Investigating the Regulation and Function of RNA-binding Protein HuR under Genotoxic Stress

Guha, Abhishek (2020) Investigating the Regulation and Function of RNA-binding Protein HuR under Genotoxic Stress. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD theses of Abhishek Guha (11IP04)) 11IP04.pdf - Submitted Version Restricted to Repository staff only Download (3MB)

Abstract

RNA-binding protein HuR is a pivotal regulator of mRNA stability and/or translation in response to stress stimuli. HuR prevents miR-125b-mediated repression of p53 mRNA translation in response to UV-irradiation. HuR showed pulsatile expression post UV irradiation which was opposite in phase to miR-125b expression. We found a putative target site of miR-125b in the HuR mRNA 3’UTR and therefore hypothesized miR-125b as an inhibitor of HuR mRNA translation. We found that miRISC-125b binds to the 3’UTR of HuR and represses its translation. However, treating cells with antagomiR-125b did not cause a linear increase in HuR levels post UV-irradiation. Thus, translation repression of HuR by miR-125b could not solely explain the reduction in HuR observed under UV-irradiation. We found UV-irradiation also triggered polyubiquitination and proteasomal degradation of HuR. Lys-182 was determined as the site of polyubiquitination of HuR under UV-irradiation. Co-immunoprecipitation of HuR post UV, followed by mass spectrometry, identified TRIM21, a novel E3-ubiquitin ligase. Polyubiquitination of HuR post UV-irradiation was reduced upon TRIM21 knockdown and a linear increase in HuR protein in response to UV-irradiation was observed when treated with antagomiR-125b and siRNA against TRIM21. Therefore, the integrated action of miR-125b and TRIM21 gives rise to an intricate regulatory system that controls pulsatile expression of HuR and p53 in response to DNA damage damage. Furthermore, we found that HuR binds to the 3’UTR of TRIM21 and activates its translation. Finally, TRIM21 was found to reverse the decrease in cell proliferation and colony formation caused by HuR overexpression or UV-mediated DNA damage, thereby elucidating a pro-oncogenic property of TRIM21. Together, these observations have provided new insights into the regulatory processes that controls the cell’s response to DNA damage.

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