Beyond RNA binding domains: determinants of protein-RNA binding.

RNA binding proteins (RBPs) are composed of RNA binding domains (RBDs) often linked via intrinsically disordered regions (IDRs). Structural and biochemical analysis have shown that disordered linkers contribute to RNA binding by orienting the adjacent RBDs and also characterized certain disordered repeats that directly contact the RNA. However, the relative contribution of IDRs and predicted RBDs to the in-vivo binding pattern is poorly explored. Here, we upscaled the RNA tagging method to map the transcriptome-wide binding of sixteen RBPs in budding yeast. We then performed extensive sequence mutations to distinguish binding determinants within predicted RBDs and the surrounding IDRs in eight of these. The majority of the predicted RBDs tested were not individually essential for mRNA binding, while multiple IDRs that lacked predicted RNA binding potential appeared essential for binding affinity or specificity. Our results provide new insights into the function of poorly studied RBPs and emphasize the complex and distributed encoding of RBP-RNA interaction in-vivo.

C2H2-zinc-finger transcription factors bind RNA and function in diverse post-transcriptional regulatory processes.

Cys2-His2 zinc-finger proteins (C2H2-ZNFs) constitute the largest class of DNA-binding transcription factors (TFs) yet remain largely uncharacterized. Although certain family members, e.g., GTF3A, have been shown to bind both DNA and RNA, the extent to which C2H2-ZNFs interact with-and regulate-RNA-associated processes is not known. Using UV crosslinking and immunoprecipitation (CLIP), we observe that 148 of 150 analyzed C2H2-ZNFs bind directly to RNA in human cells. By integrating CLIP sequencing (CLIP-seq) RNA-binding maps for 50 of these C2H2-ZNFs with data from chromatin immunoprecipitation sequencing (ChIP-seq), protein-protein interaction assays, and transcriptome profiling experiments, we observe that the RNA-binding profiles of C2H2-ZNFs are generally distinct from their DNA-binding preferences and that they regulate a variety of post-transcriptional processes, including pre-mRNA splicing, cleavage and polyadenylation, and m6A modification of mRNA. Our results thus define a substantially expanded repertoire of C2H2-ZNFs that bind RNA and provide an important resource for elucidating post-transcriptional regulatory programs.

The Biological Mechanisms and Clinical Roles of RNA-Binding Proteins in Cardiovascular Diseases.

RNA-binding proteins (RBPs) have pivotal roles in cardiovascular biology, influencing various molecular mechanisms underlying cardiovascular diseases (CVDs). This review explores the significant roles of RBPs, focusing on their regulation of RNA alternative splicing, polyadenylation, and RNA editing, and their impact on CVD pathogenesis. For instance, RBPs are crucial in myocardial injury, contributing to disease progression and repair mechanisms. This review systematically analyzes the roles of RBPs in myocardial injury, arrhythmias, myocardial infarction, and heart failure, revealing intricate interactions that influence disease outcomes. Furthermore, the potential of RBPs as therapeutic targets for cardiovascular dysfunction is explored, highlighting the advances in drug development and clinical research. This review also discusses the emerging role of RBPs as biomarkers for cardiovascular diseases, offering insights into their diagnostic and prognostic potential. Despite significant progress, current research faces several limitations, which are critically examined. Finally, this review identifies the major challenges and outlines future research directions to advance the understanding and application of RBPs in cardiovascular medicine.

RNA binding protein ELAVL1-mediated USP33 stabilizes HIF1A to promote pathological proliferation, migration and angiogenesis of RECs.

BACKGROUND: Dysfunction of retinal vascularization plays pathogenic roles in retinopathy of prematurity (ROP). Hypoxia-inducible factor 1 alpha (HIF1A) is activated by hypoxia and contributes to ROP progression. Herein, we clarified the mechanism underlying HIF1A activation in human retinal vascular endothelial cells (HRECs) under hypoxia. METHODS: Protein expression was assayed by immunoblot analysis. Cell migration, microtubule formation, invasion, proliferation, and viability were detected by wound-healing, tube formation, transwell, EdU, and CCK-8 assays, respectively. Bioinformatics was used to predict the deubiquitinase-HIF1A interactions and RNA binding proteins (RBPs) bound to USP33. The impact of USP33 on HIF1A deubiquitination was validated by immunoprecipitation (IP) assay. RNA stability analysis was performed with actinomycin D (Act D) treatment. The ELAVL1/USP33 interaction was assessed by RNA immunoprecipitation experiment. RESULTS: In hypoxia-exposed HRECs, HIF1A and USP33 protein levels were upregulated. Deficiency of HIF1A or USP33 suppressed cell migration, proliferation and microtubule formation of hypoxia-exposed HRECs. Mechanistically, USP33 deficiency led to an elevation in HIF1A ubiquitination and degradation. USP33 deficiency reduced HIF1A protein levels to suppress the proliferation and microtubule formation of hypoxia-induced HRECs. Moreover, the RBP ELAVL1 stabilized USP33 mRNA to increase USP33 protein levels. ELAVL1 decrease repressed the proliferation and microtubule formation of hypoxia-induced HRECs by reducing USP33. CONCLUSION: Our study identifies a novel ELAVL1/USP33/HIF1A regulatory cascade with the ability to affect hypoxia-induced pathological proliferation, angiogenesis, and migration in HRECs.

SORBS2-Mediated inhibition of malignant behaviors in esophageal squamous cell carcinoma through TIMP3.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is characterized by high invasiveness and poor prognosis. The role of Sorbin and SH3 domain-containing protein 2 (SORBS2) in ESCC remains largely unexplored. METHODS: The expression levels of SORBS2 in ESCC were detected using RNA-seq and proteomics data. The biological functions of SORBS2 in ESCC were investigated through in vivo and in vitro experiments. The mechanism of SORBS2 was explored using RIP-seq technology, which identified the key downstream molecule metalloproteinase-3 (TIMP3). The interaction between SORBS2 and TIMP3, including specific binding sites, was validated through RIP-qPCR and RNA pull-down assays. The impact of altered SORBS2 expression in ESCC on HUVECs was assessed using endothelial tube formation assays. RESULTS: SORBS2 expression was significantly downregulated in ESCC tissues, and its decreased expression was associated with poor prognosis. Overexpression of SORBS2 in ESCC cell lines inhibited cell proliferation, migration, and invasion both in vitro and in vivo. Mechanistically, SORBS2 bound to the 3' UTR of TIMP3 mRNA, enhancing its stability and thereby regulating TIMP3 expression. Rescue experiments demonstrated that increased TIMP3 expression could reverse the promotive effects of SORBS2 knockdown on ESCC, confirming TIMP3 as a critical downstream molecule of SORBS2. Furthermore, downregulation of SORBS2 in ESCC cells was associated with activation of HUVEC functions, whereas upregulation of TIMP3 could reverse this effect. The SORBS2/TIMP3 axis may exert tumor suppressive effects by influencing extracellular matrix degradation. CONCLUSION: This study confirms that SORBS2 inhibits ESCC tumor progression by regulating extracellular matrix degradation through TIMP3, providing a potential therapeutic target for future treatment interventions.

RBPs: an RNA editor's choice.

RNA-binding proteins (RBPs) play a key role in gene expression and post-transcriptional RNA regulation. As integral components of ribonucleoprotein complexes, RBPs are susceptible to genomic and RNA Editing derived amino acid substitutions, impacting functional interactions. This article explores the prevalent RNA Editing of RBPs, unravelling the complex interplay between RBPs and RNA Editing events. Emphasis is placed on their influence on single amino acid variants (SAAVs) and implications for disease development. The role of Proteogenomics in identifying SAAVs is briefly discussed, offering insights into the RBP landscape. RNA Editing within RBPs emerges as a promising target for precision medicine, reshaping our understanding of genetic and epigenetic variations in health and disease.

Cellular RNA-binding proteins LARP4 and PABPC1 synergistically facilitate viral translation of coronavirus PEDV.

Coronaviruses are causing epizootic diseases and thus are a substantial threat for both domestic and wild animals. These viruses depend on the host translation machinery to complete their life cycle. The current paper identified cellular RNA-binding proteins (RBPs), La-related protein 4 (LARP4) and polyadenylate-binding protein cytoplasmic 1 (PABPC1), as critical regulators of efficient translation of the coronavirus porcine epidemic diarrhea virus (PEDV) mRNA. In Vero cells, PEDV infection caused LARP4 to migrate from the nucleus to the cytoplasm in a chromosome region maintenance1 (CRM1)-independent pathway. In the absence of the nuclear export signal of LARP4, viral translation was not promoted by LARP4. A further study unveiled that the cytoplasmic LARP4 binds to the 3'-terminal untranslated region (3'UTR) of PEDV mRNA with the assistance of PABPC1 to facilitate viral translation. LARP4 knockdown reduced the promotion of the PABPC1-induced 3'UTR translation activity. Moreover, the rabbit reticulocyte lysate (RRL) system revealed that the prokaryotic expressed protein LARP4 and PABPC1 enhance PEDV mRNA translation. To our knowledge, this is the first study demonstrating that PEDV induces nucleo-cytoplasmic shuttling of LARP4 to enhance its own replication, which broadens our insights into how viruses use host's RBPs for the efficient translation of viral mRNA.

RNA-binding proteins in bone pathophysiology.

Bone remodelling is a highly regulated process that maintains mineral homeostasis and preserves bone integrity. During this process, intricate communication among all bone cells is required. Indeed, adapt to changing functional situations in the bone, the resorption activity of osteoclasts is tightly balanced with the bone formation activity of osteoblasts. Recent studies have reported that RNA Binding Proteins (RBPs) are involved in bone cell activity regulation. RBPs are critical effectors of gene expression and essential regulators of cell fate decision, due to their ability to bind and regulate the activity of cellular RNAs. Thus, a better understanding of these regulation mechanisms at molecular and cellular levels could generate new knowledge on the pathophysiologic conditions of bone. In this Review, we provide an overview of the basic properties and functions of selected RBPs, focusing on their physiological and pathological roles in the bone.

Diagnosis and Molecular Characterization of Potential RNA Binding Protein Involved in the Pathogenesis of Liver Ischemia Reperfusion Injury.

Background: Liver ischemia-reperfusion is one of the common complications after liver surgery. Uncontrolled liver ischemia-reperfusion will lead to many serious consequences such as surgical failure. It is an urgent clinical problem to search for diagnostic markers and explore its potential pathogenesis. Methods: In this study, we focus on 1411 candidate RNA binding protein. Through several GEO (Gene Expression Omnibus) online datasets, we construct a diagnostic model and perform interactive validation. We evaluate the efficacy of the prognostic model. Using bioinformatics methods, we predicted the relevant signaling pathways of liver ischemia-reperfusion and key genes. We also evaluated the association of RNA binding protein with immune cell infiltration. Single cell sequencing datasets were used to explore the expression profiles of key genes at the single cell level. Machine learning algorithm is used to predict key gene RNA binding domains. Results: ROC (Receiver Operating Characteristic) and DCA (Decision Curve Analysis) curves showed that the above diagnostic model had good and stable diagnostic efficacy and clinical practicability. We identified three key genes (BTG2, CCNL1 and DNAJB1) in liver ischemia-reperfusion. DNAJB1, BTG2 and CCNL1 are mainly expressed in immune cells such as macrophages and T cells, and are closely related to inflammatory pathways such as TNF-α, highlighting their importance in hepatic ischemia reperfusion. We identified RNA-binding domains of the above three genes. We found that the expression of DNAJB1, CCNL1 and BTG2 in the ischemia-reperfusion group were significantly higher than those in the sham operation group. Conclusion: Our study revealed the importance of the candidate RNA binding protein in liver ischemia reperfusion injury and provided new insights into the therapeutic of hepatic ischemia-reperfusion injury.

Single-cell transcriptome revealed dysregulated RNA-binding protein expression patterns and functions in human ankylosing spondylitis.

Objective: To explore the expression characteristics and regulatory patterns of RBPs in different immune cell types of AS, and to clarify the potential key role of RBPs in the occurrence and development of AS disease. Methods: PBMC sample data from scRNA-seq (HC*29, AS*10) and bulk RNA-seq (NC*3, AS*5) were selected for correlation analysis. Results: (1) Compared with the HC group, the numbers of B, DC (dendritic cells), CD14+ Mono and CD8+ T cells were increased in AS group, while the numbers of platelet (platelets), CD8+ NKT, CD16+ Mono (non-classical monocytes), Native CD4+ T and NK were decreased. (2) Through the analysis of RBP genes in B cells, some RBPs were found to play an important role in B cell differentiation and function, such as DDX3X, SFPQ, SRRM1, UPF2. (3) It may be related to B-cell receptor, IgA immunity, NOD-like receptor and other signaling pathways; Through the analysis of RBP genes in CD8+ T cells, some RBPs that play an important role in the immune regulation of CD8+ T were found, such as EIF2S3, EIF4B, HSPA5, MSL3, PABPC1 and SRSF7; It may be related to T cell receptor, TNF, IL17 and other signaling pathways. (4) Based on bulk RNA-seq, it was found that compared with HC and AS patients, differentially expressed variable splicing genes (RASGs) may play an important role in the occurrence and development of AS by participating in transcriptional regulation, protein phosphorylation and ubiquitination, DNA replication, angiogenesis, intracellular signal transduction and other related pathways. Conclusion: RBPs has specific expression characteristics in different immune cell types of AS patients, and has important regulatory functions. Its abnormal expression and regulation may be closely related to the occurrence and development of AS.

RNA binding proteins as mediators of pathological cardiac remodeling.

RNA binding proteins (RBPs) play a central in the post-transcriptional regulation of gene expression, which can account for up to 50% of all variations in protein expression within a cell. Following their binding to target RNAs, RBPs most typically confer changes in gene expression through modulation of alternative spicing, RNA stabilization/degradation, or ribosome loading/translation rate. All of these post-transcriptional regulatory processes have been shown to play a functional role in pathological cardiac remodeling, and a growing body of evidence is beginning to identify the mechanistic contribution of individual RBPs and their cardiac RNA targets. This review highlights the mechanisms of RBP-dependent post-transcriptional gene regulation in cardiomyocytes and fibroblasts and our current understanding of how RNA binding proteins functionally contribute to pathological cardiac remodeling.

Deep Learning for Elucidating Modifications to RNA-Status and Challenges Ahead.

RNA-binding proteins and chemical modifications to RNA play vital roles in the co- and post-transcriptional regulation of genes. In order to fully decipher their biological roles, it is an essential task to catalogue their precise target locations along with their preferred contexts and sequence-based determinants. Recently, deep learning approaches have significantly advanced in this field. These methods can predict the presence or absence of modification at specific genomic regions based on diverse features, particularly sequence and secondary structure, allowing us to decipher the highly non-linear sequence patterns and structures that underlie site preferences. This article provides an overview of how deep learning is being applied to this area, with a particular focus on the problem of mRNA-RBP binding, while also considering other types of chemical modification to RNA. It discusses how different types of model can handle sequence-based and/or secondary-structure-based inputs, the process of model training, including choice of negative regions and separating sets for testing and training, and offers recommendations for developing biologically relevant models. Finally, it highlights four key areas that are crucial for advancing the field.

Regulatory role of RNA-binding proteins in microRNA biogenesis.

MicroRNAs (miRNAs) are small non-coding RNAs that silence gene expression through their interaction with complementary sequences in the 3' untranslated regions (UTR) of target mRNAs. miRNAs undergo a series of steps during their processing and maturation, which are tightly regulated to fine-tune their abundance and ability to function in post-transcriptional gene silencing. miRNA biogenesis typically involves core catalytic proteins, namely, Drosha and Dicer, and several other RNA-binding proteins (RBPs) that recognize and interact with miRNA precursors and/or their intermediates, and mature miRNAs along with their interacting proteins. The series of RNA-protein and protein-protein interactions are critical to maintaining miRNA expression levels and their function, underlying a variety of cellular processes. Throughout this article, we review RBPs that play a role in miRNA biogenesis and focus on their association with components of the miRNA pathway with functional consequences in the processing and generation of mature miRNAs.

Human antigen R: Exploring its inflammatory response impact and significance in cardiometabolic disorders.

RNA-binding proteins (RBPs) play a crucial role in the regulation of posttranscriptional RNA networks, which can undergo dysregulation in many pathological conditions. Human antigen R (HuR) is a highly researched RBP that plays a crucial role as a posttranscriptional regulator. HuR plays a crucial role in the amplification of inflammatory signals by stabilizing the messenger RNA of diverse inflammatory mediators and key molecular players. The noteworthy correlations between HuR and its target molecules, coupled with the remarkable impacts reported on the pathogenesis and advancement of multiple diseases, position HuR as a promising candidate for therapeutic intervention in diverse inflammatory conditions. This review article examines the significance of HuR as a member of the RBP family, its regulatory mechanisms, and its implications in the pathophysiology of inflammation and cardiometabolic illnesses. Our objective is to illuminate potential directions for future research and drug development by conducting a comprehensive analysis of the existing body of research on HuR.

Role of Post-Transcriptional Regulation in Learning and Memory in Mammals.

After many decades, during which most molecular studies on the regulation of gene expression focused on transcriptional events, it was realized that post-transcriptional control was equally important in order to determine where and when specific proteins were to be synthesized. Translational regulation is of the most importance in the brain, where all the steps of mRNA maturation, transport to different regions of the cells and actual expression, in response to specific signals, constitute the molecular basis for neuronal plasticity and, as a consequence, for structural stabilization/modification of synapses; notably, these latter events are fundamental for the highest brain functions, such as learning and memory, and are characterized by long-term potentiation (LTP) of specific synapses. Here, we will discuss the molecular bases of these fundamental events by considering both the role of RNA-binding proteins (RBPs) and the effects of non-coding RNAs involved in controlling splicing, editing, stability and translation of mRNAs. Importantly, it has also been found that dysregulation of mRNA metabolism/localization is involved in many pathological conditions, arising either during brain development or in the adult nervous system.

Predicting circRNA-RBP Binding Sites Using a Hybrid Deep Neural Network.

Circular RNAs (circRNAs) are non-coding RNAs generated by reverse splicing. They are involved in biological process and human diseases by interacting with specific RNA-binding proteins (RBPs). Due to traditional biological experiments being costly, computational methods have been proposed to predict the circRNA-RBP interaction. However, these methods have problems of single feature extraction. Therefore, we propose a novel model called circ-FHN, which utilizes only circRNA sequences to predict circRNA-RBP interactions. The circ-FHN approach involves feature coding and a hybrid deep learning model. Feature coding takes into account the physicochemical properties of circRNA sequences and employs four coding methods to extract sequence features. The hybrid deep structure comprises a convolutional neural network (CNN) and a bidirectional gated recurrent unit (BiGRU). The CNN learns high-level abstract features, while the BiGRU captures long-term dependencies in the sequence. To assess the effectiveness of circ-FHN, we compared it to other computational methods on 16 datasets and conducted ablation experiments. Additionally, we conducted motif analysis. The results demonstrate that circ-FHN exhibits exceptional performance and surpasses other methods. circ-FHN is freely available at https://github.com/zhaoqi106/circ-FHN .

Interaction between lncRNAs and RNA-binding proteins (RBPs) influences DNA damage response in cancer chemoresistance.

The DNA damage response (DDR) is a crucial cellular signaling pathway activated in response to DNA damage, including damage caused by chemotherapy. Chemoresistance, which refers to the resistance of cancer cells to the effects of chemotherapy, poses a significant challenge in cancer treatment. Understanding the relationship between DDR and chemoresistance is vital for devising strategies to overcome this resistance and improve treatment outcomes. Long non-coding RNAs (lncRNAs) are a class of RNA molecules that do not code for proteins but play important roles in various biological processes, including cancer development and chemoresistance. RNA-binding proteins (RBPs) are a group of proteins that bind to RNA molecules and regulate their functions. The interaction between lncRNAs and RBPs has been found to regulate gene expression at the post-transcriptional level, thereby influencing various cellular processes, including DDR signaling pathways. Multiple studies have demonstrated that lncRNAs can interact with RBPs to modulate the expression of genes involved in cancer chemoresistance by impacting DDR signaling pathways. Conversely, RBPs can regulate the expression and function of lncRNAs involved in DDR. Exploring these interactions can provide valuable insights for the development of innovative therapeutic approaches to overcome chemoresistance in cancer patients. This review article aims to summarize recent research on the interaction between lncRNAs and RBPs during cancer chemotherapy, with a specific focus on DDR pathways.

Circular RNA-mediated miRNA sponge & RNA binding protein in biological modulation of breast cancer.

Circular RNAs (circRNAs) and small non-coding RNAs of the head-to-junction circle in the construct play critical roles in gene regulation and are significantly associated with breast cancer (BC). Numerous circRNAs are potential cancer biomarkers that may be used for diagnosis and prognosis. Widespread expression of circRNAs is regarded as a feature of gene expression in highly diverged eukaryotes. Recent studies show that circRNAs have two main biological modulation models: sponging and RNA-binding. This review explained the biogenesis of circRNAs and assessed emerging findings on their sponge function and role as RNA-binding proteins (RBPs) to better understand how their interaction alters cellular function in BC. We focused on how sponges significantly affect the phenotype and progression of BC. We described how circRNAs exercise the translation functions in ribosomes. Furthermore, we reviewed recent studies on RBPs, and post-protein modifications influencing BC and provided a perspective on future research directions for treating BC.

Cryo-EM structures of functional and pathological amyloid ribonucleoprotein assemblies.

Amyloids are implicated in neurodegenerative and systemic diseases, yet they serve important functional roles in numerous organisms. Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of RNA-binding proteins (RBPs) that control central events of RNA biogenesis in normal and diseased cellular conditions. Many of these proteins contain prion-like sequences of low complexity, which not only assemble into functional fibrils in response to cellular cues but can also lead to disease when missense mutations arise in their sequences. Recent advances in cryo-electron microscopy (cryo-EM) have provided unprecedented high-resolution structural insights into diverse amyloid assemblies formed by hnRNPs and structurally related RBPs, including TAR DNA-binding protein 43 (TDP-43), Fused in Sarcoma (FUS), Orb2, hnRNPA1, hnRNPA2, and hnRNPDL-2. This review provides a comprehensive overview of these structures and explores their functional and pathological implications.

RNA-Binding Protein-Mediated mRNA Deadenylation in Mammalian Cell Extracts.

Removal of the poly(A) tail, or deadenylation, is a crucial step in destabilizing mRNAs in eukaryotes. In this chapter, we describe a cell-free deadenylation assay that uses cytoplasmic cell extracts from human HEK293 cells transiently transfected with DNA encoding RNA-binding proteins (RBP), and in vitro-transcribed, radiolabeled, RNA probes. We include methods to evaluate the effects of RBPs or deadenylases on various in vitro-transcribed probes, with or without poly(A) tails. Finally, we also demonstrate the adaptability of these assays to test purified protein components in our cell-free deadenylation assay. In our experience, these methods are well suited for the initial assessment of the effects of RBPs on the deadenylation of mRNAs.