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:The 3’ untranslated regions (3’UTRs) of mRNAs play a critical role in controlling gene expression and function because they contain binding sites for microRNAs and RNA binding proteins (RBPs) that alter mRNA stability, localization, and translation. Most mRNA 3’ ends contain multiple polyadenylation sites (PAS) that can be utilized in condition-specific manners, a process known as alternative polyadenylation (APA), however the mechanisms driving the regulation of APA remain poorly characterized. By integrating a large set of over 500 RNA binding protein (RBP) depletion and binding experiments across two cell lines generated by the ENCODE consortium, we uncovered a number of RBPs in each cell type whose depletion leads to widespread alteration of 3’ UTR patterns. These include not only known regulators of APA, but also many putative novel regulators of 3’UTR isoform expression. We focused analysis on the largely unstudied DEAD box RNA helicase, DDX55, and validate its novel role in 3’UTR isoform regulation using molecular assays and targeted 3’ end sequencing experiments (QuantSeq REV V2, Lexogen). Our findings identify DDX55 as a new regulator of APA, particularly at PAS that contain features of RNA secondary structure. Our data also suggest additional previously unrecognized regulators of 3’ UTR processing and differential stability.
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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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