Investigating the Role of the E3 Ubiquitin Ligase TRIM21 in Regulating the RNA-binding Protein HuR under Heat Shock

Nag, Sharanya (2023) Investigating the Role of the E3 Ubiquitin Ligase TRIM21 in Regulating the RNA-binding Protein HuR under Heat Shock. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Sharanya Nag (15IP013)) 15IP013.pdf - Submitted Version Restricted to Repository staff only Download (6MB)

Abstract

Cellular stress responses call for rapid and selective gene expression under conditions of global translation inhibition. Post-transcriptional gene regulation by RNA-binding proteins is of paramount importance in cellular stress responses. RNA-binding protein HuR is a pivotal regulator of mRNA stability and/or translation in response to stress stimuli. It has been earlier reported that HuR is subjected to proteasomal degradation following polyubiquitination at Lys-182 (K182) residue by E3 ligase TRIM21 in response to UVC irradiation. Also, HuR is degraded in response to heat stress consequent to the ubiquitination of the K182 residue. We found that HuR was proteasomaly degraded following polyubiquitination at K182 by TRIM21 under heat shock. Further, TRIM21 was found to enhance cell viability and adaptation to heat shock, which was imparted at least partly by its ability to degrade HuR. A combined approach of MD simulation and systematic experimentation led us to identify two key amino acid residues, namely S100 and E101, in the linker region of HuR that acts as molecular determinants of recognition of HuR by TRIM21 under heat shock. We found that HuR S100A/E101A mutant retained the ability to bind with and stabilize HuR target mRNAs which were otherwise destabilized in heat-stressed cells upon HuR degradation. S100 in HuR was found to be phosphorylated in response to heat shock. HuR S100 phosphorylation acted as a pre-requisite for TRIM21 binding to HuR and its subsequent degradation. AKT1 was found to be activated under heat shock and directly bind to HuR causing S100 phosphorylation. This allowed the recognition of HuR by TRIM21. Therefore, sequential phosphorylation by AKT1 at S100 and ubiquitination by TRIM21 at K182 determine a novel “phosphodegron” in HuR that is required for regulating the cellular level of HuR under heat shock, thereby enabling a crucial adaptive mechanism allowing cell survival in response to heat stress.

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