CircZKSCAN1 suppresses stemness in hepatocellular carcinoma (HCC) by competing against RNA-binding protein FMRP
ABSTRACT: We found that Quaking5 protein, a RNA splicing protein was downregulated in HCC. Collectively, this study uncovers the mechanisms underlying the regulating role of circZKSCAN1 in HCC cancer stem cells and reveals that the new discovered Qki5-circZKSCAN1-FMRP-CCAR1-Wnt signaling axis is of important therapeutic value in improving the treatment of HCC. Overall design: identify the regulating role of circZKSCAN1 in HCC
Project description:Circular RNA (circRNA) possesses great pre-clinical diagnostic and therapeutic potentials in multiple cancers. It has been reported playing roles in multiple malignant behaviors including proliferation, migration, metastasis and chemoresistance. However, the underlying correlation between circRNAs and cancer stem cells (CSCs) has not been reported yet. Methods: circZKSCAN1 level was detected in HCC tissue microarrays to clarify its prognostic values. Gain and loss function experiments were applied to investigate the role of circZKSCAN1 in HCC stemness. Bioinformatic analysis was used to predict the possible downstream RNA binding protein and further RNA immunoprecipitation sequencing was carried out to identify the RBP-regulated genes. Results: The absence of circZKSCAN1 endowed several malignant properties including cancer stemness and tightly correlated with worse overall and recurrence-free survival rate in HCC. Bioinformatics analysis and RNA immunoprecipitation-sequencing (RIP-seq) results revealed that circZKSCAN1 exerted its inhibitive role by competitively binding FMRP, therefore, block the binding between FMRP and ?-catenin-binding protein-cell cycle and apoptosis regulator 1 (CCAR1) mRNA, and subsequently restrain the transcriptional activity of Wnt signaling. In addition, RNA-splicing protein Quaking 5 was found downregulated in HCC tissues and responsible for the reduction of circZKSCAN1. Conclusion: Collectively, this study revealed the mechanisms underlying the regulatory role of circZKSCAN1 in HCC CSCs and identified the newly discovered Qki5-circZKSCAN1-FMRP-CCAR1-Wnt signaling axis as a potentially important therapeutic target for HCC treatment.
Project description:Amyloid precursor protein (APP) regulates neuronal synapse function, and its cleavage product Abeta is linked to Alzheimer's disease. Here, we present evidence that the RNA-binding proteins (RBPs) heterogeneous nuclear ribonucleoprotein (hnRNP) C and fragile X mental retardation protein (FMRP) associate with the same APP mRNA coding region element, and they influence APP translation competitively and in opposite directions. Silencing hnRNP C increased FMRP binding to APP mRNA and repressed APP translation, whereas silencing FMRP enhanced hnRNP C binding and promoted translation. Repression of APP translation was linked to colocalization of FMRP and tagged APP RNA within processing bodies; this colocalization was abrogated by hnRNP C overexpression or FMRP silencing. Our findings indicate that FMRP represses translation by recruiting APP mRNA to processing bodies, whereas hnRNP C promotes APP translation by displacing FMRP, thereby relieving the translational block.
Project description:Matrix stiffness as an important physical attribute of extracellular matrix exerts significant impacts on biological behaviors of cancer cells such as growth, proliferation, motility, metabolism and invasion. However, its influence on cancer stemness still remains elusive. Here, we explore whether matrix stiffness-mediated effects on stemness characteristics occur in HCC cells. As the substrate stiffness increased, HCC cells exhibited high proportion of cells with CD133(+)/EpCAM(+), high expression levels of CD133, EpCAM, Nanog and SOX2, greater self-renewing ability and oxaliplatin resistance. Simultaneously, their phosphorylation levels of Akt and mTOR, as well as p-4E-BP and SOX2 expressions were also obviously upregulated. Conversely, knockdown of integrin ?1 partially attenuated higher stiffness-mediated stemness characteristics in HCC cells, and reversed the phosphorylation levels of Akt and mTOR, and expressions of p-4E-BP and SOX2, suggesting that integrin ?1 may deliver higher stiffness signal into HCC cells and activate mTOR signaling pathway. Additionally, mTOR inhibitor suppressed the mTOR phosphorylation level and expression levels of p-4E-BP and SOX2 in HCC cells grown on higher stiffness substrate, as well as depressed their stemness properties significantly, favoring a regulating role of mTOR signaling pathway in matrix stiffness-mediated effects on stemness. In summary, matrix stiffness may be involved in the process of stemness regulation via activating integrin ?1/Akt/mTOR/SOX2 signaling pathway. To the best of our knowledge, this study first reveals a novel regulating pathway to direct the stemness characteristics in HCC cells.
Project description:N6-methyladenosine (m6A) modification of mRNA is emerging as a vital mechanism regulating RNA function. Here, we show that fragile X mental retardation protein (FMRP) reads m6A to promote nuclear export of methylated mRNA targets during neural differentiation. Fmr1 knockout (KO) mice show delayed neural progenitor cell cycle progression and extended maintenance of proliferating neural progenitors into postnatal stages, phenocopying methyltransferase Mettl14 conditional KO (cKO) mice that have no m6A modification. RNA-seq and m6A-seq reveal that both Mettl14cKO and Fmr1KO lead to the nuclear retention of m6A-modified FMRP target mRNAs regulating neural differentiation, indicating that both m6A and FMRP are required for the nuclear export of methylated target mRNAs. FMRP preferentially binds m6A-modified RNAs to facilitate their nuclear export through CRM1. The nuclear retention defect can be mitigated by wild-type but not nuclear export-deficient FMRP, establishing a critical role for FMRP in mediating m6A-dependent mRNA nuclear export during neural differentiation.
Project description:Outside of Fragile X syndrome (FXS), the role of Fragile-X Mental Retardation Protein (FMRP) in mediating neuropsychological abnormalities is not clear. FMRP, p70-S6 kinase (S6K) and protein phosphatase 2A (PP2A) are thought to cooperate as a dynamic signaling complex. In our prior work, adult rats have enhanced CA1 hippocampal long-term depression (LTD) following an early life seizure (ELS). We now show that mGluR-mediated LTD (mLTD) is specifically enhanced following ELS, similar to FMRP knock-outs. Total FMRP expression is unchanged but S6K is hyperphosphorylated, consistent with S6K overactivation. We postulated that either disruption of the FMRP-S6K-PP2A complex and/or removal of this complex from synapses could explain our findings. Using subcellular fractionation, we were surprised to find that concentrations of FMRP and PP2A were undisturbed in the synaptosomal compartment but reduced in parallel in the cytosolic compartment. Following ELS FMRP phosphorylation was reduced in the cytosolic compartment and increased in the synaptic compartment, in parallel with the compartmentalization of S6K activation. Furthermore, FMRP and PP2A remain bound following ELS. In contrast, the interaction of S6K with FMRP is reduced by ELS. Blockade of PP2A results in enhanced mLTD; this is occluded by ELS. This suggests a critical role for the location and function of the FMRP-S6K-PP2A signaling complex in limiting the amount of mLTD. Specifically, non-synaptic targeting and the function of the complex may influence the "set-point" for regulating mLTD. Consistent with this, striatal-enriched protein tyrosine phosphatase (STEP), an FMRP "target" which regulates mLTD expression, is specifically increased in the synaptosomal compartment following ELS. Further, we provide behavioral data to suggest that FMRP complex dysfunction may underlie altered socialization, a symptom associated and observed in other rodent models of autism, including FXS.
Project description:The fragile X mental retardation protein FMRP regulates translation of its bound mRNAs through incompletely defined mechanisms. FMRP has been linked to the microRNA pathway, and we show here that it associates with the RNA helicase MOV10, also associated with the microRNA pathway. FMRP associates with MOV10 directly and in an RNA-dependent manner and facilitates MOV10's association with RNAs in brain and cells, suggesting a cooperative interaction. We identified the RNAs recognized by MOV10 using RNA immunoprecipitation and iCLIP. Examination of the fate of MOV10 on RNAs revealed a dual function for MOV10 in regulating translation: it facilitates microRNA-mediated translation of some RNAs, but it also increases expression of other RNAs by preventing AGO2 function. The latter subset was also bound by FMRP in close proximity to the MOV10 binding site, suggesting that FMRP prevents MOV10-mediated microRNA suppression. We have identified a mechanism for FMRP-mediated translational regulation through its association with MOV10.
Project description:Loss of the Fragile X mental retardation protein (FMRP) is associated with presumed postsynaptic deficits in mouse models of Fragile X syndrome. However, the possible presynaptic roles of FMRP in learning-related plasticity have received little attention. As a result, the mechanisms whereby FMRP influences synaptic function remain poorly understood. To investigate the cellular locus of the effects of FMRP on synaptic plasticity, we cloned the Aplysia homolog of FMRP and find it to be highly expressed in neurons. By selectively down-regulating FMRP in individual Aplysia neurons at the sensory-to-motor neuron synapse reconstituted in co-cultures, we demonstrate that FMRP functions both pre- and postsynaptically to constrain the expression of long-term synaptic depression induced by repeated pulses of FMRF-amide. In contrast, FMRP has little to no effect on long-term synaptic facilitation induced by repeated pulses of serotonin. Since other components of signaling pathways involved in plasticity appear to be conserved between Aplysia and mammalian neurons, our findings suggest that FMRP can participate in both pre- and postsynaptic regulation of enduring synaptic plasticity that underlies the storage of certain types of long-term memory.
Project description:The fragile X mental retardation protein (FMRP) is an mRNA-binding regulator of protein translation that associates with 4-6% of brain transcripts and is central to neurodevelopment. Autism risk genes' transcripts are overrepresented among FMRP-binding mRNAs, and FMRP loss-of-function mutations are responsible for fragile X syndrome, the most common cause of monogenetic autism. It is thought that FMRP-dependent translational repression is governed by the phosphorylation of serine residue 499 (S499). However, recent evidence suggests that S499 phosphorylation is not modulated by metabotropic glutamate receptor class I (mGluR-I) or protein phosphatase 2A (PP2A), two molecules shown to regulate FMRP translational repression. Moreover, the mammalian FMRP S499 kinase remains unknown. We found that casein kinase II (CK2) phosphorylates murine FMRP S499. Further, we show that phosphorylation of FMRP S499 permits phosphorylation of additional, nearby residues. Evidence suggests that these nearby residues are modulated by mGluR-I and PP2A pathways. These data support an alternative phosphodynamic model of FMRP that is harmonious with prior studies and serves as a framework for further investigation.
Project description:Fragile X syndrome is a common form of cognitive deficit caused by the functional absence of fragile X mental retardation protein (FMRP), a dendritic RNA-binding protein that represses translation of specific messages. Although FMRP is phosphorylated in a group I metabotropic glutamate receptor (mGluR) activity-dependent manner following brief protein phosphatase 2A (PP2A)-mediated dephosphorylation, the kinase regulating FMRP function in neuronal protein synthesis is unclear. Here we identify ribosomal protein S6 kinase (S6K1) as a major FMRP kinase in the mouse hippocampus, finding that activity-dependent phosphorylation of FMRP by S6K1 requires signaling inputs from mammalian target of rapamycin (mTOR), ERK1/2, and PP2A. Further, the loss of hippocampal S6K1 and the subsequent absence of phospho-FMRP mimic FMRP loss in the increased expression of SAPAP3, a synapse-associated FMRP target mRNA. Together these data reveal a S6K1-PP2A signaling module regulating FMRP function and place FMRP phosphorylation in the mGluR-triggered signaling cascade required for protein-synthesis-dependent synaptic plasticity.