Project description:P63, a p53 family tumor suppressor, is involved in many cellular processes, including growth suppression and differentiation. Thus, p63 activity needs to be tightly controlled. Here, we found that RNPC1, a RNA-binding protein and a target of the p53 family, regulates p63 mRNA stability and consequently p63 activity. Specifically, we showed that overexpression of RNPC1 decreases, whereas knockdown of RNPC1 increases, the half-life of p63 transcript, which leads to altered p63 expression. Consistent with this, we showed that RNPC1 binds the AU-/U-rich elements in p63 3' UTR in vitro and in vivo and the RRM domain in RNPC1 is required for binding, and regulating the stability of, p63 transcript. Furthermore, we showed that RNPC1 promotes keratinocyte differentiation by repressing p63 expression. Together, we uncovered a previously undetected mechanism by which p63 expression is regulated via mRNA stability and a novel regulatory feedback loop between RNPC1 and p63.
Project description:Regulator of G protein signaling 4 (RGS4) regulates the strength and duration of G protein signaling and plays an important role in smooth muscle contraction, cardiac development, and psychiatric disorders. Little is known about the posttranscriptional regulation of RGS4 expression. We cloned the full-length cDNA of rabbit RGS4, which contains a long 3'-untranslated region (UTR) with several AU-rich elements (AREs). We determined whether RGS4 mRNA stability is mediated by the RNA-binding protein human antigen R (HuR) and contributes to IL-1β-induced upregulation of RGS4 expression. We show that IL-1β treatment in colonic smooth muscle cells doubled the half-life of RGS4 mRNA. Addition of RGS4 3'-UTR to the downstream of Renilla luciferase reporter induced dramatic reduction in the enzyme activity and mRNA expression of luciferase, which was attenuated by the site-directed mutation of the two 3'-most ARE sites. IL-1β increased luciferase mRNA stability in a UTR-dependent manner. Knockdown of HuR significantly aggravated UTR-mediated instability of luciferase and inhibited IL-1β-induced upregulation of RGS4 mRNA. In addition, IL-1β increased cytosolic translocation and RGS4 mRNA binding of HuR. These findings suggest that 3'-most ARE sites within RGS4 3'-UTR are essential for the instability of RGS4 mRNA and IL-1β promotes the stability of RGS4 mRNA through HuR.
Project description:Both genetic and microenvironmental detrimental factors are involved in ovarian dysfunction, leading to the increasing rate of involuntary childlessness in recent years. Oxidative stress (OS), which is characterized by the imbalance of redox system with redundant reactive oxygen species (ROS) overwhelming the antioxidant defense, is regarded as one of the culprits of ovarian dysfunction. OS causes damage to various types of ovarian cells including granulosa cells (GCs), jeopardizing the ovarian microenvironment, disturbing follicular development and participating in various female reproductive disorders. However, the specific molecular pathological mechanisms underlying this process have not been fully elucidated. In this study, we found that 3-nitropropionic acid (3-NP) treatment led to significant IGF2BP1 downregulation via, at least partially, inducing ROS overproduction. IGF2BP1 regulates GCs viability, proliferation, cell cycle and cellular senescence by enhancing MDM2 mRNA stability in an m6A-dependant manner. IGF2BP1 overexpression partially rescued 3-NP induced GCs damages, while ectopically expressed MDM2 alleviated both 3-NP or IGF2BP1-knockdown induced GCs dysfunction. These results reveal an epigenetic molecular mechanism underlying OS-related GCs disorders, which may help to establish a novel potential clinical marker for predicting the GCs status as well as the follicular developmental potential.
Project description:BackgroundThe mechanisms underlying the occurrence and development of esophageal squamous cell carcinoma (ESCC) remains to be elucidated. The present study aims to investigate the roles and implications of IGF2BP1 overexpression in ESCC.MethodsIGF2BP1 protein expression in ESCC samples was assessed by immunohistochemistry (IHC), and the mRNA abundance of IGF2BP1 and INHBA was analyzed with TCGA datasets and by RNA in situ hybridization (RISH). The methylation level of the IGF2BP1 promoter region was detected by methylation-specific PCR (MSP-PCR). Cell viability, migration, invasion and in vivo metastasis assays were performed to explore the roles of IGF2BP1 overexpression in ESCC. RNA immunoprecipitation sequencing (RIP-seq) and mass spectrometry were applied to identify the target RNAs and interacting proteins of IGF2BP1, respectively. RIP-PCR, RNA pulldown, immunofluorescence (IF), gene-specific m6A PCR and RNA stability assays were used to uncover the molecular mechanisms underlying the malignant phenotypes of ESCC cells caused by IGF2BP1 dysregulation. BTYNB, a small molecular inhibitor of IGF2BP1, was evaluated for its inhibitory effect on the malignant phenotypes of ESCC cells.ResultsIGF2BP1 overexpression was detected in ESCC tissues and associated with the depth of tumor invasion. In addition, IGF2BP1 mRNA expression in ESCC cells was negatively correlated with the level of its promoter methylation. Knockdown of IGF2BP1 inhibited ESCC cell invasion and migration as well as tumor metastasis. Mechanistically, we observed that IGF2BP1 bound and stabilized INHBA mRNA and then resulted in higher protein expression of INHBA, leading to the activation of Smad2/3 signaling, thus promoting malignant phenotypes. The mRNA level of INHBA was upregulated in ESCC tissues as well. Furthermore, IGF2BP1 interacted with G3BP stress granule assembly factor 1 (G3BP1). Knockdown of G3BP1 also down-regulated the INHBA-Smad2/3 signaling. BTYNB abolished this activated signaling and significantly attenuated the malignant phenotypes of ESCC cells.ConclusionsElevated expression of IGF2BP1 is a frequent event in ESCC tissues and might be a candidate biomarker for the disease. IGF2BP1 overexpression promotes the invasion and migration of ESCC cells by activating the INHBA-Smad2/3 pathway, providing a potential therapeutic target for ESCC patients with high expression of IGF2BP1.
Project description:Posttranscriptional regulation is a major mechanism to rewire transcriptomes during differentiation. Heterogeneous nuclear RNA-binding protein LL (hnRNPLL) is specifically induced in terminally differentiated lymphocytes, including effector T cells and plasma cells. To study the molecular functions of hnRNPLL at a genome-wide level, we identified hnRNPLL RNA targets and binding sites in plasma cells through integrated Photoactivatable-Ribonucleoside-Enhanced Cross-Linking and Immunoprecipitation (PAR-CLIP) and RNA sequencing. hnRNPLL preferentially recognizes CA dinucleotide-containing sequences in introns and 3' untranslated regions (UTRs), promotes exon inclusion or exclusion in a context-dependent manner, and stabilizes mRNA when associated with 3' UTRs. During differentiation of primary B cells to plasma cells, hnRNPLL mediates a genome-wide switch of RNA processing, resulting in loss of B-cell lymphoma 6 (Bcl6) expression and increased Ig production--both hallmarks of plasma-cell maturation. Our data identify previously unknown functions of hnRNPLL in B-cell to plasma-cell differentiation and demonstrate that the RNA-binding protein hnRNPLL has a critical role in tuning transcriptomes of terminally differentiating B lymphocytes.
Project description:The quaking viable (qk(v)) mice represent an animal model of dysmyelination. The absence of expression of the QKI-6 and QKI-7 cytoplasmic isoforms in oligodendrocytes (OLs) during CNS myelination causes the qk(v) mouse phenotype. The QKI RNA-binding proteins are known to regulate RNA metabolism of cell cycle proteins and myelin components in OLs; however, little is known of their role in reorganizing the cytoskeleton or process outgrowth during OL maturation and differentiation. Here, we identify the actin-interacting protein (AIP)-1 mRNA as a target of QKI-6 by using two-dimensional differential gel electrophoresis. The AIP-1 mRNA contains a consensus QKI response element within its 3'-untranslated region that, when bound by QKI-6, decreases the half-life of the AIP-1 mRNA. Although the expression of QKI-6 is known to increase during OL differentiation and CNS myelination, we show that this increase is paralleled with a corresponding decrease in AIP-1 expression in rat brains. Furthermore, qk(v)/qk(v) mice that lack QKI-6 and QKI-7 within its OLs had an increased level of AIP-1 in OLs. Moreover, primary rat OL precursors harboring an AIP-1 small interfering RNA display defects in OL process outgrowth. Our findings suggest that the QKI RNA-binding proteins regulate OL differentiation by modulating the expression of AIP-1.
Project description:Vascular endothelial growth factor (VEGF) is a key angiogenic factor expressed under restricted nutrient and oxygen conditions in most solid tumors. The expression of VEGF under hypoxic conditions requires transcription through activated hypoxia-inducible factor 1 (HIF-1), increased mRNA stability, and facilitated translation. This study identified double-stranded RNA-binding protein 76/NF90 (DRBP76/NF90), a specific isoform of the DRBP family, as a VEGF mRNA-binding protein which plays a key role in VEGF mRNA stability and protein synthesis under hypoxia. The DRBP76/NF90 protein binds to a human VEGF 3' untranslated mRNA stability element. RNA interference targeting the DRBP76/NF90 isoform limited hypoxia-inducible VEGF mRNA and protein expression with no change in HIF-1-dependent transcriptional activity. Stable repression of DRBP76/NF90 in MDA-MB-435 breast cancer cells demonstrated reduced polysome-associated VEGF mRNA levels under hypoxic conditions and reduced mRNA stability. Transient overexpression of the DRBP76/NF90 protein increased both VEGF mRNA and protein levels synthesized under normoxic and hypoxic conditions. Cells with stable repression of the DRBP76/NF90 isoform showed reduced tumorigenic and angiogenic potential in an orthotopic breast tumor model. These data demonstrate that the DRBP76/NF90 isoform facilitates VEGF expression by promoting VEGF mRNA loading onto polysomes and translation under hypoxic conditions, thus promoting breast cancer growth and angiogenesis in vivo.
Project description:Lipids play a critical role in many cellular processes by serving as structural components of cell membranes or functioning as energy fuel and signaling molecules. The RNA-binding proteins RBM24 and RBM38 share an identical RNA-binding domain and thereby, regulate a group of same targets, such as p21. However, it is not certain whether RBM24 and RBM38 participates in lipid homeostasis. Here, lipidomic analysis showed that a deficiency in RBM24 or RBM38 leads to altered lipid metabolism, with more profound alteration by loss of RBM24 in MCF7 cells. We also showed that mice deficient in RBM24 were prone to chronic inflammation and liver steatosis, but not spontaneous tumors. These data let us speculate whether RBM24 regulates ferroptosis, a programmed cell death that links inflammation and liver steatosis via lipid peroxidation. Indeed, we found that over-expression of RBM24 protected, whereas knockout of RBM24 sensitized, cells to Erastin-induced ferroptosis by modulating the mRNA stability of SLC7A11, a ferroptosis inhibitor. Moreover, we showed that knockdown of SLC7A11 reversed the effect of RBM24 on ferroptosis. Together, our study revealed that RBM24 regulates lipid metabolism and SLC7A11 mRNA stability to modulate ferroptosis and inflammatory response.
Project description:The insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1) plays essential roles in embryogenesis and carcinogenesis. IGF2BP1 serves as a post-transcriptional fine-tuner regulating the expression of some essential mRNA targets required for the control of tumor cell proliferation and growth, invasion, and chemo-resistance, associating with a poor overall survival and metastasis in various types of human cancers. Therefore, IGF2BP1 has been traditionally regarded as an oncogene and potential therapeutic target for cancers. Nevertheless, a few studies have also demonstrated its tumor-suppressive role. However, the details about the contradictory functions of IGF2BP1 are unclear. The growing numbers of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) have been identified as its direct regulators, during tumor cell proliferation, growth, and invasion in multiple cancers. Thus, the mechanisms of post-transcriptional modulation of gene expression mediated by IGF2BP1, miRNAs, and lncRNAs in determining the fate of the development of tissues and organs, as well as tumorigenesis, need to be elucidated. In this review, we summarized the tissue distribution, expression, and roles of IGF2BP1 in embryogenesis and tumorigenesis, and focused on modulation of the interconnectivity between IGF2BP1 and its targeted mRNAs or non-coding RNAs (ncRNAs). The potential use of inhibitors of IGF2BP1 and its related pathways in cancer therapy was also discussed.