipulating the Akt signalling pathway in PD. This review study suggested that miR‐221 has the potential to be used as a clinical biomarker for PD diagnosis and stage assignment. [ABSTRACT FROM AUTHOR]

Motivation On-target gene knockdown, using siRNA, ideally results from binding fully complementary regions in mRNA transcripts to induce direct cleavage. Off-target siRNA gene knockdown can occur through several modes, one being a seed-mediated mechanism mimicking miRNA gene regulation. Seed-mediated off-target effects occur when the ∼8 nucleotides at the 5' end of the guide strand, called a seed region, bind the 3' untranslated regions of mRNA, causing reduced translation. Experiments using siRNA knockdown paired with RNA-seq can be used to detect siRNA sequences with off-target effects driven by the seed region. However, there are limited computational tools designed specifically for detecting siRNA off-target effects mediated by the seed region in differential gene expression experiments. Results SeedMatchR is an R package developed to provide users a single, unified resource for detecting and visualizing seed-mediated off-target effects of siRNA using RNA-seq experiments. SeedMatchR is designed to extend current differential expression analysis tools, such as DESeq2 , by annotating results with predicted seed matches. Using publicly available data, we demonstrate the ability of SeedMatchR to detect cumulative changes in differential gene expression attributed to siRNA seed region activity. Availability SeedMatchR is available on CRAN. Documentation and example workflows are available through the SeedMatchR GitHub page at https://github.com/tacazares/SeedMatchR. [ABSTRACT FROM AUTHOR]

One of the most frequent epigenetic modifications of RNA in eukaryotes is N6 methyladenosine (m6A), which is mostly present in messenger RNAs. Through the influence of several RNA processing stages, m6Amodification is a crucial approach for controlling gene expression, especially in cancer progression. It is universally acknowledged that numerous non-coding RNAs (ncRNAs), such as microRNAs, circular RNAs, long non-coding RNAs, and piRNAs, are also significantly affected by m6A modification, and the complex genetic regulatory relationship between m6A and ncRNAs plays a pivotal role in the development of cancer. The connection between m6A modifications and ncRNAs offers an opportunity to explore the oncogene potential regulatory mechanisms and suggests that m6A modifications and ncRNAs could be vital biomarkers for multiple cancers. In this review, we discuss the mechanisms of interaction between m6Amethylation and ncRNAs in cancer, and we also summarize diagnostic and prognostic biomarkers for clinical cancer detection. Furthermore, our article includes some methodologies for identifying m6A sites when assessing biomarker potential. [ABSTRACT FROM AUTHOR]

Co‐expression of two or more genes at the single‐cell level is usually associated with functional co‐regulation. While mRNA co‐expression—measured as the correlation in mRNA levels—can be influenced by both transcriptional and post‐transcriptional events, transcriptional regulation is typically considered dominant. We review and connect the literature describing transcriptional and post‐transcriptional regulation of co‐expression. To enhance our understanding, we integrate four datasets spanning single‐cell gene expression data, single‐cell promoter activity data and individual transcript half‐lives. Confirming expectations, we find that positive co‐expression necessitates promoter coordination and similar mRNA half‐lives. Surprisingly, negative co‐expression is favored by differences in mRNA half‐lives, contrary to initial predictions from stochastic simulations. Notably, this association manifests specifically within clusters of genes. We further observe a striking compensation between promoter coordination and mRNA half‐lives, which additional stochastic simulations suggest might give rise to the observed co‐expression patterns. These findings raise intriguing questions about the functional advantages conferred by this compensation between distal kinetic steps. [ABSTRACT FROM AUTHOR]

The hairpin element (HpE) near the start codon in the 5′ untranslated region (UTR) was developed to tune the mRNA translation in mammalian cells. The parameters of HpEs including thermodynamic stability, the content of guanine and cytosine, and distance between HpEs and the 5′ cap were investigated. These parameters influenced the mRNA expression level. In addition, the start codon and the upstream open reading frame sequestered within the structures of HpEs also reduced the translation initiation. In summary, this study shows that the simple engineering HpE structure can be efficiently adopted for gene expression regulation. The predictable controllability of this simple cloning strategy can potentially achieve precise gene expression regulation in different mammalian cell types. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that play a role in post-transcriptional gene regulation. It is generally accepted that their main mechanism of action is the negative regulation of gene expression, through binding to specific regions in messenger RNA (mRNA) and repressing protein translation. By interrupting protein synthesis, miRNAs can effectively turn genes off and influence many basic processes in the body, such as developmental and apoptotic behaviours of cells and cardiac organogenesis. Their importance is highlighted by inhibiting or overexpressing certain miRNAs, which will be discussed in the context of coronary artery disease, atrial fibrillation, bradycardia, and heart failure. Dysregulated levels of miRNAs in the body can exacerbate or alleviate existing disease, and their omnipresence in the body makes them reliable as quantifiable markers of disease. This review aims to provide a summary of miRNAs as biomarkers and their interactions with targets that affect cardiac health, and intersperse it with current therapeutic knowledge. It intends to succinctly inform on these topics and guide readers toward more comprehensive works if they wish to explore further through a wide-ranging citation list. [ABSTRACT FROM AUTHOR]

Background: Long intergenic non-coding RNA, is one type of lncRNA, exerting various cellular activities, as does ncRNA, including the regulation of gene expression and chromatin remodeling. The abnormal expression of lincRNAs can induce or suppress carcinogenesis. Main body: LincRNAs can regulate cancer progression through different mechanisms and are considered as potential drug targets. Genetic variations such as single nucleotide polymorphisms (SNPs) in lincRNAs may affect gene expression and messenger ribonucleic acid (mRNA) stability. SNPs in lincRNAs have been found to be associated with different types of cancer, as well. Specifically, LINC00511 has been known to promote the progression of multiple malignancies such as breast cancer, colorectal cancer, lung cancer, hepatocellular carcinoma, and others, making it a promising cancer prognostic molecular marker. Conclusion: LincRNAs have been proved to be associated with different cancer types through various pathways. Herein, we performed a comprehensive literature and in silico databases search listing lncRNAs, lincRNAs including LINC00511, lncRNAs' SNPs, as well as LINC00511 SNPs in different cancer types, focusing on their role in various cancer types and mechanism(s) of action. [ABSTRACT FROM AUTHOR]

The article reports on the relationship between mRNA stability and coding sequence length, highlighting a switch-like emergence of differential mRNA stability in yeast. It also discusses how untranslated regions (UTRs) can influence this threshold length, providing insights into the regulation of mRNA stability and translation in the context of gene expression control.

Translational control is critical for cell fate transitions during development, lineage specification, and tumorigenesis. Here, we show that the transcription factor double homeobox protein 4 (DUX4), and its previously characterized transcriptional program, broadly regulates translation to change the cellular proteome. DUX4 is a key regulator of zygotic genome activation in human embryos, whereas misexpression of DUX4 causes facioscapulohumeral muscular dystrophy (FSHD) and is associated with MHC-I suppression and immune evasion in cancer. We report that translation initiation and elongation factors are disrupted downstream of DUX4 expression in human myoblasts. Genome-wide translation profiling identified mRNAs susceptible to DUX4-induced translation inhibition, including those encoding antigen presentation factors and muscle lineage proteins, while DUX4-induced mRNAs were robustly translated. Endogenous expression of DUX4 in human FSHD myotubes and cancer cell lines also correlated with reduced protein synthesis and MHC-I presentation. Our findings reveal that DUX4 orchestrates translational reprogramming by suppressing the cellular proteome while maintaining translation of DUX4-induced mRNAs to promote an early developmental program. Cellular reprogramming requires multifaceted regulation of gene expression. This study shows that DUX4 modulates multiple translational regulators to broadly suppress protein synthesis while promoting expression of DUX4-induced genes, suggesting a natural role in cell state conversion during development and a pathological role when expressed in cancer or muscular dystrophy. [ABSTRACT FROM AUTHOR]

The localization of messenger RNAs (mRNAs) is a frequently observed phenomenon and a crucial aspect of gene expression regulation. It is also a mechanism for targeting proteins to a specific cellular region. Moreover, prior research and studies have shown the significance of intracellular RNA positioning during embryonic and neural dendrite formation. Incorrect RNA localization, which can be caused by a variety of factors, such as mutations in trans-regulatory elements, has been linked to the development of certain neuromuscular diseases and cancer. In this study, we introduced NN-RNALoc, a neural network-based method for predicting the cellular location of mRNA using novel features extracted from mRNA sequence data and protein interaction patterns. In fact, we developed a distance-based subsequence profile for RNA sequence representation that is more memory and time-efficient than well-known k-mer sequence representation. Combining protein-protein interaction data, which is essential for numerous biological processes, with our novel distance-based subsequence profiles of mRNA sequences produces more accurate features. On two benchmark datasets, CeFra-Seq and RNALocate, the performance of NN-RNALoc is compared to powerful predictive models proposed in previous works (mRNALoc, RNATracker, mLoc-mRNA, DM3Loc, iLoc-mRNA, and EL-RMLocNet), and a ground neural (DNN5-mer) network. Compared to the previous methods, NN-RNALoc significantly reduces computation time and also outperforms them in terms of accuracy. This study's source code and datasets are freely accessible at https://github.com/NeginBabaiha/NN-RNALoc. [ABSTRACT FROM AUTHOR]

Chemical modifications of nucleotides expand the complexity and functional properties of genomes and transcriptomes. A handful of modifications in DNA bases are part of the epigenome, wherein DNA methylation regulates chromatin structure, transcription, and co-transcriptional RNA processing. In contrast, more than 150 chemical modifications of RNA constitute the epitranscriptome. Ribonucleoside modifications comprise a diverse repertoire of chemical groups, including methylation, acetylation, deamination, isomerization, and oxidation. Such RNA modifications regulate all steps of RNA metabolism, including folding, processing, stability, transport, translation, and RNA's intermolecular interactions. Initially thought to influence all aspects of the post-transcriptional regulation of gene expression exclusively, recent findings uncovered a crosstalk between the epitranscriptome and the epigenome. In other words, RNA modifications feedback to the epigenome to transcriptionally regulate gene expression. The epitranscriptome achieves this feat by directly or indirectly affecting chromatin structure and nuclear organization. This review highlights how chemical modifications in chromatin-associated RNAs (caRNAs) and messenger RNAs (mRNAs) encoding factors involved in transcription, chromatin structure, histone modifications, and nuclear organization affect gene expression transcriptionally. [ABSTRACT FROM AUTHOR]

HTLV-1 is an oncogenic human retrovirus and the etiologic agent of the highly aggressive ATL malignancy. Two viral genes, Tax and Hbz, are individually linked to oncogenic transformation and play an important role in the pathogenic process. Consequently, regulation of HTLV-1 gene expression is a central feature in the viral lifecycle and directly contributes to its pathogenic potential. Herein, we identified the cellular transcription factor YBX1 as a binding partner for HBZ. We found YBX1 activated transcription and enhanced Tax-mediated transcription from the viral 5′ LTR promoter. Interestingly, YBX1 also interacted with Tax. shRNA-mediated loss of YBX1 decreased transcript and protein abundance of both Tax and HBZ in HTLV-1-transformed T-cell lines, as well as Tax association with the 5′ LTR. Conversely, YBX1 transcriptional activation of the 5′ LTR promoter was increased in the absence of HBZ. YBX1 was found to be associated with both the 5′ and 3′ LTRs in HTLV-1-transformed and ATL-derived T-cell lines. Together, these data suggest that YBX1 positively influences transcription from both the 5′ and 3′ promoter elements. YBX1 is able to interact with Tax and help recruit Tax to the 5′ LTR. However, through interactions with HBZ, YBX1 transcriptional activation of the 5′ LTR is repressed. [ABSTRACT FROM AUTHOR]

Transcription is an essential process in biology whereby gene-specific transcription factors target sites on DNA to recruit the basal transcription machinery that will produce messenger RNA (mRNA). It is a highly regulated multi-step process that involves many proteins and protein complexes. Transcription factors, the proteins that mark genes for activation, and other transcriptional regulators are highly enriched in low-complexity disordered regions, which are strongly linked to multivalent binding and phase separation. These disordered regions can form multivalent dynamic complexes that are essential for many aspects of transcription. Many of these proteins can phase separate in vitro and show evidence of phase separation in vivo. Whether these interactions represent biologically relevant phase separation in vivo is controversial. However, what these events do demonstrate is that many transcriptional proteins co-cluster with other factors in vivo , forming multivalent dynamic clusters that contribute to transcriptional events. We review some of these recently investigated events and consider how they contribute to our understanding of transcription. Proteins that regulate transcription are highly intrinsically disordered, which is strongly linked to multivalent binding and liquid–liquid phase separation. The biological relevance of widespread phase separation is controversial, but many transcriptional proteins co-cluster with other factors, forming multiprotein–DNA complexes and multivalent dynamic hubs to target DNA and initiate transcription. Nucleic acids and other biopolymers (green, purple) and transcription factors (blue) form multivalent networks. TFs (blue) make direct interactions with DNA (orange). Multiple TFs (multiple colours) interact with DNA (orange) to initiate transcription. Dark green indicates concentrated liquid droplets surrounded by a low concentration liquid phase. [ABSTRACT FROM AUTHOR]

RNA‐binding protein with serine‐rich domain 1 (RNPS1) gets deposited on the mRNA during the process of splicing and concomitantly associates with the exon junction complex (EJC). RNPS1 participates in post‐transcriptional gene regulation, including constitutive and alternative splicing, transcriptional regulation and nonsense‐mediated mRNA decay. In this study, we found that the tethering of RNPS1 or its isolated serine‐rich domain (S domain) causes exon inclusion of an HIV‐1 splicing substrate. In contrast, overexpressing the RRM domain of RNPS1 acts in a dominant negative manner and leads to the exon skipping of endogenous apoptotic pre‐mRNAs (Bcl‐X and MCL‐1). Further, tethering of core EJC proteins, eIF4A3, MAGOH, or Y14, does not lead to exon inclusion of an HIV substrate. Together, our results demonstrate how RNPS1 and its domains are differentially involved in alternative splicing activity. [ABSTRACT FROM AUTHOR]

Significance: Stress granules (SGs) are biomolecular condensates that form upon global translation suppression during stress. SGs are enriched in translation factors and messenger RNAs (mRNAs), which they may sequester away from the protein synthesis machinery. While this is hypothesized to remodel the functional transcriptome during stress, it remains unclear whether SGs are a cause, or simply a consequence, of translation repression. Understanding the function of SGs is particularly important because they are implicated in numerous diseases including viral infections, cancer, and neurodegeneration. Recent Advances: We synthesize recent SG research spanning biological scales, from observing single proteins and mRNAs within one cell to measurements of the entire transcriptome or proteome of SGs in a cell population. We use the emerging understanding from these studies to suggest that SGs likely have less impact on global translation, but instead may strongly influence the translation of individual mRNAs localized to them. Critical Issues: Development of a unified model that links stress-induced RNA-protein condensation to regulation of downstream gene expression holds promise for understanding the mechanisms of cellular resilience. Future Directions: Therefore, upcoming research should clarify what influence SGs exert on translation at all scales as well as the molecular mechanisms that enable this. The resulting knowledge will be required to drive discovery in how SGs allow organisms to adapt to challenges and support health or go awry and lead to disease. Antioxid. Redox Signal. 39, 390–409. [ABSTRACT FROM AUTHOR]

Couch's spadefoot toad (Scaphiopus couchii) spends most of the year underground in a hypometabolic state known as estivation. During this time, they overcome significant dehydration and lack of food through many mechanisms including employing metabolic rate depression (MRD), increasing urea concentration, switching to lipid oxidation as the primary energy source, and decreasing their breathing and heart rate. MicroRNA (miRNA) are known to regulate translation by targeting messenger RNA (mRNA) for degradation or temporary storage, with several studies having reported that miRNA is differentially expressed during MRD, including estivation. Thus, we hypothesized that miRNA would be involved in gene regulation during estivation in S. couchii heart. Next-generation sequencing and bioinformatic analyses were used to assess changes in miRNA expression in response to two-month estivation and to predict the downstream effects of this expression. KEGG and GO analyses indicated that ribosome and cardiac muscle contraction are among the pathways predicted to be upregulated, whereas cell signaling and fatty acid metabolism were predicted to be downregulated. Together these results suggest that miRNAs contribute to the regulation of gene expression related to cardiac muscle physiology and energy metabolism during estivation. [Display omitted] • Spadefoot toads undergo estivation for ∼10 months in response to drought conditions. • 4 miRNAs increase and 11 miRNAs decrease in S. couchii heart during estivation. • miRNAs predict upregulation of ribosome and cardiac muscle contraction. • miRNAs predict downregulation of cell signaling and fatty acid metabolism. [ABSTRACT FROM AUTHOR]

Background: Insulin like growth factor II mRNA binding protein 3 (IGF2BP3) is an RNA binding protein with multiple roles in regulation of gene expression at the post-transcriptional level and is implicated in tumorigenesis and progression of numerous cancers including gastric cancer (GC). Circular RNAs (circRNAs) are a diverse endogenous noncoding RNA population that have important regulatory roles in cancer. However, circRNAs that regulate the expression of IGF2BP3 in GC is largely unknown. Methods: CircRNAs that bound to IGF2BP3 were screened in GC cells using RNA immunoprecipitation and sequencing (RIP-seq). The identification and localization of circular nuclear factor of activated T cells 3 (circNFATC3) were identified using Sanger sequencing, RNase R assays, qRT-PCR, nuclear-cytoplasmic fractionation and RNA-FISH assays. CircNFATC3 expression in human GC tissues and adjacent normal tissues were measured by qRT-PCR and ISH. The biological role of circNFATC3 in GC was confirmed by in vivo and in vitro experiments. Furthermore, RIP, RNA-FISH/IF, IP and rescue experiments were performed to uncover interactions between circNFATC3, IGF2BP3 and cyclin D1 (CCND1). Results: We identified a GC-associated circRNA, circNFATC3, that interacted with IGF2BP3. CircNFATC3 was significantly overexpressed in GC tissues and was positively associated with tumor volume. Functionally, the proliferation of GC cells decreased significantly after circNFATC3 knockdown in vivo and in vitro. Mechanistically, circNFATC3 bound to IGF2BP3 in the cytoplasm, which enhanced the stability of IGF2BP3 by preventing ubiquitin E3 ligase TRIM25-mediated ubiquitination, thereby enhancing the regulatory axis of IGF2BP3-CCND1 and promoting CCND1 mRNA stability. Conclusions: Our findings demonstrate that circNFATC3 promotes GC proliferation by stabilizing IGF2BP3 protein to enhance CCND1 mRNA stability. Therefore, circNFATC3 is a potential novel target for the treatment of GC. [ABSTRACT FROM AUTHOR]

Simple Summary: Alzheimer's disease (AD) is a progressive, age-related neurodegenerative disorder representing the most common cause of senile dementia and neurological dysfunction in our aging domestic population. MicroRNAs (miRNAs) are a small family of non-coding single-stranded RNAs (ssRNAs) that function in complex interactive networks to direct the post-transcriptional repression of messenger RNA (mRNA)-encoded genetic information. Current evidence suggests that these ssRNAs perform an important regulatory role in the expression of genes in the AD-affected brain and central nervous system (CNS). This review paper will focus on recent studies, developments and advancements in our appreciation and understanding of the complexity of miRNA signaling in AD-affected brain hippocampal CA1 compared to age- and gender-matched controls. AD is a complex, progressive, age-related neurodegenerative disorder representing the most common cause of senile dementia and neurological dysfunction in our elderly domestic population. The widely observed heterogeneity of AD is a reflection of the complexity of the AD process itself and the altered molecular-genetic mechanisms operating in the diseased human brain and CNS. One of the key players in this complex regulation of gene expression in human pathological neurobiology are microRNAs (miRNAs) that, through their actions, shape the transcriptome of brain cells that normally associate with very high rates of genetic activity, gene transcription and messenger RNA (mRNA) generation. The analysis of miRNA populations and the characterization of their abundance, speciation and complexity can further provide valuable clues to our molecular-genetic understanding of the AD process, especially in the sporadic forms of this common brain disorder. Current in-depth analyses of high-quality AD and age- and gender-matched control brain tissues are providing pathophysiological miRNA-based signatures of AD that can serve as a basis for expanding our mechanistic understanding of this disorder and the future design of miRNA- and related RNA-based therapeutics. This focused review will consolidate the findings from multiple laboratories as to which are the most abundant miRNA species, both free and exosome-bound in the human brain and CNS, which miRNA species appear to be the most prominently affected by the AD process and review recent developments and advancements in our understanding of the complexity of miRNA signaling in the hippocampal CA1 region of AD-affected brains. [ABSTRACT FROM AUTHOR]

Across all kingdoms of life, gene regulatory mechanisms underlie cellular adaptation to ever-changing environments. Regulation of gene expression adjusts protein synthesis and, in turn, cellular growth. Messenger RNAs are key molecules in the process of gene expression. Our ability to quantitatively measure mRNA expression in single cells has improved tremendously over the past decades. This revealed an unexpected coordination between the steps that control the life of an mRNA, from transcription to degradation. Here, we provide an overview of the state-of-the-art imaging approaches for measurement and quantitative understanding of gene expression, starting from the early visualizations of single genes by electron microscopy to current fluorescence-based approaches in single cells, including live-cell RNA-imaging approaches to FISH-based spatial transcriptomics across model organisms. We also highlight how these methods have shaped our current understanding of the spatiotemporal coupling between transcriptional and post-transcriptional events in prokaryotes. We conclude by discussing future challenges of this multidisciplinary field. Abbreviations: mRNA: messenger RNA; rRNA: ribosomal rDNA; tRNA: transfer RNA; sRNA: small RNA; FISH: fluorescence in situ hybridization; RNP: ribonucleoprotein; smFISH: single RNA molecule FISH; smiFISH: single molecule inexpensive FISH; HCR-FISH: Hybridization Chain-Reaction-FISH; RCA: Rolling Circle Amplification; seqFISH: Sequential FISH; MERFISH: Multiplexed error robust FISH; UTR: Untranslated region; RBP: RNA binding protein; FP: fluorescent protein; eGFP: enhanced GFP, MCP: MS2 coat protein; PCP: PP7 coat protein; MB: Molecular beacons; sgRNA: single guide RNA. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) occurs in all organisms and modulates most, if not all, biological pathways. It is the process by which non-coding RNAs, including microRNAs (miRNAs), regulate gene transcription and post-transcriptional processing of messenger RNA (mRNA). A single miRNA can modulate several genes within a cell, and several miRNAs can regulate expression of the same gene, adding tiers of complexity to the regulation of gene expression. miRNAs and other RNAi approaches have been successfully used in vitro and in vivo to selectively manipulate gene transcription, making them pivotal agents for basic science research and candidates for targeted therapeutics. This review focuses on miRNAs and their potential as biomarkers and novel therapeutics for glomerular disease. [ABSTRACT FROM AUTHOR]

Post‐transcriptional regulation of messenger RNAs (mRNAs) (i.e., mechanisms that control translation, stability and localization) is a critical focal point in spatiotemporal regulation of gene expression in response to changes in environmental conditions. The human genome encodes ~ 2000 microRNAs (miRNAs), each of which could control the expression of hundreds of protein‐coding mRNAs by inducing translational repression and/or promoting mRNA decay. While mRNA degradation is a terminal event, translational repression is reversible and can be employed for rapid response to internal or external cues. Recent years have seen significant progress in our understanding of how miRNAs induce degradation or translational repression of the target mRNAs. Here, we review the recent findings that illustrate the cellular machinery that contributes to miRNA‐induced silencing, with a focus on the factors that could influence translational repression vs. decay. [ABSTRACT FROM AUTHOR]

Simple Summary: The objective of this investigation was to elucidate the association of miRNA-mediated genes in the regulation of dog testes development from immature to adult form by in-silico analysis. In silico analysis of differentially expressed (DE) testis miRNAs between healthy immature and mature dogs were performed using miRNet, STRING, and ClueGo programs. The determination of mRNA and protein expressions of predicted pivotal genes and their association with miRNA were studied. The predicted genes are involved in the governing of several key biological functions (cell cycle, cell proliferation, growth, maturation, survival, and apoptosis) in the testis as they evolve from immature to adult forms, mediated by several key signaling pathways (ErbB, p53, PI3K-Akt, VEGF, and JAK-STAT), cytokines and hormones (estrogen, GnRH, relaxin, thyroid hormone, and prolactin). Elucidation of DE-miRNA predicted genes' specific roles, signal transduction pathways, and mechanisms, by mimics and inhibitors, which could perhaps offer diagnostic and therapeutic targets for infertility, cancer, and birth control. High-throughput in-silico techniques help us understand the role of individual proteins, protein–protein interaction, and their biological functions by corroborating experimental data as epitomized biological networks. The objective of this investigation was to elucidate the association of miRNA-mediated genes in the regulation of dog testes development from immature to adult form by in-silico analysis. Differentially expressed (DE) canine testis miRNAs between healthy immature (2.2 ± 0.13 months; n = 4) and mature (11 ± 1.0 months; n = 4) dogs were utilized in this investigation. In silico analysis was performed using miRNet, STRING, and ClueGo programs. The determination of mRNA and protein expressions of predicted pivotal genes and their association with miRNA were studied. The results showed protein–protein interaction for the upregulated miRNAs, which revealed 978 enriched biological processes GO terms and 127 KEGG enrichment pathways, and for the down-regulated miRNAs revealed 405 significantly enriched biological processes GO terms and 72 significant KEGG enrichment pathways (False Recovery Rate, p < 0.05). The in-silico analysis of DE-miRNA's associated genes revealed their involvement in the governing of several key biological functions (cell cycle, cell proliferation, growth, maturation, survival, and apoptosis) in the testis as they evolve from immature to adult forms, mediated by several key signaling pathways (ErbB, p53, PI3K-Akt, VEGF and JAK-STAT), cytokines and hormones (estrogen, GnRH, relaxin, thyroid hormone, and prolactin). Elucidation of DE-miRNA predicted genes' specific roles, signal transduction pathways, and mechanisms, by mimics and inhibitors, which could perhaps offer diagnostic and therapeutic targets for infertility, cancer, and birth control. [ABSTRACT FROM AUTHOR]

Alternative splicing (AS) in plants plays a key role in regulating the expression of numerous transcripts from a single gene in a regulatory pathway. Variable concentrations of growth regulatory hormones and external stimuli trigger alternative splicing to switch among different growth stages and adapt to environmental stresses. In the AS phenomenon, a spliceosome causes differential transcriptional modifications in messenger RNA (mRNAs), resulting in partial or complete retention of one or more introns as compared to fully spliced mRNA. Differentially expressed proteins translated from intron-retaining messenger RNA (mRNAir) perform vital functions in the feedback mechanism. At the post-transcriptional level, AS causes the remodeling of transcription factors (TFs) by the addition or deletion of binding domains to activate and/or repress transcription. In this study, we have summarized the specific role of AS in the regulation of gene expression through repression and activation of the transcriptional regulatory network under external stimuli and switch among developmental stages. [ABSTRACT FROM AUTHOR]

Background: Alfalfa is one of the most important legume forages in the world; it includes three fall dormancy (FD) types. There is previous difference between FD alfalfa and non-FD alfalfa. However, the molecular basis of these differences remains largely dim. MicroRNAs (miRNAs) have critical roles in the regulation of gene expression; the reverse differential expression of genes and miRNAs can help us to reveal the regulation mechanism of fall dormancy in alfalfa. Methods: We carried out integrative analysis of miRNA and mRNA in dormant alfalfa under different growth conditions based on miRNA and transcriptome sequencing analysis. The differential genes were obtained by transcriptome analysis and the differential miRNAs were obtained by small RNA analysis. Then the integration analysis of miRNAs and mRNAs was performed to screen reverse different expressions between genes and miRNAs to construct the key miRNA-mRNA regulatory network. Result: Our results indicated the biological process was the key factor in the fall dormancy of alfalfa, 24 miRNAs downregulated with transcript genes upregulated and 24 miRNAs upregulated with transcript genes downregulated, respectively. Key factors of the regulatory network showed that MiR5287b and miR2643a had a more complex network. MiR5287b had 22 corresponding regulatory transcript factors and miR2643a had 17 corresponding regulatory transcript factors and some conserved miRNAs (e.g., miR172a, miR156e and miRNA169h). Most of them play the vital role in plant growth and development and also participate in regulating fall dormancy in alfalfa. [ABSTRACT FROM AUTHOR]

Trichomonas vaginalis is one of the most common sexually transmitted parasites in humans. This protozoan has high iron requirements for growth, metabolism, and virulence. However, iron concentrations also differentially modulate T. vaginalis gene expression as in the genes encoding cysteine proteinases TvCP4 and TvCP12. Our goal was to identify the regulatory mechanism mediating the upregulation of tvcp12 under iron-restricted (IR) conditions. Here, we showed by RT-PCR, Western blot, and immunocytochemistry assays that IR conditions increase mRNA stability and amount of TvCP12. RNA electrophoretic mobility shift assay (REMSA), UV cross-linking, and competition assays demonstrated that a non-canonical iron-responsive element (IRE)-like structure at the 3′-untranslated region of the tvcp12 transcript (IRE-tvcp12) specifically binds to human iron regulatory proteins (IRPs) and to atypical RNA-binding cytoplasmic proteins from IR trichomonads, such as HSP70 and α-Actinin 3. These data were confirmed by REMSA supershift and Northwestern blot assays. Thus, our findings show that a positive gene expression regulation under IR conditions occurs at the posttranscriptional level possibly through RNA-protein interactions between atypical RNA-binding proteins and non-canonical IRE-like structures at the 3′-UTR of the transcript by a parallel mechanism to the mammalian IRE/IRP system that can be applied to other iron-regulated genes of T. vaginalis. [ABSTRACT FROM AUTHOR]

Background: Subcellular localization of messenger RNA (mRNAs) plays a pivotal role in the regulation of gene expression, cell migration as well as in cellular adaptation. Experiment techniques for pinpointing the subcellular localization of mRNAs are laborious, time-consuming and expensive. Therefore, in silico approaches for this purpose are attaining great attention in the RNA community. Methods: In this article, we propose MSLP, a machine learning-based method to predict the subcellular localization of mRNA. We propose a novel combination of four types of features representing k-mer, pseudo k-tuple nucleotide composition (PseKNC), physicochemical properties of nucleotides, and 3D representation of sequences based on Z-curve transformation to feed into machine learning algorithm to predict the subcellular localization of mRNAs. Results: Considering the combination of the above-mentioned features, ennsemble-based models achieved state-of-the-art results in mRNA subcellular localization prediction tasks for multiple benchmark datasets. We evaluated the performance of our method in ten subcellular locations, covering cytoplasm, nucleus, endoplasmic reticulum (ER), extracellular region (ExR), mitochondria, cytosol, pseudopodium, posterior, exosome, and the ribosome. Ablation study highlighted k-mer and PseKNC to be more dominant than other features for predicting cytoplasm, nucleus, and ER localizations. On the other hand, physicochemical properties and Z-curve based features contributed the most to ExR and mitochondria detection. SHAP-based analysis revealed the relative importance of features to provide better insights into the proposed approach. Availability: We have implemented a Docker container and API for end users to run their sequences on our model. Datasets, the code of API and the Docker are shared for the community in GitHub at: https://github.com/smusleh/MSLP. [ABSTRACT FROM AUTHOR]

Dissection of regulatory network that control wood structure is highly challenging in functional genomics. Nevertheless, due to the availability of genomic, transcriptomic and proteomic sequences, a large amount of information is available for use in achieving this goal. MicroRNAs, which compose a class of small non-coding RNA molecules that inhibit protein translation by targeting mRNA cleavage sites and thus regulate a wide variety of developmental and physiological processes in plants, are important parts of this regulatory network. These findings and the availability of sequence information have made it possible to carry out an in silico analysis to predict and annotate miRNAs and their target genes associated with an important factor affecting wood rigidity, microfibril angle (MFA), throughout the Populus trichocarpa Hook. genome. Our computational approach revealed miRNAs and their targets via ESTs, sequences putatively associated with microfibril angle. In total, 250 miRNAs were identified as RNA molecules with roles in the silencing and post-transcriptional regulation of the expression of nine genes. We found SHY2, IAA4 (ATAUX2–11), BZIP60, AP2, MYB15, ABI3, MYB17, LAF1 and MYB28 as important nodes in a network with possible role in MFA determination. Other co-expressed genes putatively involved in this regulatory system were also identified by construction of a co-expression network. The candidate genes from this study may help unravel the regulatory networks putatively linked to microfibril angle. [ABSTRACT FROM AUTHOR]

Simple Summary: In eukaryotes, the elimination of the protective cap from 5′ end of messenger RNA (mRNA) by the decapping complex is an important mechanism of mRNA decay and translation, affecting several cellular processes. In the nematode C. elegans, perturbations in decapping activity slow development and shorten the lifespan. A microarray analysis performed in mid-aged nematodes with reduced decapping activity revealed a specific role for decapping in the suppression of spermatogenic genes in germ cells during ageing. In addition, the expression of many innate immunity and detoxification genes were upregulated in the somatic cells of the mutant nematodes, despite the lack of pathogens. This upregulation appears to be mediated by the effect of aberrant decapping on both the mRNA stability and the nuclear translocation of the transcription factor PQM-1, which controls the expression of those immunity/detoxification genes. Although the decapping-mediated induction of immunity was found to be detrimental for the normal lifespan, it mitigates the paralysis in a C. elegans model of polyglutamine toxicity. These results reinforce the selectivity of decapping in the regulation of gene expression and provide a link between mRNA decapping and innate immunity in older ages that could serve as a protective mechanism for disturbed proteostasis, commonly associated with human neurodegenerative diseases. Removal of the 5′ cap structure of RNAs (termed decapping) is a pivotal event in the life of cytoplasmic mRNAs mainly catalyzed by a conserved holoenzyme, composed of the catalytic subunit DCP2 and its essential cofactor DCP1. While decapping was initially considered merely a step in the general 5′-3′ mRNA decay, recent data suggest a great degree of selectivity that plays an active role in the post-transcriptional control of gene expression, and regulates multiple biological functions. Studies in Caenorhabditis elegans have shown that old age is accompanied by the accumulation of decapping factors in cytoplasmic RNA granules, and loss of decapping activity shortens the lifespan. However, the link between decapping and ageing remains elusive. Here, we present a comparative microarray study that was aimed to uncover the differences in the transcriptome of mid-aged dcap-1/DCP1 mutant and wild-type nematodes. Our data indicate that DCAP-1 mediates the silencing of spermatogenic genes during late oogenesis, and suppresses the aberrant uprise of immunity gene expression during ageing. The latter is achieved by destabilizing the mRNA that encodes the transcription factor PQM-1 and impairing its nuclear translocation. Failure to exert decapping-mediated control on PQM-1 has a negative impact on the lifespan, but mitigates the toxic effects of polyglutamine expression that are involved in human disease. [ABSTRACT FROM AUTHOR]

The field of RNA modification, also referred to as "epitranscriptomics," is gaining more and more interest from the scientific community. More than 160 chemical modifications have been identified in RNA molecules, but the functional significance of most of them still needs to be clarified. In this review, we discuss the role of N6,2′-O-dimethyladenosine (m6Am) in gene expression regulation. m6Am is present in the first transcribed nucleotide close to the cap in many mRNAs and snRNAs in mammals and as internal modification in the snRNA U2. The writer and eraser proteins for these modifications have been recently identified and their deletions have been utilized to understand their contributions in gene expression regulation. While the role of U2 snRNA-m6Am in splicing regulation has been reported by different independent studies, conflicting data were found for the role of cap-associated m6Am in mRNA stability and translation. However, despite the open debate on the role of m6Am in mRNA expression, the modulation of regulators produced promising results in cancer cells. We believe that the investigation on m6Am will continue to yield relevant results in the future. [ABSTRACT FROM AUTHOR]

Orchids are among the most precious flowers in the world. Regulation of flowering time is one of the most important targets to enhance their ornamental value. The beauty of Arundina graminifolia is its year-round flowering, although the molecular mechanism of this flowering ability remains masked. Therefore, we performed a comprehensive assessment to integrate transcriptome and miRNA sequencing to disentangle the genetic regulation of flowering in this valuable species. Clustering analyses provided a set of molecular regulators of floral transition and floral morphogenesis. We mined candidate floral homeotic genes, including FCA, FPA, GI, FT, FLC, AP2, SOC1, SVP, GI, TCP, and CO, which were targeted by a variety of miRNAs. MiR11091 targeted the highest number of genes, including candidate regulators of phase transition and hormonal control. The conserved miR156-miR172 pathway of floral time regulation was evident in our data, and we found important targets of these miRNAs in the transcriptome. Moreover, endogenous hormone levels were determined to decipher the hormonal control of floral buds in A. graminifolia. The qRT-PCR analysis of floral and hormonal integrators validated the transcriptome expression. Therefore, miRNA-mediated mining of candidate genes with hormonal regulation forms the basis for comprehending the complex regulatory network of perpetual flowering in precious orchids. The findings of this study can do a great deal to broaden the breeding programs for flowering time manipulation of orchids. [ABSTRACT FROM AUTHOR]