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The complex interplay of RNA-binding proteins and RISC in neurons
The complex interplay of RNA-binding proteins and RISC in neurons
An extensive network of RNA-binding proteins (RBPs) is at the center of posttranscriptional gene regulation. Importantly, different RBPs – including microRNA-loaded Argonaute (Ago) – can bind to a single mRNA resulting in antagonistic or cooperative mode of actions, thereby determining the fate and function of an mRNA. Here, I investigated the impact of two different RBPs, HuR and Staufen2 (Stau2), on microRNA/Ago-dependent gene expression homeostasis. The results of my thesis allow me to present a working model how these three RBPs might control neuronal function in a novel RNA-structure dependent manner. HuR protein binds to AU-rich elements within mRNAs. In the case of Regulator of G-protein signaling (Rgs4) mRNA, I find that HuR binding occurs close to a miR-26/RISC binding site, resulting in Rgs4 destabilization. As both binding sites are in close proximity within a predicted RNA hairpin structure, only synergistic action of HuR and miR-26 results in Rgs4 repression. I propose a novel mechanism involving the trifold combination of HuR, miR-26-loaded Ago and RNA secondary structure in governing functional regulation of Rgs4 mRNA in neurons. Certain RBPs such as Stau2 protein bind to double-stranded RNAs (dsRNAs), thereby shaping local and global secondary structures of mRNAs. Based on preliminary data linking Stau2 and the miRNA pathway, I investigated Stau2-dependent expression, localization and function of the miRNA-induced silencing complex (RISC) in neurons. Proteome and small RNA transcriptome analysis in Stau2 deficient primary neurons revealed significant upregulation of several RISC associated proteins, including Ago1/2, while global miRNA levels were unaffected. This upregulation was accompanied by decreased global translation and translocation of Ago2 from Processing-bodies, sites of mRNA storage, to translating polysomes. Phenotypically, depletion of Ago1/2 reduced dendritic branching. This effect could be rescued by simultaneous knockdown of Stau2, suggesting that Ago1/2 and Stau2 functionally counterbalance each other in neurons. I hypothesize that Stau2’s ability to bind to dsRNA stabilizes defined mRNA structures thereby governing association of RISC and mRNAs. Based on Stau2 hiCLIP experiments by our collaborator Jernej Ule, I was able to define a long-range RNA duplex in the 3’-untranslated region of Rgs4 mRNA bound by Stau2 in vivo. This RNA duplex is necessary and sufficient to drive Stau2-dependent ribonucleoprotein particle (RNP) assembly as well as dendritic RNA localization in neurons. Together, the data presented in my thesis support a model, in which balanced expression and interdependent action of RBPs, RISC and RNA structure shapes RNP assembly and gene expression homeostasis, important for neuronal function.
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Ehses, Janina
2021
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Ehses, Janina (2021): The complex interplay of RNA-binding proteins and RISC in neurons. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

An extensive network of RNA-binding proteins (RBPs) is at the center of posttranscriptional gene regulation. Importantly, different RBPs – including microRNA-loaded Argonaute (Ago) – can bind to a single mRNA resulting in antagonistic or cooperative mode of actions, thereby determining the fate and function of an mRNA. Here, I investigated the impact of two different RBPs, HuR and Staufen2 (Stau2), on microRNA/Ago-dependent gene expression homeostasis. The results of my thesis allow me to present a working model how these three RBPs might control neuronal function in a novel RNA-structure dependent manner. HuR protein binds to AU-rich elements within mRNAs. In the case of Regulator of G-protein signaling (Rgs4) mRNA, I find that HuR binding occurs close to a miR-26/RISC binding site, resulting in Rgs4 destabilization. As both binding sites are in close proximity within a predicted RNA hairpin structure, only synergistic action of HuR and miR-26 results in Rgs4 repression. I propose a novel mechanism involving the trifold combination of HuR, miR-26-loaded Ago and RNA secondary structure in governing functional regulation of Rgs4 mRNA in neurons. Certain RBPs such as Stau2 protein bind to double-stranded RNAs (dsRNAs), thereby shaping local and global secondary structures of mRNAs. Based on preliminary data linking Stau2 and the miRNA pathway, I investigated Stau2-dependent expression, localization and function of the miRNA-induced silencing complex (RISC) in neurons. Proteome and small RNA transcriptome analysis in Stau2 deficient primary neurons revealed significant upregulation of several RISC associated proteins, including Ago1/2, while global miRNA levels were unaffected. This upregulation was accompanied by decreased global translation and translocation of Ago2 from Processing-bodies, sites of mRNA storage, to translating polysomes. Phenotypically, depletion of Ago1/2 reduced dendritic branching. This effect could be rescued by simultaneous knockdown of Stau2, suggesting that Ago1/2 and Stau2 functionally counterbalance each other in neurons. I hypothesize that Stau2’s ability to bind to dsRNA stabilizes defined mRNA structures thereby governing association of RISC and mRNAs. Based on Stau2 hiCLIP experiments by our collaborator Jernej Ule, I was able to define a long-range RNA duplex in the 3’-untranslated region of Rgs4 mRNA bound by Stau2 in vivo. This RNA duplex is necessary and sufficient to drive Stau2-dependent ribonucleoprotein particle (RNP) assembly as well as dendritic RNA localization in neurons. Together, the data presented in my thesis support a model, in which balanced expression and interdependent action of RBPs, RISC and RNA structure shapes RNP assembly and gene expression homeostasis, important for neuronal function.