MicroRNAs binding with 3'UTR of tapasin
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1004588 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR25619068.fastq.gz | Fastqsanger.gz | |||
| SRR25619069.fastq.gz | Fastqsanger.gz | |||
| SRR25619070.fastq.gz | Fastqsanger.gz | |||
| SRR25619071.fastq.gz | Fastqsanger.gz |
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Project description:Background Dysregulation of major histocompatibility complex (MHC) class I antigen processing and presentation machinery (APM) components in the tumor as one main molecular mechanism of immune escape leading to deactivation of T cell immune surveillance could be due to post-transcriptional regulation via immune-modulatory microRNAs (miRNA). It is now well established from a variety of studies that several miRNAs could effectively modulate the expression of some MHC class I APM components in tumors. Tapasin is an important APM molecule involved in the association of MHC class I with transporter associated with antigen processing (TAP) and peptide loading. Since so far no detailed investigation of the posttranscriptional regulation of tapasin exists, the aim of this study is to identify and functionally characterize miRNAs targeting tapasin in melanoma. Methods Using miRNA trapping by RNA in vitro affinity purification (miTRAP) and in silico as well as small RNA sequencing, miRNAs will be identified, which bind to the 3'untranslated region (3' UTR) of tapasin. Dual luciferase assays will be performed to determine binding of the miRNA. In silico analysis was performed to predict the effect of miRNAs on the survival of melanoma patients in correlation to tapasin. RT-qPCR, Western blot, flow cytometry and other functional assays were performed after transfecting miRNA mimics in three melanoma cell lines. Results Using the combination strategy of miTRAP and RNA seq we identified miR-155-5p to bind to the 3’UTR of tapasin, which was further confirmed by in silico analysis and dual luciferase reporter assay. Transfection of miR-155-5p mimics demonstrated that miR-155-5p upregulate tapasin protein level, which was accompanied by an upregulation of the MHC class I (HLA-ABC) surface expression. Simultaneously, in several different types of cancer, including melanoma, the expression of miR-155-5p is significantly positively correlated with the patient's survival and HLA-A protein. Conclusion Our data revealed for the first time a positive role of miR-155-5p in the posttranscriptional regulation of tapasin in melanoma and provide further insights into the miR-155-5p-mediated induction of HLA-ABC surface expression. This might lead to a better T cell response, avoidance tumor cell escape, improvement of patients' survival and thus might be a potential therapeutic target.
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Project description:Using affinity purification for the detection of RNA-binding proteins in tapasin 3'UTR
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Project description:Nrf2 transcript factor plays an important role in cellular defense against oxidative stress due to its control for the expression of antioxidant and detoxification genes. We have found that Nrf2 undergoes de novo protein translation when mammalian cells encounter oxidative stress. Here, we report the discovery of DHX9 as a binding protein for Nrf2 5’UTR. H2O2 treatment causes dose- or time- dependent increases of DHX9 binding to Nrf2 5’UTR, in parallel with elevation of Nrf2 protein. Inhibiting DHX9 expression with siRNA or its activity with YK-4-279 blocked H2O2 from inducing Nrf2 mRNA recruitment to the ribosomes or Nrf2 protein elevation. As a nuclear protein, DHX9 was found to increase its abundance in the cytosol with oxidative stress. An increase of DHX9 was detected in total ribosomal collections in cells treated with H2O2, most significantly with 100 uM H2O2, and at 60 mins. Ribosomal fractionation revealed an increase of DHX9 protein at 43/48S and 80S fractions in H2O2 treated cells. H2O2 treatment caused an RNA dependent increase of DHX9 interaction with eIF3 The binding of DHX9 to Nrf2 5’UTR probe was enhanced by H2O2 treated cells or by deducting the length of Nrf2 5’UTR, suggesting a negative element normally prohibiting DHX9 binding to Nrf2 5’UTR. RNase digestion enhanced DHX9 association with the ribosomes. Our data have revealed a novel mechanism of de novo Nrf2 protein translation under oxidative stress involving DHX9 binding to Nrf2 5’UTR, perhaps via removal of a negative RNA element, to recruit 43S pre-initiation complex for translation initiation.
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Project description:The 3' untranslated region (3'UTR) of mRNA plays several important roles in post-transcriptional gene regulation. Some of its functions include regulating mRNA stability by polyadenylation and microRNAs. However, the overall function of the Gata1 3’UTR in mammals has not been defined. In this study, we used CRISPR/CAS9 technology to knock out the sequence of the mouse Gata1 3’UTR. We found a defect in erythropoiesis in mutant mice, evidenced by macrocytic anemia at the baseline. Ablation of Gata1 3’UTR also resulted in a reduced number of erythroid precursors that might be associated with the cell cycle, especially G2/M disruption in fetal livers. Mechanistically, deletion of the Gata1 3’UTR destabilizes the Gata1 mRNA and ultimately reduces Gata1 protein levels. The low stability of the Gata1 mRNA is unlikely to be caused by the loss of binding of microRNAs or lack of polyadenylation; rather, in part, by the dissociation of AU-rich elements in the 3’UTR with a trans-activating factor Elavl1. Specifically, we transcribed Gata1 3’UTR in vitro and performed an RNA pulldown assay followed by mass spectrometry to profile the proteins that bind the 3’UTR. Gene Ontology analysis demonstrated that several proteins specifically targeting the 3’UTR were found to potentially bind Gata1 3’UTR, among which Elavl1 was in almost all categories related to mRNA stabilization. Western blotting and RNA immunoprecipitation confirmed the direct interaction of Gata1 3’UTR with Elavl1. Manipulation of Elavl1 activity and protein levels by the small molecule inhibitor Dihydro-tanshinone-I and Elavl1 overexpression in fetal liver erythroblasts confirmed Elavl1 as a stabilizing factor for Gata1 mRNA. Our findings shed light on the functional significance of the 3’UTR of Gata1 mRNA in the context of erythroid development. More importantly, our findings highlight the complexity and diversity of regulatory mechanisms that govern Gata1 mRNA stability and precise expression at the post-transcriptional levels. In addition, our findings prove that miRNAs are not always essential in vivo for controlling transcription factor levels to maintain body homeostasis.
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