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Role of RNA binding protein HuR in antagonizing the microRNA-mediated repression

Kundu, Pradipta. Role of RNA binding protein HuR in antagonizing the microRNA-mediated repression. 2011, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_9709

Abstract

MicroRNAs (miRNAs) are ~21-nt-long non-coding RNAs regulating gene expression in eukaryotes. In metazoa, miRNAs control gene expression by base-pairing to target mRNAs, bringing about their translational repression and/or deadenylation resulting in mRNA degradation. MiRNA-mediated translational repression is a reversible process in mammalian cells. It was previously demonstrated that target mRNAs containing AU-rich regulatory elements (AREs) in the 3’UTR, can be relieved from miRNA repression in human hepatoma Huh7 or HeLa cells in response to different forms of cellular stress. The derepression required binding of the ELAV family protein HuR to the mRNA 3’UTR (Bhattacharyya et al., Cell 125, 111-1124, 2006). However, whether stress-induced factors other than HuR participate in the process and the actual mechanism of HuR action remain unknown.
In the present study, we have addressed these questions, using different cell-based and in vitro assays. Using mutants of HuR accumulating in the cytoplasm in the absence of stress and tumor cell lines constitutively accumulating endogenous HuR in the cytoplasm, we were able to uncouple the HuR effect on miRNA repression from stress. We also found that Ago2 and HuR do not interact with each other and that their binding to target mRNA appears to be largely mutually exclusive. Using an in vitro system with purified miRISC and recombinant HuR and its mutants, we demonstrate that HuR, by oligomerizing along RNA, leads to displacement of miRISC from RNA, even when miRISC is positioned at a distance from the primary HuR-binding site. Further, we show that HuR association with AREs can inhibit RISC-mediated endonucleolytic cleavage of target RNAs both in vivo and in vitro, and also miRNA-mediated deadenylation of RNA in the Krebs-2 ascites extract.
Advisors:Filipowicz, Witold
Committee Members:Muehlemann, Oliver
Faculties and Departments:09 Associated Institutions > Friedrich Miescher Institut FMI
Item Type:Thesis
Thesis no:9709
Bibsysno:Link to catalogue
Number of Pages:105 S.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:42
Deposited On:07 Feb 2012 13:21

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