AU-rich Elements Research Articles

The short conserved region-2 of LARP4 interacts with ribosome-associated RACK1 and promotes translation.

LARP4 interacts with poly(A)-binding protein (PABP) to protect messenger RNAs (mRNAs) from deadenylation and decay, and recent data indicate it can direct the translation of functionally related mRNA subsets. LARP4 was known to bind RACK1, a ribosome-associated protein, although the specific regions involved and relevance had been undetermined. Here, through a combination of in-cell and in vitro methodologies, we identified positions 615-625 in conserved region-2 (CR2) of LARP4 (and 646-656 in LARP4B) as directly binding RACK1. Consistent with these results, AlphaFold2-Multimer predicted high-confidence interaction of CR2 with RACK1 propellers 5 and6. CR2 mutations strongly decreased LARP4 association with cellular RACK1 and ribosomes by multiple assays, whereas PABP association was less affected, consistent with independent interactions. The CR2 mutations decreased LARP4's ability to stabilize a β-globin mRNA reporter containing an AU-rich element (ARE) to higher degree than β-globin and GFP (green fluorescent protein)mRNAs lacking the ARE. We showLARP4 robustly increases translation of β-glo-ARE mRNA, whereas the LARP4 CR2 mutant is impaired. Analysis of nanoLuc-ARE mRNA for production of luciferase activity confirmedLARP4 promotes translation efficiency, while CR2 mutations are disabling. Thus, LARP4 CR2-mediated interaction with RACK1 can promote translational efficiency of some mRNAs.

Development of an attenuated glutamine synthetase (GS) selection system for the stable expression of tissue plasminogen activator in CHO-K1 cells

Chinese hamster ovary (CHO) cells represent the most common host system for the expression of high-quality recombinant proteins. The development of stable CHO cell lines used in industrial recombinant protein production often relies on dihydrofolate reductase (DHFR) and glutamine synthetase (GS) amplification systems. Conventional approaches to develop stable cell lines lead to heterogeneous cell populations. Consequently, it is desirable to adopt innovative strategies to increase the efficiency of clone selection to reduce the time and effort invested in the cell line development process. Attenuating the selection marker gene is an effective strategy for isolating high-producing cells. In this study, we evaluated the efficiency of an attenuated glutamine synthetase selection system for the expression of human tissue plasminogen activator (t-PA) in CHO cells. We introduced an AU-rich element (ARE) at the 3’UTR of the glutamine synthetase coding sequence and employed a weak promoter (mSV40) for the expression of this gene. Subsequently, we analyzed the effect of ARE on the GS RNA levels, and recombinant t-PA expression. Our results demonstrate that the use of ARE significantly enhances the detection of high expressing cells compared to the control. Additionally, the t-PA expression level in GS-ARE clones was approximately 900-fold greater than those without the ARE.

P4HA3 depletion induces ferroptosis and inhibits colorectal cancer growth by stabilizing ACSL4 mRNA.

Colorectal cancer (CRC) is a malignancy with high global incidence and mortality rates, posing a serious threat to human health. Despite favorable outcomes following early detection and surgical intervention, the asymptomatic nature of CRC often results in delayed diagnoses, limiting surgical treatment options. Furthermore, effective therapeutic drugs for CRC remain lacking in clinical practice, highlighting an urgent need to identify novel therapeutic targets. In this study, we identified that prolyl 4-hydroxylase subunit alpha 3 (P4HA3) is significantly upregulated in CRC and is associated with poor prognosis in patients. Both in vitro and in vivo experiments demonstrated that knockdown of P4HA3 induces ferroptosis, thereby inhibiting tumor growth. This ferroptosis induction is closely linked to increased lipid peroxidation, and P4HA3 knockdown promotes ferroptosis by upregulating acyl-CoA synthetase long-chain family member 4 (ACSL4), which regulates polyunsaturated fatty acid-containing phospholipids (PUFA-PLs) biosynthesis. Mechanistically, P4HA3 knockdown stabilizes ACSL4 mRNA by downregulating AUF1, an important RNA-binding protein (RBP) that binds to AU-rich elements (AREs) in the ACSL4 mRNA 3' untranslated region (UTR), thereby preventing its degradation. Additionally, given the lack of research on P4HA3 inhibitors, we employed virtual screening and identified Tubuloside A as a potential therapeutic agent. Tubuloside A promotes the ubiquitin-proteasome degradation of P4HA3, exerting anti-CRC effects. In summary, our findings demonstrate that P4HA3 protects CRC cells from ferroptosis by regulating ACSL4 mRNA stability via AUF1, and Tubuloside A serves as a potential P4HA3 degrader, offering a promising therapeutic strategy for CRC treatment.

PTH-dependent stabilization of RANKL mRNA is associated with increased phosphorylation of the KH-Type Splicing Regulatory Protein

Parathyroid hormone (PTH) receptor agonists promote bone formation but also increase osteoclastogenesis, in part by increasing expression of the receptor activator of nuclear factor kappa-Β ligand (RANKL). In addition to activation of transcription, regulation of mRNA stability is another important molecular mechanism controlling mRNA abundance. PTH treatment for 6 hrs. resulted in a 7.4-fold elevation in RANKL mRNA expression in UAMS-32P cells, despite prior inhibition of cellular transcription by thiophosphoryl (TPL). RANKL mRNA, like other TNF family members, contains AU-Rich Elements (AREs) in the 3’ UTR. AU-Rich Element Binding Proteins (ABPs including KSRP, TTP, AUF1 and HuR) bind to AREs and regulate mRNA stability. There was significantly more KSRP bound to RANKL mRNA than any of the other ABPs. PTH did not increase the amount of ABPs bound to the RANKL transcript. However, the level of cellular phosphorylated KSRP was significantly increased in UAMS-32P cells pre-treated with TPL followed by PTH exposure, compared to cells treated with vehicle following TPL. The extent of phosphorylation of cellular AUF1, HuR, and TTP did not increase with PTH treatment. There were no significant changes in the cellular content of total Pin1 and phospho-Pin1 protein with PTH treatment. We conclude that increases in cellular phospho-KSRP following PTH treatment, together with fact that the total amount of the KSRP bound to the RANKL mRNA did not change with PTH-treatment, may indicate that phospho-KSRP plays some role in stabilizing the RANKL transcript.

RPRD1B's direct interaction with phosphorylated RNA polymerase II regulates polyadenylation of cell cycle genes and drives cancer progression.

RNA polymerase II (Pol II) regulates eukaryotic gene expression through dynamic phosphorylation of its C-terminal domain (CTD). Phosphorylation at Ser2 and Thr4 on the CTD is crucial for RNA 3' end processing and facilitating the recruitment of cleavage and termination factors. However, the transcriptional roles of most CTD-binding proteins remain poorly understood. In this study, we focus on RPRD1B, a transcriptional regulator that interacts with the phosphorylated CTD and has been implicated in various cancers. We investigated its molecular mechanism during transcription and found that RPRD1B modulates alternative polyadenylation of cell growth transcripts by directly interacting with the CTD. RPRD1B is recruited to transcribing Pol II near the 3' end of the transcript, specifically in response to Ser2 and Thr4 phosphorylation, but only after flanking Ser5 phosphorylation is removed. Transcriptomic analysis of RPRD1B knockdown cells revealed its role in cell proliferation via termination of the key cell growth genes at upstream polyadenylation sites, leading to the production of tumor suppressor transcripts that lack AU-rich elements (AREs) with increased mRNA stability. Overall, our study uncovers previously unrecognized connections between the Pol II CTD and CID, highlighting their influence on 3' end processing and their contribution to abnormal cell growth in cancer.

Identification of Three POMCa Genotypes in Largemouth Bass (Micropterus salmoides) and Their Differential Physiological Responses to Feed Domestication.

Diverse feeding habits in teleosts involve a wide range of appetite-regulating factors. As an appetite-suppressing gene, the polymorphisms of POMCa in largemouth bass (Micropterus salmoides) were validated via sequencing and high-resolution melting (HRM). The frequency distribution of different POMCa genotypes were analyzed in two populations, and physiological responses of different POMCa genotypes to feed domestication were investigated. The indel of an 18 bp AU-rich element (ARE) in the 3' UTR and four interlocked SNP loci in the ORF of 1828 bp of POMCa cDNA sequence were identified in largemouth bass and constituted three genotypes of POMC-A I, II, and III, respectively. POMC-A I and Allele I had increased frequencies in the selection population than in the non-selection population (p < 0.01), 63.55% vs. 43.33% and 0.7850 vs. 0.6778, respectively. POMC-A I possessed the lowest value of POMCa mRNA during fasting (p < 0.05) and exhibited growth and physiological advantages under food deprivation and refeeding according to the levels of body mass and four physiological indicators, i.e., cortisol (Cor), growth hormone (GH), insulin-like growth factor-1 (IGF-1), and glucose (Glu). The identification of three POMCa genotypes, alongside their varying physiological responses during feed domestication, suggests a selective advantage that could be leveraged in molecular marker-assisted breeding of largemouth bass that are adapted to feeding on formula diet.

Regulation of AUF1 alternative splicing by hnRNPA1 and SRSF2 modulate the sensitivity of ovarian cancer cells to cisplatin.

Clarification of cisplatin resistance may provide new targets for therapy in cisplatin resistant ovarian cancer. The current study aims to explore involvement of isoforms of AU-rich element RNA-binding protein 1 (AUF1) in cisplatin resistance in ovarian cancer. The cancer stem cell-like features were analyzed using colony formation assay, tumor sphere formation assay and nude mouse xenograft experiments. AUF1 isoforms expression was analyzed using immunoblotting, qRT-PCR, and immunohistochemistry. RIP and Biotin pulldown was used to analyze the interaction of SRSF2 and hnRNPA1 with AUF1 transcript. Transcriptome regulated by AUF1 isoforms was analyzed by RNA-seq. The current study demonstrated differential expression of AUF1 isoforms in cisplatin sensitive and resistant ovarian cancer tissues and cells. P37 isoform promoted proliferation, while p45 isoform enhanced responsiveness of ovarian cancer cells to cisplatin. the clonal formation capacity of the cells, and the restoration of p45 expression reduced the capacity with cisplatin treatment. The competitive binding of phosphorylated hnRNPA1 and O-GlcNAc-modified SRSF2 on AUF1 exon 2 and exon 7 regulated the alternative splicing of AUF1. The competitive binding of phosphorylated hnRNPA1 and O-GlcNAc modified SRSF2 on exon 2 and exon 7 regulated the alternative splicing of AUF1 and subsequent isoform expression. P37 isoform played a "cancer promoter" role, p42 and p45, especially p45 played a "cancer suppressor" role in ovarian cancer. This study provides a new target for exploring the drug resistance mechanism of ovarian cancer.

LIX1L aggravates MASH-HCC progression by reprogramming of hepatic metabolism and microenvironment via CD36.

Limb expression 1-like protein (LIX1L) is an essential player in liver disorders, but its function in metabolic dysfunction-associated steatohepatitis (MASH) and associated hepatocellular carcinoma (HCC) progression remains obscure. Here, we identify LIX1L as a key integrative regulator linking lipid metabolism and inflammation, adipose tissue and hepatic microenvironment, which promotes MASH progression. LIX1L significantly upregulates in MASH patients, mouse models, and palmitic acid-stimulated hepatocytes. Lix1l deletion inhibits hepatic lipid accumulation, inflammation, and fibrosis as well as adipocyte differentiation by downregulating CD36, alleviating MASH and associated HCC progression in mice. Mechanistically, metabolic stress promotes PARP1-mediated poly-ADP-ribosylation of LIX1L to increase stability and RNA binding ability of LIX1L. Subsequently, LIX1L binds to AU-rich element in the 3'UTR and CDS of CD36 mRNA, thus mitigating CD36 mRNA decay. Furthermore, LIX1L deficiency-mediated downregulation of CD36 reprograms the tumor-prone liver microenvironment with increased cytotoxic T lymphocytes and reduced immunosuppressive cell proportions. These data indicate a systematic function of LIX1L in the pathogenesis of MASH and underscore targeting PARP1/LIX1L/CD36 axis as a feasible strategy for treatment of MASH and associated HCC.

The short conserved region-2 of LARP4 interacts with ribosome-associated RACK1 and promotes translation.

LARP4 interacts with poly(A)-binding protein (PABP) to protect mRNAs from deadenylation and decay, and recent data indicate it can direct the translation of functionally related mRNA subsets. LARP4 was known to bind RACK1, a ribosome-associated protein, although the specific regions involved, and relevance had been undetermined. Here, yeast two-hybrid domain mapping followed by other methods identified positions 615-625 in conserved region-2 (CR2) of LARP4 (and LARP4B) as directly binding RACK1 region 200-317. Consistent with these results, AlphaFold2-multimer predicted high confidence interaction of CR2 with RACK1 propellers 5-6. CR2 mutations strongly decreased LARP4 association with cellular RACK1 and ribosomes by multiple assays, whereas less effect was observed for PABP association, consistent with independent interactions. CR2 mutations decreased LARP4 ability to optimally stabilize a β-globin mRNA reporter containing an AU-rich element (ARE) more significantly than a β-globin and other reporters lacking this element. While polysome profiles indicate the β-glo-ARE mRNA is inefficiently translated, consistent with published data, we show that LARP4 increases its translation whereas the LARP4-CR2 mutant is impaired. Analysis of nanoLuc-ARE mRNA for production of luciferase activity confirmed LARP4 promotes translation efficiency while CR2 mutations are disabling. Thus, LARP4 CR2-mediated interaction with RACK1 can promote translational efficiency of some mRNAs.

Sex-specific role of RNA-binding protein, pAUF1, on prolonged hypersensitivity after repetitive ischemia with reperfusion injury.

Repetitive ischemia with reperfusion (I/R) injury is a common cause of myalgia. Ischemia with reperfusion injuries occur in many conditions that differentially affect males and females including complex regional pain syndrome and fibromyalgia. Our preclinical studies have indicated that primary afferent sensitization and behavioral hypersensitivity caused by I/R injury may be due to sex-specific gene expression in the dorsal root ganglia (DRG) and distinct upregulation of growth factors and cytokines in the affected muscles. To determine how these unique gene expression programs may be established in a sex-dependent manner in a model that more closely mimics clinical scenarios, we used a developed prolonged ischemic myalgia model in mice whereby animals experience repeated I/R injuries and compared behavioral results with unbiased and targeted screening strategies in male and female DRG. Several distinct proteins were found to be differentially expressed in male and female DRG, including phosphorylated AU-rich element RNA-binding protein (pAUF1), which is known to regulate gene expression. Nerve-specific siRNA-mediated knockdown of AUF1 inhibited prolonged hypersensitivity in females only, whereas overexpression of AUF1 in male DRG neurons increased pain-like responses. AUF1 knockdown was able to specifically inhibit repeated I/R-induced gene expression in females potentially downstream of prolactin receptor signaling. Data suggest RNA-binding proteins such as pAUF1 may underlie the sex-specific effects on DRG gene expression that modulates behavioral hypersensitivity after repeated I/R injury through prolactin signaling. This study may aid in finding distinct receptor differences related to the evolution of acute to chronic ischemic muscle pain development between sexes.

Super-enhancer-driven ZFP36L1 promotes PD-L1 expression in infiltrative gastric cancer

Gastric cancer (GC) is a major cause of cancer-related mortality worldwide. Despite the widespread recognition of tumor immunotherapy in treating unresectable GC, challenges, including ineffective immunotherapy and drug resistance, persist. Therefore, understanding the regulatory mechanisms of PD-L1, particularly in the context of super-enhancers (SEs) and zinc finger protein 36 ring finger protein-like 1 (ZFP36L1) RNA-binding protein, is crucial. In this study, we performed H3K27ac Cleavage Under Targets and Tagmentation (CUT&amp;Tag) sequencing, investigated the heterogeneity of SEs between two GC subtypes with differential growth patterns, and revealed the immune escape signatures driven by ZFP36L1-SE in infiltrative GC through SEs inhibitors treatment. The regulation of ZFP36L1 to PD-L1 was evaluated by quantitative PCR, western blot, flow cytometry, and immunohistochemistry. Furthermore, we explored its regulatory mechanisms using a combination of molecular biology techniques, including luciferase reporter assay, GST/RNA pull-down, chromatin immunoprecipitation (ChIP)/RIP experiments, and in vivo functional assays. We demonstrated that ZFP36L1, driven by an SE, enhances IFN-γ-induced PD-L1 expression, with SPI1 identified as the specific transcription factor binding to ZFP36L1-SE. Mechanistically, ZFP36L1 binds to the adenylate uridylate-rich element in the 3ʹ untranslated region (3ʹUTR) of HDAC3 mRNA, exacerbating its mRNA decay, and thereby facilitating PD-L1 abnormal transcriptional activation. Collectively, our findings provide mechanistic insights into the role of the SPI1-ZFP36L1-HDAC3-PD-L1 signaling axis in orchestrating immune escape mechanisms in GC, thereby offering valuable insights into the potential targets for immune checkpoint therapy in GC management.

Super-enhancer-driven ZFP36L1 promotes PD-L1 expression in infiltrative gastric cancer.

Gastric cancer (GC) is a major cause of cancer-related mortality worldwide. Despite the widespread recognition of tumor immunotherapy in treating unresectable GC, challenges, including ineffective immunotherapy and drug resistance, persist. Therefore, understanding the regulatory mechanisms of PD-L1, particularly in the context of super-enhancers (SEs) and zinc finger protein 36 ring finger protein-like 1 (ZFP36L1) RNA-binding protein, is crucial. In this study, we performed H3K27ac Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing, investigated the heterogeneity of SEs between two GC subtypes with differential growth patterns, and revealed the immune escape signatures driven by ZFP36L1-SE in infiltrative GC through SEs inhibitors treatment. The regulation of ZFP36L1 to PD-L1 was evaluated by quantitative PCR, western blot, flow cytometry, and immunohistochemistry. Furthermore, we explored its regulatory mechanisms using a combination of molecular biology techniques, including luciferase reporter assay, GST/RNA pull-down, chromatin immunoprecipitation (ChIP)/RIP experiments, and in vivo functional assays. We demonstrated that ZFP36L1, driven by an SE, enhances IFN-γ-induced PD-L1 expression, with SPI1 identified as the specific transcription factor binding to ZFP36L1-SE. Mechanistically, ZFP36L1 binds to the adenylate uridylate-rich element in the 3' untranslated region (3'UTR) of HDAC3 mRNA, exacerbating its mRNA decay, and thereby facilitating PD-L1 abnormal transcriptional activation. Collectively, our findings provide mechanistic insights into the role of the SPI1-ZFP36L1-HDAC3-PD-L1 signaling axis in orchestrating immune escape mechanisms in GC, thereby offering valuable insights into the potential targets for immune checkpoint therapy in GC management.

Copy number amplification-induced overexpression of lncRNA LOC101927668 facilitates colorectal cancer progression by recruiting hnRNPD to disrupt RBM47/p53/p21 signaling

BackgroundSomatic copy number alterations (SCNAs) are pivotal in cancer progression and patient prognosis. Dysregulated long non-coding RNAs (lncRNAs), modulated by SCNAs, significantly impact tumorigenesis, including colorectal cancer (CRC). Nonetheless, the functional significance of lncRNAs induced by SCNAs in CRC remains largely unexplored.MethodsThe dysregulated lncRNA LOC101927668, induced by copy number amplification, was identified through comprehensive bioinformatic analyses utilizing multidimensional data. Subsequent in situ hybridization was employed to ascertain the subcellular localization of LOC101927668, and gain- and loss-of-function experiments were conducted to elucidate its role in CRC progression. The downstream targets and signaling pathway influenced by LOC101927668 were identified and validated through a comprehensive approach, encompassing RNA sequencing, RT-qPCR, Western blot analysis, dual-luciferase reporter assay, evaluation of mRNA and protein degradation, and rescue experiments. Analysis of AU-rich elements (AREs) within the mRNA 3’ untranslated region (UTR) of the downstream target, along with exploration of putative ARE-binding proteins, was conducted. RNA pull-down, mass spectrometry, RNA immunoprecipitation, and dual-luciferase reporter assays were employed to elucidate potential interacting proteins of LOC101927668 and further delineate the regulatory mechanism between LOC101927668 and its downstream target. Moreover, subcutaneous xenograft and orthotopic liver xenograft tumor models were utilized to evaluate the in vivo impact of LOC101927668 on CRC cells and investigate its correlation with downstream targets.ResultsSignificantly overexpressed LOC101927668, driven by chr7p22.3-p14.3 amplification, was markedly correlated with unfavorable clinical outcomes in our CRC patient cohort, as well as in TCGA and GEO datasets. Moreover, we demonstrated that enforced expression of LOC101927668 significantly enhanced cell proliferation, migration, and invasion, while its depletion impeded these processes in a p53-dependent manner. Mechanistically, nucleus-localized LOC101927668 recruited hnRNPD and translocated to the cytoplasm, accelerating the destabilization of RBM47 mRNA, a transcription factor of p53. As a nucleocytoplasmic shuttling protein, hnRNPD mediated RBM47 destabilization by binding to the ARE motif within RBM47 3'UTR, thereby suppressing the p53 signaling pathway and facilitating CRC progression.ConclusionsThe overexpression of LOC101927668, driven by SCNAs, facilitates CRC proliferation and metastasis by recruiting hnRNPD, thus perturbing the RBM47/p53/p21 signaling pathway. These findings underscore the pivotal roles of LOC101927668 and highlight its therapeutic potential in anti-CRC interventions.

The role of the Arabidopsis tandem zinc-finger C3H15 protein in metal homeostasis

Living organisms have developed finely regulated homeostatic networks to mitigate the effects of environmental fluctuations in transition metal micronutrients, including iron, zinc, and copper. In Saccharomyces cerevisiae, the tandem zinc-finger protein Cth2 post-transcriptionally regulates gene expression under conditions of iron deficiency by controlling the levels of mRNAs that code for non-essential ferroproteins. The molecular mechanism involves Cth2 binding to AU-rich elements present in the 3′ untranslated region of target mRNAs, negatively affecting their stability and translation. Arabidopsis thaliana has two TZF proteins homologous to yeast Cth2, C3H14 and C3H15, which participate in cell wall remodelling. The present work examines the expression of representative metal homeostasis genes with putative AREs in plants with altered levels of C3H14 and C3H15 grown under varying metal availabilities. The results suggest that C3H15 may act as a post-transcriptional plant modulator of metal adequacy, as evidenced by the expression of SPL7, the main transcriptional regulator under copper deficiency, and PETE2, which encodes plastocyanin. In contrast to S. cerevisiae, the plant C3H15 affects copper and zinc homeostasis rather than iron. When grown under copper-deficient conditions, adult C3H15OE plants exhibit lower chlorophyll content and photosynthetic efficiency compared to control plants, suggesting accelerated senescence. Likewise, metal content in C3H15OE plants under copper deficiency shows altered mobilization of copper and zinc to seeds. These data suggest that the C3H15 protein plays a role in modulating both cell wall remodelling and metal homeostasis. The interaction between these processes may be the cause of altered metal translocation.

Regulation of Zfp36 by ISGF3 and MK2 restricts the expression of inflammatory cytokines during necroptosis stimulation

Necrosome activation following TLR- or cytokine receptor-signaling results in cell death by necroptosis which is characterized by the rupture of cell membranes and the consequent release of intracellular contents to the extracellular milieu. While necroptosis exacerbates various inflammatory diseases, the mechanisms through which the inflammatory responses are regulated are not clear. We show that the necrosome activation of macrophages results in an upregulation of various pathways, including the mitogen-activated protein kinase (MAPK) cascade, which results in an elevation of the inflammatory response and consequent expression of several cytokines and chemokines. Programming for this upregulation of inflammatory response occurs during the early phase of necrosome activation and proceeds independently of cell death but depends on the activation of the receptor-interacting protein kinase-1 (RipK1). Interestingly, necrosome activation also results in an upregulation of IFNβ, which in turn exerts an inhibitory effect on the maintenance of inflammatory response through the repression of MAPK-signaling and an upregulation of Zfp36. Activation of the interferon-induced gene factor-3 (ISGF3) results in the expression of ZFP36 (TTP), which induces the post-transcriptional degradation of mRNAs of various inflammatory cytokines and chemokines through the recognition of AU-rich elements in their 3’UTR. Furthermore, ZFP-36 inhibits IFNβ-, but not TNFα- induced necroptosis. Overall, these results reveal the molecular mechanism through which IFNβ, a pro-inflammatory cytokine, induces the expression of ZFP-36, which in turn inhibits necroptosis and halts the maintenance of the inflammatory response.

ZFP36L1 and ZFP36L2 reduce cyclin D1 expression by decreasing expression of E2F1 and long 3'UTR isoform of CCND1 transcripts.

The CCND1 mRNA possesses at least two distinct lengths of the 3'-untranslated region (3'UTR), with the long isoform containing multiple AU-rich elements (AREs). The tandem zinc finger (TZF) domains of human ZFP36 family members have the capacity to bind to AREs and promote mRNA degradation. Our previous study demonstrated that mutations in the TZF domain of ZFP36L1 or ZFP36L2 increased the CCND1 expression. In this study, we investigated whether ZFP36L1 and ZFP36L2 could downregulate the expression of the long 3'UTR isoform of CCND1 mRNA in human colorectal cancer (CRC) cells. Firstly, the Gene Expression Profiling Interactive Analysis 2 database indicated downregulation of ZFP36 and ZFP36L1, while E2F1 and CCND1 were upregulated in human CRC tissues compared to normal colorectal tissues. Overexpression of ZFP36L1 and/or ZFP36L2 in T-REx-293, DLD-1, and HCT116 cells led to a decrease in the total CCND1, long isoform ratio of CCND1 mRNA, and E2F1 expression. Conversely, knockdown of ZFP36L1 and ZFP36L2 in HCT116 cells resulted in an increase in total CCND1, long isoform ratio of CCND1 mRNA, and E2F1 expression. Knockdown of E2F1 decreased CCND1 expression, indicating a potential role for E2F1 in regulating CCND1 expression at the transcriptional level. These findings suggest that ZFP36L1 and ZFP36L2 play a negative role in CCND1 expression. The underlying mechanisms might involve the reduction of E2F1 transactivation at the transcriptional level and the promotion of AREs-mediated decay of the long 3'UTR isoform of CCND1 through posttranscriptional processes.

Ribonucleotide reductase small subunit M2 promotes the proliferation of esophageal squamous cell carcinoma cells via HuR-mediated mRNA stabilization

Esophageal squamous cell carcinoma (ESCC), a malignancy of the digestive system, is highly prevalent and the primary cause of cancer-related deaths worldwide due to the lack of early diagnostic biomarkers and effective therapeutic targets. Dysregulated ribonucleotide reductase (RNR) expression has been confirmed to be causally linked to tumorigenesis. This study demonstrated that ribonucleotide reductase small subunit M2 (RRM2) is significantly upregulated in ESCC tissue and that its expression is negatively correlated with clinical outcomes. Mechanistically, HuR promotes RRM2 mRNA stabilization by binding to the adenine/uridine (AU)-rich elements (AREs) within the 3′UTR, resulting in persistent overexpression of RRM2. Furthermore, bifonazole is identified as an inhibitor of HuR via computational screening and molecular docking analysis. Bifonazole disrupts HuR-ARE interactions by competitively binding to HuR at F65, R97, I103, and R153 residues, resulting in reduced RRM2 expression. Furthermore, bifonazole exhibited antitumor effects on ESCC patient-derived xenograft (PDX) models by decreasing RRM2 expression and the dNTP pool. In summary, this study reveals the interaction network among HuR, RRM2, and bifonazole and demonstrated that bifonazole is a potential therapeutic compound for ESCC through inhibition of the HuR/RRM2 axis.

AUF1-mediated inhibition of autophagic lysosomal degradation contributes to CagA stability and Helicobacter pylori-induced inflammation

ABSTRACT CagA, a virulence factor of Helicobacter pylori (H. pylori), is known to drive inflammation in gastric epithelial cells and is typically degraded through autophagy. However, the molecular mechanism by which CagA evades autophagy-mediated degradation remains elusive. This study found that H. pylori inhibits autophagic flux by upregulating the expression of AU-rich element RNA-binding factor 1 (AUF1). We confirmed that AUF1 does not affect autophagy initiation but instead hampers lysosomal clearance, as evidenced by treatments with 3-MA, CQ and BafA1. Upregulated AUF1 stabilizes CagA protein levels by inhibiting the autolysosomal degradation of intracellular CagA in H. pylori-infected gastric epithelial cells. Knocking down AUF1 promotes CagA degradation, an effect that can be reversed by the lysosome inhibitor BafA1 and CQ. Transcriptome analysis of AUF1-knockdown gastric epithelial cells infected with H. pylori indicated that AUF1 regulates the expression of lysosomal-associated hydrolase genes, specifically CTSD, to inhibit autolysosomal degradation. Moreover, we observed that knockdown of AUF1 enhanced the stability of CTSD mRNA and identified AUF1 binding to the 3’UTR region of CTSD mRNA. AUF1-mediated downregulation of CTSD expression contributes to CagA stability, and AUF1 overexpression leads to an increase in CagA levels in exosomes, thus promoting extracellular inflammation. In clinical gastric mucosa, the expression of AUF1 and its cytoplasmic translocation are associated with H. pylori-associated gastritis, with CagA being necessary for the translocation of AUF1 into the cytoplasm. Our findings suggest that AUF1 is a novel host-positive regulator of CagA, and dysregulation of AUF1 expression increases the risk of H. pylori-associated gastritis.

HuR facilitates miR-93-5p-induced activation of MAP3K2 translation via MAP3K2 3′UTR ARE2 in hepatocellular carcinoma

MicroRNAs (miRNAs) can positively regulate gene expression through an unconventional RNA activation mechanism involving direct targeting 3′ untranslated regions (UTRs). Our prior study found miR-93-5p activates mitogen-activated protein kinase kinase kinase 2 (MAP3K2) in hepatocellular carcinoma (HCC) via its 3′UTR. However, the underlying mechanism remains elusive. Here, we identified two candidate AU-rich element (ARE) motifs (ARE1 and ARE2) adjacent to the miR-93-5p binding site located within the MAP3K2 3′UTR using AREsite2. Luciferase reporter and translation assays validated that only ARE2 participated in MAP3K2 activation. Integrative analysis revealed that human antigen R (HuR), an ARE2-associated RNA-binding protein (RBP), physically and functionally interacted with the MAP3K2 3′UTR. Consequently, an HuR-ARE2 complex was shown to facilitate miR-93-5p-mediated upregulation of MAP3K2 expression. Furthermore, bioinformatics analysis and studies of HCC cells and specimens highlighted an oncogenic role for HuR and positive HuR-MAP3K2 expression correlation. HuR is also an enhancing factor in the positive feedback circuit comprising miR-93-5p, MAP3K2, and c-Jun demonstrated in our prior study. The newly identified HuR-ARE2 involvement enriches the mechanism of miR-93-5p-driven MAP3K2 activation and suggests new therapeutic strategies warranted for exploration in HCC.

A non-canonical mechanism of antiviral activity of Oligoadenylate Synthetase 1

Abstract The translational arrest of protein synthesis is often a cellular response to the virus infection. However, some antiviral proteins continue to be translated through unknown mechanisms during this translational shutdown. Here, we report a mechanism by which antiviral effectors are upregulated during this translational shutdown. We found that interferon (IFN) stimulated gene, Oligoadenylate Synthetase 1 (OAS1), binds AU-rich elements (ARE) of specific mRNA, including IFNβ, prolonging the half-life and continued expression. This increased IFN expression protects from WNV infection in vitro and in vivo via downstream IFNAR signaling. This mechanism is common between human OAS1 and mouse Oas1b, independent of OAS enzyme activity and RNase L. However, human OAS1 inhibits SARS-CoV-2 replication through its canonical enzyme activity via RNase L, thus establishing two different mechanisms of OAS1 antiviral activity. These results establish OAS1 as an ARE-binding protein with a broader non-canonical function that protects IFN expression from translational shutdown.