Identification of RNA-binding protein residues using machine learning approaches

ATF3 expression is induced in cells exposed to a variety of stress conditions, including nutrient limitation. Here we demonstrated that the mechanism by which the ATF3 mRNA content is increased following amino acid limitation of human HepG2 hepatoma cells is mRNA stabilization. Analysis of ATF3 mRNA turnover revealed that the half-life was increased from about 1 h in control cells to greater than 8 h in the histidine-deprived state, demonstrating mRNA stabilization in response to nutrient deprivation. Treatment of HepG2 cells with thapsigargin, which causes endoplasmic reticulum stress, also increased the half-life of ATF3 mRNA. HuR is an RNA-binding protein that regulates both the stability and cytoplasmic/nuclear localization of mRNA species containing AU-rich elements. Another RNA-binding protein, AUF1, regulates target mRNA molecules by enhancing their decay. Amino acid limitation caused a slightly elevated mRNA level for HuR and AUF1 mRNA. The nuclear HuR protein content was unchanged, and AUF1 protein increased slightly after amino acid limitation, whereas the cytoplasmic levels of both HuR and AUF1 protein increased. Immunoprecipitation of HuR-RNA complexes followed by reverse transcriptase-PCR analysis showed that HuR interacted with ATF3 mRNA in vivo and that this interaction increased following amino acid limitation. In contrast, the interaction of AUF1 with the ATF3 mRNA is decreased in histidine-deprived cells relative to control cells. Suppression of HuR expression by RNA interference partially blocked the accumulation of ATF3 mRNA following amino acid deprivation. The results demonstrated that coordinated regulation of mRNA stability by HuR and AUF1 proteins contributes to the observed increase in ATF3 expression following amino acid limitation.

Breaking the protein-RNA recognition code

Post-transcriptional regulation of gene expression is a critical process for adapting to and surviving Trypanosoma cruzi, a parasite with a complex life cycle. RNA-binding proteins (RBPs) are key players in this regulation, forming ribonucleoprotein complexes (messenger ribonucleoproteins) and RNA granules that control transcript stability, localization, degradation, and translation modulation. Understanding the specific roles of individual RBPs is crucial for unraveling the details of this regulatory network. In this study, we generated null mutants of the TcZC3HTTP gene, a specific RBP in the Trypanosoma family characterized by a C3H zinc finger and a DNAJ domain associated with RNA and protein binding, respectively. Through cell growth assays, we demonstrated that the absence of TcZC3HTTP or the expression of an additional tagged version impacted epimastigote growth, indicating its contribution to cell proliferation. TcZC3HTTP was found to associate with mRNAs involved in cell cycle and division in epimastigotes, while in nutritionally stressed parasites it exhibited associations with mRNAs coding for other RBPs and rRNA. Furthermore, our analysis identified that TcZC3HTTP protein partners were different during normal growth conditions compared to starvation conditions, with the latter showing enrichment of ribosomal proteins and other RBPs. Therefore, this study provides insights into TcZC3HTTP's role in the post-transcriptional regulation of gene expression during normal growth and nutritional stress in T. cruzi, uncovering its versatile functions in different cellular contexts.IMPORTANCEUnderstanding how Trypanosoma cruzi, the causative agent of Chagas disease, regulates gene expression is crucial for developing targeted interventions. In this study, we investigated the role of TcZC3HTTP, an RNA-binding protein, in post-transcriptional regulation. Our findings demonstrate that TcZC3HTTP is relevant for the growth and proliferation of epimastigotes, a stage of the parasite's life cycle. We identified its associations with specific mRNAs involved in cell cycle and division and its interactions with enzymes and other RNA-binding proteins (RBPs) under normal and starvation conditions. These insights shed light on the regulatory network underlying gene expression in T. cruzi and reveal the multifaceted functions of RBPs in this parasite. Such knowledge enhances our understanding of the parasite's biology and opens avenues for developing novel therapeutic strategies targeting post-transcriptional gene regulation in T. cruzi.

SelB is an elongation factor needed for the co-translational incorporation of selenocysteine. Selenocysteine is coded by a UGA stop codon in combination with a specific downstream mRNA hairpin. In bacteria, the C-terminal part of SelB recognizes this hairpin, while the N-terminal part binds GTP and tRNA in analogy with elongation factor Tu (EF-Tu). We present the crystal structure of a C-terminal fragment of SelB (SelB-C) from Moorella thermoacetica at 2.12 A resolution, solved by a combination of selenium and yttrium multiwavelength anomalous dispersion. This 264 amino acid fragment contains the entire C-terminal extension beginning after the EF-Tu-homologous domains. SelB-C consists of four similar winged-helix domains arranged into the shape of an L. This is the first example of winged-helix domains involved in RNA binding. The location of conserved basic amino acids, together with data from the literature, define the position of the mRNA-binding site. Steric requirements indicate that a conformational change may occur upon ribosome interaction. Structural observations and data in the literature suggest that this change happens upon mRNA binding.

Encyclopedia of RNA binding protein expression and domain annotations.- RNA localization and RNA binding proteins that control localization.- Genome-wide approaches to studying alternative polyadenylation events and the RBPs that control them.- RNA binding proteins, PRC complex and long ncRNAs.- Gene and genome regulation by piwi proteins.- Engineering Artificial RBP interactions.- Querying functional polymorphisms by high throughput binding and functional assays.- RNA binding protein networks in EMT/MET.- Therapies against diseases associated with RNA processing defects and RNA binding proteins.- Repeat-disorders and the RNA binding proteins implicated in them.- RNA binding proteins in heart development.- Identification of miRNAs and their targets in C elegans.- Splicing code.- Index.

PTB-AS, a Novel Natural Antisense Transcript, Promotes Glioma Progression by Improving PTBP1 mRNA Stability with SND1.

The regulation of gene expression in trypanosomatids occurs mainly at the post-transcriptional level. Despite the importance of this type of control in Trypanosoma cruzi, few RNA binding proteins have been characterized. The RRM domain (RNA Recognition Motif) is one of the most abundant domains found in RNA-binding proteins in higher eukaryotes. Proteins containing the RRM domain are involved in the majority of post-transcriptional processes regulating gene expression. In this work, we aimed to characterize the protein TcNRBD1 from T. cruzi. TcNRBD1 is an RNA-binding protein that contains 2 RRM domains and is the ortholog of the P34 and P37 proteins from Trypanosoma brucei. The TcNRBD1 protein is expressed in all developmental stages of T. cruzi, and its localization pattern is concentrated at the perinuclear region. TcNRBD1 is associated with polysomes and with the 80S monosomes. Furthermore, sequencing of the mRNAs bound to TcNRBD1 allowed the identification of several transcripts that encode ribosomal proteins. Immunoprecipitation assays followed by mass spectrometry showed that the protein complexes with several ribosomal proteins from both the 40S and 60S subunits. In summary, the results indicate that TcNRBD1 is associated with different parts of the translation process, either by regulating mRNAs that encode ribosomal proteins or by acting in some step of ribosome assembly in T. cruzi.

RNA-binding proteins (RBPs) are key post-transcriptional regulators of gene expression, and thus underlie many important biological processes. Here, we developed a strategy that entails extracting a “hotspot pharmacophore” from the structure of a protein-RNA complex, to create a template for designing small-molecule inhibitors and for exploring the selectivity of the resulting inhibitors. We demonstrate this approach by designing inhibitors of Musashi proteins MSI1 and MSI2, key regulators of mRNA stability and translation that are upregulated in many cancers. We report this novel series of MSI1/MSI2 inhibitors is specific and active in biochemical, biophysical, and cellular assays. This study extends the paradigm of “hotspots” from protein-protein complexes to protein-RNA complexes, supports the “druggability” of RNA-binding protein surfaces, and represents one of the first rationally-designed inhibitors of non-enzymatic RNA-binding proteins. Owing to its simplicity and generality, we anticipate that this approach may also be used to develop inhibitors of many other RNA-binding proteins; we also consider the prospects of identifying potential off-target interactions by searching for other RBPs that recognize their cognate RNAs using similar interaction geometries. Beyond inhibitors, we also expect that compounds designed using this approach can serve as warheads for new PROTACs that selectively degrade RNA-binding proteins.

Glorund, a Drosophila hnRNP F/H Homolog, Is an Ovarian Repressor of nanos Translation

Cancer is a major concern worldwide; over 1,200,000 cases of cancer will be diagnosed in America this year. In recent studies, RNA‐binding motif protein (RBM3) has been shown to be a protooncogene whose expression is unregulated in various cancers; the protein functions by regulating mRNA stability and translation. Within the N‐terminal region, RBM3 encodes one RNA recognition motif (RRM) domain, a 90 amino acids sequence shared by many RNA binding proteins that is implicated in the regulation of alternative splicing. This motif's structure consists of two alpha helices and four beta strands arranged in an alpha/beta sandwich. In previous studies, RBM3 has been shown to bind to AU‐rich sequences in the 3′untranslated region of mRNAs that encode cancer promoting proteins such as cyclooxygenase‐2 (COX‐2) and vascular endothelial growth factor (VEGF). Upon binding, RBM3 was shown to increase the stability and translation of these mRNAs, which is one mechanism by which the protein promotes tumorigenesis. The Olathe North SMART Team (Students Modeling A Research Topic) used 3D printing technology to model the protein RBM3 that has been found in Homo sapiens by homology of the known protein RBM39 from Mus musculus. Supported by a grant from NIH‐SEPA

RNA-binding proteins (RBPs) have been relatively overlooked in cancer research despite their contribution to virtually every cancer hallmark. Here, we use RNA interactome capture (RIC) to characterize the melanoma RBPome and uncover novel RBPs involved in melanoma progression. Comparison of RIC profiles of a non-tumoral versus a metastatic cell line revealed prevalent changes in RNA-binding capacities that were not associated with changes in RBP levels. Extensive functional validation of a selected group of 24 RBPs using five different in vitro assays unveiled unanticipated roles of RBPs in melanoma malignancy. As proof-of-principle we focused on PDIA6, an ER-lumen chaperone that displayed a novel RNA-binding activity. We show that PDIA6 is involved in metastatic progression, map its RNA-binding domain, and find that RNA binding is required for PDIA6 tumorigenic properties. These results exemplify how RIC technologies can be harnessed to uncover novel vulnerabilities of cancer cells.

BackgroundThe RNA binding proteins (RBPs) human antigen R (HuR) and Tristetraprolin (TTP) are known to exhibit competitive binding but have opposing effects on the bound messenger RNA (mRNA). How cells discriminate between the two proteins is an interesting problem. Machine learning approaches, such as support vector machines (SVMs), may be useful in the identification of discriminative features. However, this method has yet to be applied to studies of RNA binding protein motifs.ResultsApplying the k-spectrum kernel to a support vector machine (SVM), we first verified the published binding sites of both HuR and TTP. Additional feature engineering highlighted the U-rich binding preference of HuR and AU-rich binding preference for TTP. Domain adaptation along with multi-task learning was used to predict the common binding sites.ConclusionThe distinction between HuR and TTP binding appears to be subtle content features. HuR prefers strongly U-rich sequences whereas TTP prefers AU-rich as with increasing A content, the sequences are more likely to be bound only by TTP. Our model is consistent with competitive binding of the two proteins, particularly at intermediate AU-balanced sequences. This suggests that fine changes in the A/U balance within a untranslated region (UTR) can alter the binding and subsequent stability of the message. Both feature engineering and domain adaptation emphasized the extent to which these proteins recognize similar general sequence features. This work suggests that the k-spectrum kernel method could be useful when studying RNA binding proteins and domain adaptation techniques such as feature augmentation could be employed particularly when examining RBPs with similar binding preferences.

Understanding interactions between proteins and RNA is key to deciphering the mechanisms of many important biological processes. Here we describe RNABindR, a web-based server that identifies and displays RNA-binding residues in known protein–RNA complexes and predicts RNA-binding residues in proteins of unknown structure. RNABindR uses a distance cutoff to identify which amino acids contact RNA in solved complex structures (from the Protein Data Bank) and provides a labeled amino acid sequence and a Jmol graphical viewer in which RNA-binding residues are displayed in the context of the three-dimensional structure. Alternatively, RNABindR can use a Naive Bayes classifier trained on a non-redundant set of protein–RNA complexes from the PDB to predict which amino acids in a protein sequence of unknown structure are most likely to bind RNA. RNABindR automatically displays ‘high specificity’ and ‘high sensitivity’ predictions of RNA-binding residues. RNABindR is freely available at http://bindr.gdcb.iastate.edu/RNABindR.

RNA binding proteins (RBPs) post‐transcriptionally regulate gene expression by associating with the regulatory sequences in the untranslated regions of mRNAs. RBPs are often disrupted in cancer, but current knowledge of their role in regulating cancer‐associated genes is largely based on in vitro studies. This study seeks to determine the in vivo role of cold‐inducible RBP (CIRP), which is overexpressed in some human breast cancers. CIRP promotes cell proliferation and survival and inhibits apoptosis in vitro and promotes inflammation in vivo, all hallmarks of cancer. We tested the hypothesis that CIRP promotes breast tumorigenesis in vivo by crossing a transgenic mouse overexpressing CIRP in the mammary epithelium with the PyMT mouse model of breast cancer. Effects on both early (7‐week‐old mice) and late (14‐week‐old mice) tumorigenesis were assessed. Unexpectedly, mammary glands from 7‐week‐old CIRP/PyMT mice had 50% fewer mammary intraepithelial neoplasias than PyMT mice. Consistent with this, histopathological analysis showed decreased mitosis and increased apoptosis in CIRP/PyMT vs. PyMT mammary glands. Late stage tumorigenesis was also inhibited. CIRP/PyMT mice had slower tumor growth kinetics, smaller tumors, decreased tumor burden and decreased pulmonary metastasis compared to PyMT mice. To assess the mechanism of inhibition, mammary glands from 7 and 14‐week‐old mice were incubated in medium overnight and Luminex cytokine assays were performed on the medium. Results showed a decrease in cytokines that promote inflammation, angiogenesis and metastasis. Altogether, our results suggest that CIRP inhibits proliferation and promotes apoptosis in vivo to suppress early tumorigenesis, potentially via decreasing a protumorigenic inflammatory response.Support or Funding InformationThis work was funded by the University of New Mexico Research Allocation Committee, the American Association of Anatomists Fellows Grant Award Program and by the National Institutes of Health under the Ruth L. Kirschstein National Research Service Award (1 F31 CA213933‐01A1)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Background & objective: The kidneys are vital and complex organs responsible for maintaining normal body functions. Kidney disease or a loss of kidney function and, in severe cases, complete kidney failure disrupt whole of the body systems. Tristetraprolin (TTP ZFP36) is an RNA-binding proteins (RBP), that regulates cytokine mRNAs by specific binding of its two conserved ZF domains to adenylate-uridylate-rich elements (AREs) at the untranslated region (UTR), leading to degradation of the RNA. Recently, it was suggested that S100 A8/A9 and TTP were involved in pathogenesis of renal diseases. However, the protective roles of S100 A8/A9 and TTP in renal disease is still unclear. The primary objective of this study was to explore the involvement of S100 A8/A9 and TTP in the inflammatory response among different groups of kidney diseases. These factors may hold therapeutic value in the treatment of these diseases by targeting and modulating the inflammatory response. Methodology: The study was performed between October 2022 to April 2023. We included 150 subjects, including 30 patients with chronic kidney disease (CKD), (Female = 16, Male = 14) with mean age (58.33 ± 18.33 y), 30 patients with acute kidney injury (AKI), (Female = 15, Male = 15), with mean age (54.48 ± 18.10 y), 30 patients with diabetic nephropathy (DN), (Female = 18, Male = 12), with mean age (60.84 ± 12.38 y), 30 patients with nephrotic syndrome (NS), (Female = 13, Male = 17), with mean age (52.54 ± 14.02 y), and 30 healthy persons as a control group. NLR analysis was performed directly using the hematology analyzer CBC (Sysmex, Japan) technique, serum S100 A8/A9 were measured by ELISA, and TTP gene expression was measured by Quantitative Real Time-Polymerase Chain Reaction (RT-qPCR). Results: In comparison to the healthy control group, all patient groups exhibited a significant increase in the NLR (P < 0.05). Furthermore, the findings clearly demonstrated elevated levels of S100 A8/A9 in all patient groups when compared to the control group (P ≤ 0.05). The results also demonstrated a significant down regulation in the expression of TTP in DN and nephrotic syndrome (NS) groups compared to the control (P ≤ 0.01). AKI showed highly significant increase in TTP expression in comparison to control and other patient groups (P ≤ 0.01). Non-significant decrease in TTP gene expression was indicated in CKD compared to healthy group (P ≥ 0.05). Conclusions: The activation of Ca2+-binding protein S100A8/A9 is involved in the renal damage; thus, targeting it could have a therapeutic value in the treatment of kidney diseases. Tristetraprolin is contributed to the regulation of inflammatory events in kidney diseases especially in AKI group due to a rapid and robust inflammatory response that contributes to the pathogenesis of this disease. Abbreviations: AKI- acute kidney injury ARE- Adenylate-Uridylate-Rich Element; DN- diabetic nephropathy; NS- Nephrotic Syndrome; NLR- Neutrophil-to-Lymphocyte Ratio; RT-qPCR= Quantitative Real Time-Polymerase Chain Reaction; RBP- RNA-Binding Proteins; TTP- Tristetraprolin; UTR- Untranslated Region; Key words: Kidney diseases; S100 A8/A9, Anti-inflammatory RNA binding protein; Tristetraprolin; Neutrophil/Lymphocyte Ration Citation: Mohammed Radhi FZ, Almzaie AJ, Majeed MM. Serum S100A8/A9 and RNA-binding protein, tristetraprolin as prognostic markers of renal damage. Anaesth. pain intensive care 2023;27(6):706−714; DOI: 10.35975/apic.v27i6.2342 Received: August 12, 2023; Revised: September 05, 2023; Accepted: September 07, 2023