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Characterizing functional domains of the RNA helicase RHAU involved in subcellular localization and RNA interaction

Chalupníková, Katerina. Characterizing functional domains of the RNA helicase RHAU involved in subcellular localization and RNA interaction. 2008, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_8509

Abstract

Posttranscriptional regulation of gene expression is an important and highly regulated process in response to developmental, environmental and metabolic signals. During stress conditions such as heat shock (HS), oxidative stress, ischemia or viral infection, the translation machinery of cells is reprogrammed. The majority of actively translated mRNAs is released from polysomes and driven to specific cytoplasmic foci called stress granules (SGs), where dynamic changes in proteinRNA interaction determine the subsequent fate of mRNAs. In the presented thesis, I show that the DEAH-box RNA helicase RHAU is a novel SG-associated protein and that its N-terminus is necessary and sufficient for localization of RHAU in SGs. While RHAU protein was originally identified as an ARE-associated protein involved in uPA mRNA decay, it was not clear whether RHAU directly interacts with RNA. Here, I demonstrate that RHAU physically interacts with RNA in vitro and in vivo through the N-terminus. Bioinformatic analysis of the RHAU protein sequence corroborates the experimental data, revealing that the N-terminus of RHAU harbors a unique RNA-binding domain consisting of two abutting motifs: the G-rich region containing one RGG-box and the RHAU specific motif (RSM). It is widely believed that substrate specificity and subcellular localization of RNA helicases is mediated by their less conserved flanked N-/Cterminal domains. As the unique N-terminus of RHAU is essential and sufficient for both subcellular localization and RNA interaction, it most probably determines a functional specificity of RHAU. I further show that ATPase activity is responsible for the apparent instability of RHAU-RNA complex formation and markedly influences the kinetics of RHAU retention in SGs. The striking difference in SG shuttling kinetics between fully active RHAU protein and its ATPase-deficient mutant triggers the hypothesis that its ATPase activity takes part in energy dependent dynamic remodeling of RNPs in SGs. In summary, the results presented in this thesis demonstrate that after rck/p54, DDX3 and eIF4A, RHAU is the fourth RNA helicase detected in SGs and that its association with SGs is dynamic and mediated by a RHAU-specific RNAbinding domain. Additionally, I could show that RHAU is an essential factor for P-body (PB) formation and obtained initial data that RHAU is possibly also involved in the process of translation via its interaction with translation initiation factor eIF3b.
Advisors:Nagamine, Yoshikuni
Committee Members:Stöcklin, Georg Emanuel
Faculties and Departments:09 Associated Institutions > Friedrich Miescher Institut FMI
Item Type:Thesis
Thesis no:8509
Bibsysno:Link to catalogue
Number of Pages:95
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:41
Deposited On:13 Feb 2009 16:48

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