Regulation Of mRNA Stability Research Articles

Predictive modeling of oocyte maternal mRNA features for five mammalian species reveals potential shared and species-restricted regulators during maturation.

Oocyte maturation is accompanied by changes in abundances of thousands of mRNAs, many degraded and many preferentially stabilized. mRNA stability can be regulated by diverse features including GC content, codon bias, and motifs within the 3'-untranslated region (UTR) interacting with RNA binding proteins (RBPs) and miRNAs. Many studies have identified factors participating in mRNA splicing, bulk mRNA storage, and translational recruitment in mammalian oocytes, but the roles of potentially hundreds of expressed factors, how they regulate cohorts of thousands of mRNAs, and to what extent their functions are conserved across species has not been determined. We performed an extensive in silico cross-species analysis of features associated with mRNAs of different stability classes during oocyte maturation (stable, moderately degraded, and highly degraded) for five mammalian species. Using publicly available RNA sequencing data for germinal vesicle (GV) and MII oocyte transcriptomes, we determined that 3'-UTR length and synonymous codon usage are positively associated with stability, while greater GC content is negatively associated with stability. By applying machine learning and feature selection strategies, we identified RBPs and miRNAs that are predictive of mRNA stability, including some across multiple species and others more species-restricted. The results provide new insight into the mechanisms regulating maternal mRNA stabilization or degradation.NEW & NOTEWORTHY Conservation across species of mRNA features regulating maternal mRNA stability during mammalian oocyte maturation was analyzed. 3'-Untranslated region length and synonymous codon usage are positively associated with stability, while GC content is negatively associated. Just three RNA binding protein motifs were predicted to regulate mRNA stability across all five species examined, but associated pathways and functions are shared, indicating oocytes of different species arrive at comparable physiological destinations via different routes.

Exploring N6-methyladenosine (m6A) modification in tree species: opportunities and challenges.

N 6-methyladenosine (m6A) in eukaryotes is the most common and widespread internal modification in mRNA. The modification regulates mRNA stability, translation efficiency, and splicing, thereby fine-tuning gene regulation. In plants, m6A is dynamic and critical for various growth stages, embryonic development, morphogenesis, flowering, stress response, crop yield, and biomass. Although recent high-throughput sequencing approaches have enabled the rapid identification of m6A modification sites, the site-specific mechanism of this modification remains unclear in trees. In this review, we discuss the functional significance of m6A in trees under different stress conditions and discuss recent advancements in the quantification of m6A. Quantitative and functional insights into the dynamic aspect of m6A modification could assist researchers in engineering tree crops for better productivity and resistance to various stress conditions.

Oxidation of the active site cysteine residue of glyceraldehyde-3-phosphate dehydrogenase to the hyper-oxidized sulfonic acid form is favored under crowded conditions

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key cellular enzyme, with major roles in both glycolysis, and ‘moonlighting’ activities in the nucleus (uracil DNA glycosylase activity, nuclear protein nitrosylation), as a regulator of mRNA stability, a transferrin receptor, and as an antimicrobial agent. These activities are dependent, at least in part, on the integrity of an active site Cys residue, and a second neighboring Cys. These residues are differentially sensitive to oxidation, and determine both its catalytic activity and the redox signaling capacity of the protein. Such Cys modification is critical to cellular adaptation to oxidative environments by re-routing metabolic pathways to favor NADPH generation and antioxidant defenses. Despite the susceptibility of GAPDH to oxidation, it remains a puzzle as to how this enzyme acts as a redox signaling hub for oxidants such as hydrogen peroxide (H2O2) in the presence of high concentrations of specialized high-efficiency peroxide-removing enzymes. One possibility is that crowded environments, such as the cell cytosol, alter the oxidation pathways of GAPDH. In this study, we investigated the role of crowding (induced by dextran) on H2O2- and SIN-1-induced GAPDH oxidation, with data for crowded and dilute conditions compared. LC-MS/MS data revealed a lower extent of modification of the catalytic Cys under crowded conditions (i.e. less monomer units modified), but enhanced formation of the sulfonic acid resulting from hyper-oxidation. This effect was not observed with SIN-1. These data indicate that molecular crowding can modulate the oxidation pathways of GAPDH and its extent of oxidation and inactivation.

HnRNPA1 SUMOylation promotes cold hypersensitivity in chronic inflammatory pain by stabilizing TRPA1 mRNA

TRPA1 is pivotal in cold hypersensitivity, but its regulatory mechanisms in inflammatory cold hyperalgesia remain poorly understood. We show here that the upregulation of SUMO1-conjugated protein levels in a complete Freund's adjuvant (CFA)-induced inflammatory pain model enhances TRPA1 mRNA stability, ultimately leading to increased expression levels. We further demonstrate that hnRNPA1 binds to TRPA1 mRNA, and its SUMOylation, upregulated in CFA-induced inflammatory pain, contributes to stabilizing TRPA1 mRNA by accumulating hnRNPA1 in the cytoplasm. Moreover, we find that wild-type hnRNPA1 viral infection in dorsal root ganglia neurons, and not infection with the SUMOylation-deficient hnRNPA1 mutant, can rescue the reduced ability of hnRNPA1-knockdown mice to develop inflammatory cold pain hypersensitivity. These results suggest that hnRNPA1 is a regulator of TRPA1 mRNA stability, the capability of which is enhanced upon SUMO1 conjugation at lysine 3 in response to peripheral inflammation, and the increased expression of TRPA1 in turn underlies the development of chronic inflammatory cold pain hypersensitivity.

ScTEA-db: a comprehensive database of novel terminal exon isoforms identified from human single cell transcriptomes.

The usage of alternative terminal exons results in messenger RNA (mRNA) isoforms that differ in their 3' untranslated regions (3' UTRs) and often also in their protein-coding sequences. Alternative 3' UTRs contain different sets of cis-regulatory elements known to regulate mRNA stability, translation and localization, all of which are vital to cell identity and function. In previous work, we revealed that ∼25 percent of the experimentally observed RNA 3' ends are located within regions currently annotated as intronic, indicating that many 3' end isoforms remain to be uncovered. Also, the inclusion of not yet annotated terminal exons is more tissue specific compared to the already annotated ones. Here, we present the single cell-based Terminal Exon Annotation database (scTEA-db, www.scTEA-db.org) that provides the community with 12063 so far not yet annotated terminal exons and associated transcript isoforms identified by analysing 53069 publicly available single cell transcriptomes. Our scTEA-db web portal offers an array of features to find and explore novel terminal exons belonging to 5538 human genes, 110 of which are known cancer drivers. In summary, scTEA-db provides the foundation for studying the biological role of large numbers of so far not annotated terminal exon isoforms in cell identity and function.

Early Progression of Abdominal Aortic Aneurysm is Decelerated by Improved Endothelial Barrier Function via ALDH2-LIN28B-ELK3 Signaling.

The involvement of endothelial barrier function in abdominal aortic aneurysm (AAA) and its upstream regulators remains unknown. Single-cell RNA sequencing shows that disrupted endothelial focal junction is an early (3 days) and persistent (28 days) event during Angiotensin II (Ang II)-induced AAA progression. Consistently, mRNA sequencing on human aortic dissection tissues confirmed downregulated expression of endothelial barrier-related genes. Aldehyde dehydrogenase 2 (ALDH2), a negative regulator of AAA, is found to be upregulated in the intimal media of AAA samples, leading to testing its role in early-stage AAA. ALDH2 knockdown/knockout specifically in endothelial cells (ECs) significantly increases expression of EC barrier markers related to focal adhesion and tight junction, restores endothelial barrier integrity, and suppresses early aortic dilation of AAA (7 and 14 days post-Ang II). Mechanically, ELK3acts as an ALDH2 downstream regulator for endothelial barrier function preservation. At the molecular level, ALDH2 directly binds to LIN28B, a regulator of ELK3 mRNA stability, hindering LIN28B binding to ELK3 mRNA, thereby depressing ELK3 expression and impairing endothelial barrier function. Therefore, preserving vascular endothelial barrier integrity via ALDH2-specific knockdown in ECs holds therapeutic potential in the early management of AAAs.

Research Progress on the Structural and Functional Roles of hnRNPs in Muscle Development.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a superfamily of RNA-binding proteins consisting of more than 20 members. These proteins play a crucial role in various biological processes by regulating RNA splicing, transcription, and translation through their binding to RNA. In the context of muscle development and regeneration, hnRNPs are involved in a wide range of regulatory mechanisms, including alternative splicing, transcription regulation, miRNA regulation, and mRNA stability regulation. Recent studies have also suggested a potential association between hnRNPs and muscle-related diseases. In this report, we provide an overview of our current understanding of how hnRNPs regulate RNA metabolism and emphasize the significance of the key members of the hnRNP family in muscle development. Furthermore, we explore the relationship between the hnRNP family and muscle-related diseases.

Loss of Dicer in Newborn Melanocytes Leads to Premature Hair Graying and Changes in Integrin Expression

Premature hair graying occurs owing to the depletion of melanocyte stem cells in the hair follicle, which can be accelerated by stress caused by genetic or environmental factors. However, the connection between stress and melanocyte stem cell loss is not fully understood. MicroRNAs are molecules that control gene expression by regulating mRNA stability and translation and are produced by the enzyme Dicer, which is repressed under stress. In this study, using 2 mouse genetic models and human and mouse cell lines, we found that the inactivation of Dicer in melanocytes leads to misplacement of these cells within the hair follicle, resulting in a lack of melanin transfer to keratinocytes in the growing hair and the exhaustion of the melanocyte stem cell pool. We also show that miR-92b, which regulates ItgaV mRNA and protein levels, plays a role in altering melanocyte migration. Overall, our findings suggest that the Dicer-miR92b-ItgaV pathway serves as a major signaling pathway linking stress to premature hair greying.

Conditional expression of NanoLuc luciferase through a multimodular system offers rapid detection of antimalarial drug activity

Conditional gene expression is a powerful tool to investigate putative vaccine and drug targets, especially in a haploid organism such as Plasmodium falciparum. Inducible systems based on regulation of either transcription, translation, protein or mRNA stability, among others, allow switching on an off the expression of any desired gene causing specific gain or loss of function phenotypes. However, those systems can be cumbersome involving the construction of large plasmids and generation of multiple transgenic parasite lines. In addition, the dynamic range of regulation achieved is not predictable for each individual gene and can be insufficient to generate detectable phenotypes when the genes of interest are silenced. Here, we combined up to three distinct inducible systems to regulate the expression of a single gene. Expression of the reporter NanoLuc luciferase was regulated over 40-fold, which correlates to the regulation achieved by each individual system multiplied by each other. We applied the conditionally expressed NanoLuc to evaluate the effect of fast-acting antimalarials such as chloroquine and artesunate as well as of slower-acting ones such as atovaquone. The conditionally expressed reporter allowed faster and more reliable detection of toxicity to the parasite, which correlated to the expected action of each compound. Bioluminescence achieved by the expression of this inducible highly sensitive reporter is therefore a promising tool to investigate the temporal effect of potential new antimalarials. This single plasmid combination system might also prove useful to achieve sufficient regulation of genes of interest to produce loss-of-function phenotypes.

Yeast Lsm Pro-Apoptotic Mutants Show Defects in Autophagy.

LSM4 is an essential yeast gene encoding a component of different LSM complexes involved in the regulation of mRNA splicing, stability, and translation. In previous papers, we reported that the expression in S. cerevisiae of the K. lactis LSM4 gene lacking the C-terminal Q/N-rich domain in an Lsm4 null strain S. cerevisiae (Sclsm4Δ1) restored cell viability. Nevertheless, in this transformed strain, we observed some phenotypes that are typical markers of regulated cell death, reactive oxygen species (ROS), and oxidated RNA accumulation. In this paper, we report that a similar truncation operated in the S. cerevisiae LSM4 gene confers on cells the same phenotypes observed with the K. lactis lsm4Δ1 gene. Up until now, there was no evidence of the direct involvement of LSM4 in autophagy. Here we found that the Sclsm4Δ1 mutant showed a block in the autophagic process and was very sensitive to nitrogen starvation or treatment with low doses of rapamycin, an inducer of autophagy. Moreover, both during nitrogen starvation and aging, the Sclsm4Δ1 mutant accumulated cytoplasmic autophagy-related structures, suggesting a role of Lsm4 in a later step of the autophagy process.

Identification and validation of key biomarkers based on RNA methylation genes in sepsis.

RNA methylation is closely involved in immune regulation, but its role in sepsis remains unknown. Here, we aim to investigate the role of RNA methylation-associated genes (RMGs) in classifying and diagnosing of sepsis. Five types of RMGs (m1A, m5C, m6Am, m7G and Ψ) were used to identify sepsis subgroups based on gene expression profile data obtained from the GEO database (GSE57065, GSE65682, and GSE95233). Unsupervised clustering analysis was used to identify distinct RNA modification subtypes. The CIBERSORT, WGCNA, GO and KEGG analysis were performed to explore immune infiltration pattern and biological function of each cluster. RF, SVM, XGB, and GLM algorithm were applied to identify the diagnostic RMGs in sepsis. Finally, the expression levels of the five key RMGs were verified by collecting PBMCs from septic patients using qRT-PCR, and their diagnostic efficacy for sepsis was verified in combination with clinical data using ROC analysis. Sepsis was divided into three subtypes (cluster 1 to 3). Cluster 1 highly expressed NSUN7 and TRMT6, with the characteristic of neutrophil activation and upregulation of MAPK signaling pathways. Cluster 2 highly expressed NSUN3, and was featured by the regulation of mRNA stability and amino acid metabolism. NSUN5 and NSUN6 were upregulated in cluster 3 which was involved in ribonucleoprotein complex biogenesis and carbohydrate metabolism pathways. In addition, we identified that five RMGs (NSUN7, NOP2, PUS1, PUS3 and FTO) could function as biomarkers for clinic diagnose of sepsis. For validation, we determined that the relative expressions of NSUN7, NOP2, PUS1 and PUS3 were upregulated, while FTO was downregulated in septic patients. The area under the ROC curve (AUC) of NSUN7, NOP2, PUS1, PUS3 and FTO was 0.828, 0.707, 0.846, 0.834 and 0.976, respectively. Our study uncovered that dysregulation of RNA methylation genes (m1A, m5C, m6Am, m7G and Ψ) was closely involved in the pathogenesis of sepsis, providing new insights into the classification of sepsis endotypes. We also revealed that five hub RMGs could function as novel diagnostic biomarkers and potential targets for treatment.

A lncRNA from the FTO locus acts as a suppressor of the m6A writer complex and p53 tumor suppression signaling

N6-methyladenosine (m6A) of mRNAs modulated by the METTL3-METTL14-WTAP-RBM15 methyltransferase complex and m6A demethylases such as FTO play important roles in regulating mRNA stability, splicing, and translation. Here, we demonstrate that FTO-IT1 long noncoding RNA (lncRNA) was upregulated and positively correlated with poor survival of patients with wild-type p53-expressing prostate cancer (PCa). m6A RIP-seq analysis revealed that FTO-IT1 knockout increased mRNA m6A methylation of a subset of p53 transcriptional target genes (e.g., FAS, TP53INP1, and SESN2) and induced PCa cell cycle arrest and apoptosis. We further showed that FTO-IT1 directly binds RBM15 and inhibits RBM15 binding, m6A methylation, and stability of p53 target mRNAs. Therapeutic depletion of FTO-IT1 restored mRNA m6A level and expression of p53 target genes and inhibited PCa growth in mice. Our study identifies FTO-IT1 lncRNA as a bona fide suppressor of the m6A methyltransferase complex and p53 tumor suppression signaling and nominates FTO-IT1 as a potential therapeutic target of cancer.

Role(s) of G3BPs in Human Pathogenesis.

Ras-GTPase-activating protein (SH3 domain)-binding proteins (G3BP) are RNA binding proteins that play a critical role in stress granule (SG) formation. SGs protect critical mRNAs from various environmental stress conditions by regulating mRNA stability and translation to maintain regulated gene expression. Recent evidence suggests that G3BPs can also regulate mRNA expression through interactions with RNA outside of SGs. G3BPs have been associated with a number of disease states, including cancer progression, invasion, metastasis, and viral infections, and may be useful as a cancer therapeutic target. This review summarizes the biology of G3BP including their structure, function, localization, role in cancer progression, virus replication, mRNA stability, and SG formation. We will also discuss the potential of G3BPs as a therapeutic target. SIGNIFICANCE STATEMENT: This review will discuss the molecular mechanism(s) and functional role(s) of Ras-GTPase-activating protein (SH3 domain)-binding proteins in the context of stress granule formation, interaction with viruses, stability of RNA, and tumorigenesis.

Lethal eosinophilic crystalline pneumonia in mice expressing a stabilized Csf2 mRNA.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that stimulates the proliferation and differentiation of granulocyte and macrophage precursors. The mouse gene-encoding GM-CSF, Csf2, is regulated at both transcriptional and post-transcriptional levels. An adenine-uridine-rich element (ARE) within the 3'-untranslated region of Csf2 mRNA was shown in cell transfection studies to confer instability on this transcript. To explore the physiological importance of this element in an intact animal, we generated mice with a knock-in deletion of the 75-nucleotide ARE. Mice heterozygous for this ARE deletion developed severe respiratory distress and death within about 12 weeks of age. There was dense infiltration of lung alveolar spaces by crystal-containing macrophages. Increased stability of Csf2 mRNA was confirmed in bone marrow-derived macrophages, and elevated GM-CSF levels were observed in serum and lung. These mice did not exhibit notable abnormalities in blood or bone marrow, and transplantation of bone marrow from mutant mice into lethally irradiated WT mice did not confer the pulmonary phenotype. Mice with a conditional deletion of the ARE restricted to lung type II alveolar cells exhibited an essentially identical lethal lung phenotype at the same ages as the mice with the whole-body deletion. In contrast, mice with the same conditional ARE deletion in myeloid cells, including macrophages, exhibited lesser degrees of macrophage infiltration into alveolar spaces much later in life, at approximately 9 months of age. Post-transcriptional Csf2 mRNA stability regulation in pulmonary alveolar epithelial cells appears to be essential for normal physiological GM-CSF secretion and pulmonary macrophage homeostasis.

The cytoplasmic fraction of the histone lysine methyltransferase Setdb1 is essential for embryonic stem cells

The major lysine methyltransferase (KMT) Setdb1 is essential for self-renewal and viability of mouse embryonic stem cells (mESCs). Setdb1 was primarily known to methylate the lysine 9 of histone 3 (H3K9) in the nucleus, where it regulates chromatin functions. However, Setdb1 is also massively localized in the cytoplasm, including in mESCs, where its role remains elusive. Here, we show that the cytoplasmic Setdb1 (cSetdb1) is essential for the survival of mESCs. Yeast two-hybrid analysis revealed that cSetdb1 interacts with several regulators of mRNA stability and protein translation machinery, such as the ESCs-specific E3 ubiquitin ligase and mRNA silencer Trim71/Lin41. We found that cSetdb1 is required for the integrity of Trim71 complex(es) involved in mRNA metabolism and translation. cSetdb1 modulates the abundance of mRNAs and the rate of newly synthesized proteins. Altogether, our data uncovered the cytoplasmic post-transcriptional regulation of gene expression mediated by a key epigenetic regulator.

IGF2BP family of RNA-binding proteins regulate innate and adaptive immune responses in cancer cells and tumor microenvironment.

Insulin-like growth factor 2 mRNA-binding proteins (IGF2BP1, IGF2BP2, and IGF2BP3) are a family of RNA-binding proteins that play an essential role in the development and disease by regulating mRNA stability and translation of critical regulators of cell division and metabolism. Genetic and chemical inhibition of these proteins slows down cancer cell proliferation, decreases invasiveness, and prolongs life span in a variety of animal models. The role of RNA-binding proteins in the induction of tissues' immunogenicity is increasingly recognized, but, the impact of the IGF2BPs family of proteins on the induction of innate and adaptive immune responses in cancer is not fully understood. Here we report that downregulation of IGF2BP1, 2, and 3 expression facilitates the expression of interferon beta-stimulated genes. IGF2BP1 has a greater effect on interferon beta and gamma signaling compared to IGF2BP2 and IGF2BP3 paralogs. We demonstrate that knockdown or knockout of IGF2BP1, 2, and 3 significantly potentiates inhibition of cell growth induced by IFNβ and IFNγ. Mouse melanoma cells with Igf2bp knockouts demonstrate increased expression of MHC I (H-2) and induce intracellular Ifn-γ expression in syngeneic T-lymphocytes in vitro. Increased immunogenicity, associated with Igf2bp1 inhibition, "inflames" mouse melanoma tumors microenvironment in SM1/C57BL/6 and SW1/C3H mouse models measured by a two-fold increase of NK cells and tumor-associated myeloid cells. Finally, we demonstrate that the efficiency of anti-PD1 immunotherapy in the mouse melanoma model is significantly more efficient in tumors that lack Igf2bp1 expression. Our retrospective data analysis of immunotherapies in human melanoma patients indicates that high levels of IGF2BP1 and IGF2BP3 are associated with resistance to immunotherapies and poor prognosis. In summary, our study provides evidence of the role of IGF2BP proteins in regulating tumor immunogenicity and establishes those RBPs as immunotherapeutic targets in cancer.

Transfer RNA-derived small RNAs (tsRNAs) in gastric cancer.

Transfer RNA-derived small RNAs (tsRNAs) are newly discovered noncoding RNAs (ncRNAs). According to the specific cleavage of nucleases at different sites of tRNAs, the produced tsRNAs are divided into tRNA-derived stress-inducible RNAs (tiRNAs) and tRNA-derived fragments (tRFs). tRFs and tiRNAs have essential biological functions, such as mRNA stability regulation, translation regulation and epigenetic regulation, and play significant roles in the occurrence and development of various tumors. Although the roles of tsRNAs in some tumors have been intensively studied, their roles in gastric cancer are still rarely reported. In this review, we focus on recent advances in the generation and classification of tsRNAs, their biological functions, and their roles in gastric cancer. Sixteen articles investigating dysregulated tsRNAs in gastric cancer are summarized. The roles of 17 tsRNAs are summarized, of which 9 were upregulated and 8 were downregulated compared with controls. Aberrant regulation of tsRNAs was closely related to the main clinicopathological factors of gastric cancer, such as lymph node metastasis, Tumor-Node-Metastasis (TNM) stage, tumor size, and vascular invasion. tsRNAs participate in the progression of gastric cancer by regulating the PTEN/PI3K/AKT, MAPK, Wnt, and p53 signaling pathways. The available literature suggests the potential of using tsRNAs as clinical biomarkers for gastric cancer diagnosis and prognosis and as therapeutic targets for gastric cancer treatment.

QKI shuttles internal m7G-modified transcripts into stress granules and modulates mRNA metabolism

N7-methylguanosine (m7G) modification, routinely occurring at mRNA 5' cap or within tRNAs/rRNAs, also exists internally in messenger RNAs (mRNAs). Although m7G-cap is essential for pre-mRNA processing and protein synthesis, the exact role of mRNA internal m7G modification remains elusive. Here, we report that mRNA internal m7G is selectively recognized by Quaking proteins (QKIs). By transcriptome-wide profiling/mapping of internal m7G methylome and QKI-binding sites, we identified more than 1,000 high-confidence m7G-modified and QKI-bound mRNA targets with a conserved "GANGAN (N= A/C/U/G)" motif. Strikingly, QKI7 interacts (via C terminus) with the stress granule (SG) core protein G3BP1 and shuttles internal m7G-modified transcripts into SGs to regulate mRNA stability and translation under stress conditions. Specifically, QKI7 attenuates the translation efficiency of essential genes in Hippo signaling pathways to sensitize cancer cells to chemotherapy. Collectively, we characterized QKIs as mRNA internal m7G-binding proteins that modulate target mRNA metabolism and cellular drug resistance.

Post-transcriptional checkpoints in autoimmunity.

Post-transcriptional regulation is a fundamental process in gene expression that has a role in diverse cellular processes, including immune responses. A core concept underlying post-transcriptional regulation is that protein abundance is not solely determined by transcript abundance. Indeed, transcription and translation are not directly coupled, and intervening steps occur between these processes, including the regulation of mRNA stability, localization and alternative splicing, which can impact protein abundance. These steps are controlled by various post-transcription factors such as RNA-binding proteins and non-coding RNAs, including microRNAs, and aberrant post-transcriptional regulation has been implicated in various pathological conditions. Indeed, studies on the pathogenesis of autoimmune and inflammatory diseases have identified various post-transcription factors as important regulators of immune cell-mediated and target effector cell-mediated pathological conditions. This Review summarizes current knowledge regarding the roles of post-transcriptional checkpoints in autoimmunity, as evidenced by studies in both haematopoietic and non-haematopoietic cells, and discusses the relevance of these findings for developing new anti-inflammatory therapies.

Ac4C acetylation regulates mRNA stability and translation efficiency in osteosarcoma

ObjectiveN4-acetylcytidine (ac4C) acetylation can promote target gene expression through improved mRNA stability. To explore the role of ac4C acetylation in osteosarcoma, U2OS and MG63 cell lines were treated with the N-acetyltransferase 10 (NAT10) inhibitor Remodelin. Reverse transcription–polymerase chain reaction (RT-PCR) and Western blot were used to test the gene and protein expression efficiency. MethodsThe proliferation rate of osteosarcoma cells was measured by a cell counting kit-8 (CCK8) assay. The cell cycle and apoptosis were analyzed by flow cytometry. The invasiveness of osteosarcoma cells was detected by a transwell invasion assay. The ac4C acetylation of target genes was screened by acetylated RNA immunoprecipitation and sequencing (acRIP-seq). ResultsWe found that when osteosarcoma cells were treated with Remodelin at the optimal concentration, their NAT10 expression and the cell proliferation was inhibited, the cells in the G1 phase increased (P < 0.05) but those in the S phase decreased, the apoptotic cells in the early and late stages increased, and the cells invasiveness decreased (P < 0.05). ConclusionsThe farnesyltransferase subunit beta gene (FNTB) was identified by acRIP-seq as one of the target genes of ac4C acetylation and was further verified by RT-PCR and Western blot analyses. Remodelin was demonstrated to reduce the stability and protein translation efficiency of target gene mRNA in osteosarcoma cells. In conclusion, inhibition of ac4C acetylation in osteosarcoma can block proliferation and metastasis as well as promote apoptosis and cell cycle arrest. Ac4C acetylation contributes to the stability and protein translation efficiency of the downstream target gene mRNA.