AU-rich Elements Research Articles

Post-transcriptional regulation of BIRC5/survivin expression and induction of apoptosis in breast cancer cells by tristetraprolin

ABSTRACT Inhibition of apoptosis is one of the hallmarks of cancer and is a target of various therapeutic interventions. BIRC5 is an inhibitor of apoptosis that is aberrantly expressed in cancer leading to sustained growth of tumours. Post-transcriptional control mechanisms involving RNA-binding proteins and AU-rich elements (AREs) are fundamental to many cellular processes and changes in the expression or function of these proteins can promote an aberrant and pathological phenotype. BIRC5 mRNA has an ARE in its 3’ UTR making it a candidate for regulation by the RNA binding proteins tristetraprolin (TTP) and HuR (ELAVL1). In this study, we investigated the binding of TTP and HuR by RNA-immunoprecipitation assays and found that these proteins were associated with BIRC5 mRNA to varying extents. Consequently, BIRC5 expression decreased when TTP was overexpressed and apoptosis was induced. In the absence of TTP, BIRC5 mRNA was stabilized, protein expression increased and the number of apoptotic cells declined. As an ARE-mRNA stabilizing protein, recombinant HuR led to upregulation of BIRC5 expression, whereas HuR silencing was concomitant with downregulation of BIRC5 mRNA and protein and increased cell death. Survival analyses demonstrated that increased TTP and low BIRC5 expression predicted an overall better prognosis compared to dysregulated TTP and high BIRC5. Thus, the results present a novel target of ARE-mediated post-transcriptional regulation.

Potential role of S100A8/A9 and RNA-binding protein in microvascular complications of type2 diabetes mellitus

Background & objective: Persistent hyperglycemia is the driving force for the progression of diabetic vascular complications and inflammatory response. S100A8 and S100A9 are small calcium-binding proteins involved in various cellular processes, including inflammation and immune responses. Tristetraprolin (TTP), alternatively known as zinc finger protein 36, acts as an RNA-binding molecule that has an important role in regulating the expression of messenger RNAs containing AU-rich elements. We aimed to address the involvement of inflammatory mediators like S100A8/A9 proteins and, RNA-binding proteins, in microvascular complications of type 2 diabetes mellitus (T2DM). Methodology: The study was conducted from October 2022 to April 2023. We enrolled 200 subjects in this study involved in five equal groups: T2DM, diabetic nephropathy (DN), diabetic retinopathy (DR), diabetic neuropathy (DNR) and 40 normal healthy subjects as control group. CBC 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 RT-qPCR. Results: The study's findings revealed a notable increase in neutrophil/lymphocytes ratio (NLR) and S100A8/A9 levels in patients groups compared to the healthy group (P < 0.05), while decreased TTP mRNA expression was observed in all patient groups compared to control (P < 0.05) Conclusion: An increase in S1008A/9A levels with down regulation of anti-inflammatory binding protein (TTP) in patients suffering from type 2 diabetes mellitus with diabetic nephropathy, diabetic retinopathy, or diabetic neuropathy, suggests to be the therapeutic targets to regulate inflammatory response in type 2 diabetes mellitus and its complications. Abbreviations: T2DM - Type 2 Diabetes Mellitus; DN - Diabetic Nephropathy; DR - Diabetic Retinopathy; DNR - Diabetic Neuropathy; NLR- Neutrophil/Lymphocyte Ratio; TTP - Tristetraprolin Key words: Diabetes Mellitus; Inflammation; S100A8/A9; Inflammation; RNA-binding protein; T2DM; Neutrophil/Lymphocyte Ratio Citation: Sarhan A, Almzaiel AJ, Majeed Alrufaie MM. Potential role of S100A8/A9 and RNA-binding protein in microvascular complications of type2 diabetes mellitus. Anaesth. pain intensive care 2023;27(6):673−680. DOI: 10.35975/apic.v27i6.2338 Received: August 15, 2023; Reviewed: September 03, 2023; Accepted: September 15, 2023

Serum S100A8/A9 and RNA-binding protein, tristetraprolin as prognostic markers of renal damage

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

HuR deficiency abrogated the enhanced NLRP3 signaling in experimental ischemic stroke.

Human antigen R (HuR) is a universally expressed RNA-binding protein that plays an essential role in governing the fate of mRNA transcripts. Accumulating evidence indicated that HuR is involved in the development and functions of several cell types. However, its role in cerebral ischemia/reperfusion injury (CIRI) remains unclear. In this study, we found that HuR was significantly upregulated after CIRI. Moreover, we found that silencing HuR could inhibit the inflammatory response of microglia and reduce the damage to neurons caused by oxygen-glucose deprivation/reperfusion treatment. Invivo, we found that microglial HuR deficiency significantly ameliorated CIRI and reduced NLRP3-mediated inflammasome activation. Mechanistically, we found that HuR could regulate NLRP3 mRNA stability by binding to the AU-rich element (ARE) region within the 3' untranslated region (UTR) of NLRP3 mRNA. In addition, we found that the upregulation of HuR was dependent on the upregulation of NADPH oxidase-mediated ROS accumulation. Collectively, our studies revealed that HuR could regulate NLRP3 expression and that HuR deficiency abrogated the enhanced NLRP3 signaling in experimental ischemic stroke. Targeting HuR may be a novel therapeutic strategy for cerebral ischemic stroke treatment.

Low expression of ZFP36L1 in osteosarcoma promotes lung metastasis by inhibiting the SDC4-TGF-β signaling feedback loop.

ZFP36L1, which is a negative regulator of gene transcripts, has been proven to regulate the progression of several carcinomas. However, its role in sarcoma remains unknown. Here, by using data analyses and in vivo experiments, we found that ZFP36L1 inhibited the lung metastasis of osteosarcoma (OS). Knockdown of ZFP36L1 promoted OS cell migration by activating TGF-β signaling and increasing SDC4 expression. Intriguingly, we observed a positive feedback loop between SDC4 and TGF-β signaling. SDC4 protected TGFBR3 from matrix metalloproteinase (MMP)-mediated cleavage and therefore relieved the inhibition of TGF-β signaling by soluble TGFBR3, while TGF-β signaling positively regulated SDC4 transcription. We also proved that ZFP36L1 regulated SDC4 mRNA decay through adenylate-uridylate (AU)-rich elements (AREs) in its 3'UTR. Furthermore, treatment with SB431542 (a TGF-β receptor kinase inhibitor) and MK2 inhibitor III (a MAPKAPK2 inhibitor that increases the ability of ZFP36L1 to degrade mRNA) dramatically inhibited OS lung metastasis, suggesting a promising therapeutic approach for the treatment of OS lung metastasis.

Measuring Proximity-Mediated Function of mRNA Regulatory Proteins by Engineered Tethering.

A powerful approach for studying the functional consequences of site-specific RNA-protein interactions is to artificially tether a protein to a messenger (or noncoding) RNA through a selective, high-affinity interaction. We share a strategy for evaluating the contribution of protein positioning within an mRNA on gene expression. We introduced an RNA hairpin recognition site for the MS2 coat protein into the untranslated regions or coding sequence of mRNAs expressing a luminescent reporter protein, NanoLuc. Effector proteins fused to the MS2 coat protein could thus be targeted to distinct regions across the mRNA. We illustrate this approach using ZFP36L2, which recruits the CCR4-NOT complex for poly(A) tail deadenylation. Tethering ZFP36L2 to the 3'-UTR decreased NanoLuc expression, as expected, given the known interaction of this adapter protein with adenine uridine-rich elements (AREs). Intriguingly, ZFP36L2 also decreased NanoLuc expression when bound within the coding sequence, revealing that ZFP36L2-and potentially many other mRNA regulatory proteins-can function when targeted to diverse locations within an mRNA. This multi-target tethering strategy enables exploration of the interplay between mRNA-protein proximity and gene expression.

FRI289 It Takes A Village: Musashi And Its Associated Proteins In The Adenohypophysis

Abstract Disclosure: K.L. Bronson: None. L. Hardy: None. A. Lagasse: None. A.M. MacNicol: None. Musashi is an RNA-binding protein that regulates stem and progenitor cell fates. Interestingly, we have reported expression of Musashi pituitary stem cells as expected but also in the differentiated anterior pituitary hormone-producing cell lineages as well as. The adult anterior pituitary relies on the functional cellular plasticity of its hormone-producing cell populations to maintain endocrine homeostasis when physiological demands change. Both Musashi isoforms (Musashi1 and Musashi2) are highly conserved across species and have two N-terminal RNA-recognition motifs (RRMs) and a disordered C-terminus containing two regulatory phosphorylation sites. Musashi can selectively activate or repress the translation of mRNAs depending on the transcript and cellular context. While Musashi phosphorylation is required for translational activation, Musashi does not possess inherent enzymatic activity; therefore, co-associated proteins must mediate Musashi-directed translational control. Musashi immunoprecipitation studies initially identified 50 co-associated proteins in Xenopus oocytes, but only a subset of these were directly implicated in Musashi-dependent translational activation (e.g. Elavl1 and Pabp4). The identity of Musashi-associated proteins in the anterior pituitary and their requirement for Musashi function are not known. To this end, we have immunoprecipitated Musashi from whole male mouse pituitaries for mass spectrometry analysis. 206 proteins significantly (adj p-value < 0.05) associated with Musashi1 and Musashi2, and seven of these overlapped with the Xenopus dataset: Elavl1, Pabp4, Hnrnpc, Mov10, Pura, Rps27, and Upf1. Three proteins from the mouse mass spectrometry dataset were selected for further analyses. Sam68 is a multi-functional protein that sensitizes ovaries to FSH and LH, as well as mediates the activation of leptin and insulin signaling in a variety of tissues. Ago2 is a component of the RNA-induced silencing complex that is necessary for miRNA-directed translational control and is known to associate with Musashi1. The AU-rich element binding protein Elavl1 (HuR) contributes to Musashi-dependent activation of target mRNAs in Xenopus and also stabilizes Gnrhr mRNA in LβT2 cells and sensitizes them to GnRH signaling. We will present our ongoing studies on the requirement for these protein interactions for Musashi-directed mRNA translational regulation. Presentation: Friday, June 16, 2023

Expression of maize OXS2a in Arabidopsis stunts plant growth but enhances heat tolerance

As plants encounter various environmental stresses, judicial allocation of resources to stress response is crucial for plant fitness. The plant OXS2 (OXIDATIVE STRESS 2) family has been reported to play important roles in growth regulation and stress response. Here, we report that the maize OXS2 family member ZmOXS2a when expressed in Arabidopsis retards growth including delayed flowering, but improves heat tolerance. ZmOXS2a can be found in the cytoplasm, nucleus and PBs/P bodies (mRNA processing bodies), but heat treatment induces higher accumulation in the PBs. Deletion of ARR (arginine rich region) and TZF (tandem zinc finger) domains for high-affinity RNA-binding reduced PBs accumulation of ZmOXS2a; and unlike ZmOXS2a, expression of this deletion mutant gene affected neither Arabidopsis growth nor heat tolerance. This suggests that ZmOXS2a might be involved in RNA degradation, which would also account for the larger amount of down-regulated genes found in ZmOXS2a expressing lines. Furthermore, 240 of 890 down-regulated genes contain ARE (AU-rich elements) in the mRNA 3′UTR that might be potential targets of ZmOXS2a. Expression of ZmOXS2a also disturbs the response to ABA (abscisic acid) and cytokinin, as GO (gene ontology) analysis shows that 50 and 15 DEGs (differentially expressed genes) are enriched in the GO term for ABA and cytokinin responses, respectively. ZmOXS2a expression lines are more sensitive to ABA, but less sensitive to cytokinin. It is likely that ZmOXS2a promotes the degradation of the mRNA of down-regulated genes containing ARE, which consequently perturbs the hormone pathways that affect stress response-related plant growth.

Precise Interference of RNA-Protein Interaction by CRISPR-Cas13-Mediated Peptide Competition.

RNA-protein interactions are essential nodes of cellular regulatory circuits and play critical roles in normal physiology and disease. However, the precise roles of individual RNA-protein interactions remain elusive. Here we report a method for precise interference of endogenous RNA interacting with the RNA binding protein (RBP). TTP is an RBP that recognizes the AU-rich element (ARE) of mRNA via the binding domain TZF and represses gene expression. We engineer Cas13b, a class 2 type VI CRISPR-Cas endonuclease that exclusively targets RNA, to direct the peptide of TZF to the binding site and compete with endogenous TTP. We show that this tool specifically interferes with TTP interacting with the PIM1 and IL-2 3' UTR under the guidance of the gRNA specific for the AREs. Further, precise interference with the TTP-PIM1 interaction exerts a distinct effect on cell proliferation compared to transcriptome-wide interference. Thus, our work establishes a tool for deep understanding of RNA-RBP interactions.

ZIKV induction of tristetraprolin in endothelial and Sertoli cells post-transcriptionally inhibits IFNβ/λ expression and promotes ZIKV persistence.

Our findings define a novel role for ZIKV-induced TTP expression in regulating IFNβ/IFNλ production in primary hBMECs and Sertoli cells. These cells comprise key physiological barriers subverted by ZIKV to access brain and testicular compartments and serve as reservoirs for persistent replication and dissemination. We demonstrate for the first time that the ARE-binding protein TTP is virally induced and post-transcriptionally regulates IFNβ/IFNλ secretion. In ZIKV-infected hBMEC and Sertoli cells, TTP knockout increased IFNβ/IFNλ secretion, while TTP expression blocked IFNβ/IFNλ secretion. The TTP-directed blockade of IFN secretion permits ZIKV spread and persistence in hBMECs and Sertoli cells and may similarly augment ZIKV spread across IFNλ-protected placental barriers. Our work highlights the importance of post-transcriptional ZIKV regulation of IFN expression and secretion in cells that regulate viral access to protected compartments and defines a novel mechanism of ZIKV-regulated IFN responses which may facilitate neurovirulence and sexual transmission.

Circular RNA EIF3I promotes papillary thyroid cancer progression by interacting with AUF1 to increase Cyclin D1 production.

Circular RNAs (circRNAs) play an important role in regulating the development of human cancers through diverse biological functions. However, the exact molecular mechanisms underlying the role of circRNAs in papillary thyroid cancer (PTC) remain largely unknown. Here, we found that hsa_circ_0011385, designated as circular eukaryotic translation initiation factor 3 subunit I (circEIF3I), preferentially localized in the cytoplasm of PTC cells and was more stable than its linear counterpart, EIF3I. Gain- and loss-of-function studies indicated that circEIF3I promoted PTC progression by facilitating cell proliferation, cell cycle, cell migration, and invasion in vitro, as well as PTC cell proliferation in vivo. Mechanistically, circEIF3I interacted with AU-rich element (ARE) RNA-binding factor 1 (AUF1) in the cytoplasm of PTC cells, thus reducing the degradation of Cyclin D1 mRNA and increasing Cyclin D1 protein production, ultimately resulting in PTC progression. Collectively, our results demonstrate the vital role of circEIF3I in PTC progression, supporting its significance as a potential therapeutic target.

The RNA-binding protein KSRP aggravates malignant progression of clear cell renal cell carcinoma through transcriptional inhibition and post-transcriptional destabilization of the NEDD4L ubiquitin ligase

BackgroundKH-type splicing regulatory protein (KHSRP, also called KSRP), a versatile RNA-binding protein, plays a critical role in various physiological and pathological conditions through modulating gene expressions at multiple levels. However, the role of KSRP in clear cell renal cell carcinoma (ccRCC) remains poorly understood.MethodsKSRP expression was detected by a ccRCC tissue microarray and evaluated by an in silico analysis. Cell loss-of-function and gain-of-function, colony-formation, anoikis, and transwell assays, and an orthotopic bioluminescent xenograft model were conducted to determine the functional role of KRSP in ccRCC progression. Micro (mi)RNA and complementary (c)DNA microarrays were used to identify downstream targets of KSRP. Western blotting, quantitative real-time polymerase chain reaction, and promoter- and 3-untranslated region (3'UTR)-luciferase reporter assays were employed to validate the underlying mechanisms of KSRP which aggravate progression of ccRCC.ResultsOur results showed that dysregulated high levels of KSRP were correlated with advanced clinical stages, larger tumor sizes, recurrence, and poor prognoses of ccRCC. Neural precursor cell-expressed developmentally downregulated 4 like (NEDD4L) was identified as a novel target of KSRP, which can reverse the protumorigenic and prometastatic characteristics as well as epithelial-mesenchymal transition (EMT) promotion by KSRP in vitro and in vivo. Molecular studies revealed that KSRP can decrease NEDD4L messenger (m)RNA stability via inducing mir-629-5p upregulation and directly targeting the AU-rich elements (AREs) of the 3’UTR. Moreover, KSRP was shown to transcriptionally suppress NEDD4L via inducing the transcriptional repressor, Wilm's tumor 1 (WT1). In the clinic, ccRCC samples revealed a positive correlation between KSRP and mesenchymal-related genes, and patients expressing high KSRP and low NEDD4L had the worst prognoses.ConclusionThe current findings unveil novel mechanisms of KSRP which promote malignant progression of ccRCC through transcriptional inhibition and post-transcriptional destabilization of NEDD4L transcripts. Targeting KSRP and its pathways may be a novel pharmaceutical intervention for ccRCC.

The 3' Untranslated Regions of Ebola Virus mRNAs Contain AU-Rich Elements Involved in Posttranscriptional Stabilization and Decay.

The 3' untranslated regions (UTRs) of Ebola virus (EBOV) mRNAs are enriched in their AU content and therefore represent potential targets for RNA binding proteins targeting AU-rich elements (ARE-BPs). ARE-BPs are known to fine-tune RNA turnover and translational activity. We identified putative AREs within EBOV mRNA 3' UTRs and assessed whether they might modulate mRNA stability. Using mammalian and zebrafish embryo reporter assays, we show a conserved, ARE-BP-mediated stabilizing effect and increased reporter activity with the tested EBOV 3' UTRs. When coexpressed with the prototypic ARE-BP tristetraprolin (TTP, ZFP36) that mainly destabilizes its target mRNAs, the EBOV nucleoprotein (NP) 3' UTR resulted in decreased reporter gene activity. Coexpression of NP with TTP led to reduced NP protein expression and diminished EBOV minigenome activity. In conclusion, the enrichment of AU residues in EBOV 3' UTRs makes them possible targets for cellular ARE-BPs, leading to modulation of RNA stability and translational activity.

IL-17 promotes melanoma through TRAF2 as a scaffold protein recruiting PIAS2 and ELAVL1 to induce EPHA5

An abnormal immune response induces melanoma development. IL-17 and the classical downstream signal STAT1 are associated with melanoma development. TRAF2 also mediates the downstream signaling of IL-17; however, its role in IL-17-stimulated melanoma remains unclear. Bioinformatic analysis revealed that TRAF2 can bind to PIAS2 (a SUMO E3 ligase), ELAVL1 (an RNA-binding protein), and EPHA5 (an ephrin receptor of the tyrosine kinase family). To elucidate the IL-17 downstream signal, the IL-17 receptor (R), STAT1, TRAF2, PIAS2, ELAVL1, and EPHA5 were knocked down before melanoma cells were treated with recombinant IL-17A protein. Co-immunoprecipitation and RNA immunoprecipitation were conducted to determine the interaction of TRAF2 with PIAS2, ELAVL1, and EPHA5 proteins, as well as the interaction of ELAVL1 protein with EPHA5 mRNA. STAT1 knockdown suppressed the proliferation and invasion triggered by IL-17A, but the suppressive effects were much weaker than those caused by IL-17R knockdown. This implies that another nonclassical signal mediates IL-17 effects. IL-17A induces TRAF2 recruitment of ELAVL1, PIAS2, and EPHA5 proteins. We speculated that ELAVL1 bound to the AU-rich elements in the 3′ untranslated region of the EPHA5 mRNA, thereby enhancing mRNA stability. Furthermore, PIAS2 induced EPHA5 SUMOylation, which suppressed EPHA5 ubiquitination and degradation. Through pre- and post-translational regulation, IL-17A induced EPHA5 expression in melanoma, and EPHA5 knockdown markedly suppressed IL-17A-induced proliferation and invasion. This study revealed a non-classical signaling mechanism responsible for the effects of IL-17 in melanoma.

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 yeast mRNA-binding protein Cth2 post-transcriptionally modulates ergosterol biosynthesis in response to iron deficiency

Sterol synthesis is an iron-dependent metabolic pathway in eukaryotes. Consequently, fungal ergosterol biosynthesis (ERG) is down-regulated in response to iron deficiency. In this report, we show that, upon iron limitation or overexpression of the iron-regulated mRNA-binding protein Cth2, the yeast Saccharomyces cerevisiae down-regulates the three initial enzymatic steps of ergosterol synthesis (ERG1, ERG7 and ERG11). Mechanistically, we show that Cth2 protein limits the translation and promotes the decrease in the mRNA levels of these specific ERG genes, which contain consensus Cth2-binding sites defined as AU-rich elements (AREs). Thus, expression of CTH2 leads to the accumulation of initial sterol intermediates, such as squalene, and to the drop of ergosterol levels. Changes in CTH2 expression levels disturb the response of yeast cells to stresses related to membrane integrity such as high ethanol and sorbitol concentrations. Therefore, CTH2 should be considered as a critical regulatory factor of ergosterol biosynthesis during iron deficiency.

MRNA stability and m6A are major determinants of subcellular mRNA localization in neurons

For cells to perform their biological functions, they need to adopt specific shapes and form functionally distinct subcellular compartments. This is achieved in part via an asymmetric distribution of mRNAs within cells. Currently, the main model of mRNA localization involves specific sequences called "zipcodes" that direct mRNAs to their proper locations. However, while thousands of mRNAs localize within cells, only a few zipcodes have been identified, suggesting that additional mechanisms contribute to localization. Here, we assess the role of mRNA stability in localization by combining the isolation of the soma and neurites of mouse primary cortical and mESC-derived neurons, SLAM-seq, m6A-RIP-seq, the perturbation of mRNA destabilization mechanisms, and the analysis of multiple mRNA localization datasets. We show that depletion of mRNA destabilization elements, such as m6A, AU-rich elements, and suboptimal codons, functions as a mechanism that mediates the localization of mRNAs associated with housekeeping functions to neurites in several types of neurons.

RNA activation in ticks

RNA activation (RNAa) is a burgeoning area of research in which double-stranded RNAs (dsRNAs) or small activating RNAs mediate the upregulation of specific genes by targeting the promoter sequence and/or AU-rich elements in the 3′- untranslated region (3’-UTR) of mRNA molecules. So far, studies on the phenomenon have been limited to mammals, plants, bacteria, Caenorhabditis elegans, and recently, Aedes aegypti. However, it is yet to be applied in other arthropods, including ticks, despite the ubiquitous presence of argonaute 2 protein, which is an indispensable requirement for the formation of RNA-induced transcriptional activation complex to enable a dsRNA-mediated gene activation. In this study, we demonstrated for the first time the possible presence of RNAa phenomenon in the tick vector, Haemaphysalis longicornis (Asian longhorned tick). We targeted the 3ʹ-UTR of a novel endochitinase-like gene (HlemCHT) identified previously in H. longicornis eggs for dsRNA-mediated gene activation. Our results showed an increased gene expression in eggs of H. longicornis endochitinase-dsRNA-injected (dsHlemCHT) ticks on day-13 post-oviposition. Furthermore, we observed that eggs of dsHlemCHT ticks exhibited relatively early egg development and hatching, suggesting a dsRNA-mediated activation of the HlemCHT gene in the eggs. This is the first attempt to provide evidence of RNAa in ticks. Although further studies are required to elucidate the detailed mechanism by which RNAa occurs in ticks, the outcome of this study provides new opportunities for the use of RNAa as a gene overexpression tool in future studies on tick biology, to reduce the global burden of ticks and tick-borne diseases.

Human antigen R knockdown attenuates the invasive activity of oral cancer cells through inactivation of matrix metalloproteinase-1 gene expression

Background/purposeThe RNA-binding protein human antigen R (HuR) recognizes AU-rich elements in the 3′-untranslated regions of mRNA. The expression of cytoplasmic HuR is related to the malignancy of many carcinomas. The aim of this study is investigation of effect of HuR knockdown for invasive activity of oral carcinoma. Materials and methodsProliferation, invasion, real-time PCR, and reporter gene assays were performed to confirm that the knockdown of HuR downregulates the invasive activity of cancer cells. Immunohistochemical staining was performed for high invasive carcinoma, squamous cell carcinoma (SCC) and low invasive carcinoma, verrucous carcinoma (VC), to determine if the localization of cytoplasmic HuR is related to matrix metalloproteinase-1 (MMP-1) expression. ResultsInvasive activity was significantly lower in HuR knockdown cancer cells than in control cells. A luciferase assay revealed that HuR knockdown inactivated the promoter activity of the MMP-1 gene. The mRNA levels of the transcription factors required for MMP-1 expression, including c-fos and c-jun, were decreased in HuR knockdown cancer cells. Immunohistochemical analysis revealed the level of cytoplasmic HuR and MMP-1 in invasive carcinoma to be higher than in low invasive cancer. HuR induced MMP-1 expression in the invasive front of most SCC cases. ConclusionHuR knockdown attenuated the invasive activity of cancer cells by decreasing the expression of the MMP-1, at least partially. HuR localization may help determine the invasive phenotype of cancer cells and inhibit cancer cell invasion. Furthermore, in oral SCC, HuR may be related to invasive activity through the expression of MMP-1.

HnRNPR strongly represses splicing of a critical exon associated with spinal muscular atrophy through binding to an exonic AU-rich element

BackgroundSpinal muscular atrophy (SMA) is a motor neuron disease caused by mutations of survival of motor neuron 1 (SMN1) gene, which encodes the SMN protein. SMN2, a nearly identical copy...