The RNA-binding protein HuR is a novel target of Pirh2 E3 ubiquitin ligase

Alexandra Daks,Elizaveta Tananykina,Olga Fedorova,Nickolai Barlev,Andrew Bottrill,Alena Kizenko,Oleg Shuvalov,Oleg Semenov,Elena Vasileva,Alexey Petukhov

doi:10.1038/s41419-021-03871-w

Abstract

The RING-finger protein Pirh2 is a p53 family-specific E3 ubiquitin ligase. Pirh2 also ubiquitinates several other important cellular factors and is involved in carcinogenesis. However, its functional role in other cellular processes is poorly understood. To address this question, we performed a proteomic search for novel interacting partners of Pirh2. Using the GST-pulldown approach combined with LC-MS/MS, we revealed 225 proteins that interacted with Pirh2. We found that, according to the GO description, a large group of Pirh2-associated proteins belonged to the RNA metabolism group. Importantly, one of the identified proteins from that group was an RNA-binding protein ELAVL1 (HuR), which is involved in the regulation of splicing and protein stability of several oncogenic proteins. We demonstrated that Pirh2 ubiquitinated the HuR protein facilitating its proteasome-mediated degradation in cells. Importantly, the Pirh2-mediated degradation of HuR occurred in response to heat shock, thereby affecting the survival rate of HeLa cells under elevated temperature. Functionally, Pirh2-mediated degradation of HuR augmented the level of c-Myc expression, whose RNA level is otherwise attenuated by HuR. Taken together, our data indicate that HuR is a new target of Pirh2 and this functional interaction contributes to the heat-shock response of cancer cells affecting their survival.

Highlights

Pirh[2] (p53-induced RING-H2 protein) was first described as an androgen receptor N-terminal interacting protein in 20021
Identification of novel Pirh2-interacting proteins To identify the proteins that interact with Pirh[2] and, may represent its potential targets for ubiquitination, we used the GST-pulldown approach combined with high-resolution mass spectrometry (MS) (Fig. 1)
The MS analysis has yielded 346 interacting proteins in total: 50 proteins interacted with GST only, 256 proteins interacted with Pirh2-GST, and 40 proteins interacted with both GST and Pirh2-GST

Summary

Introduction

Pirh[2] (p53-induced RING-H2 protein) was first described as an androgen receptor N-terminal interacting protein in 20021. Human Pirh[2] is coded by the RCHY1 gene (RING-finger and CHY-zinc-finger domain-containing protein 1) and belongs to the RING-finger domain-containing proteins, many of which are E3 ligases. Pirh[2] contains the centrally located RING domain responsible for the ubiquitin ligase activity, flanked by the N- and C-terminal domains responsible for protein– protein interactions[2]. Pirh[2] is a transcriptional target of the p53 tumor suppressor protein in mice. Similar to another p53-specific E3 ligase, Mdm[2], Pirh[2] is able to ubiqutinate p53 and target it for the proteasomal degradation[3]. Given the importance of the p53 for carcinogenesis, it is perhaps not surprising that Pirh[2] is being studied mostly in the context of p53 suppression

Methods

Results

Conclusion