Heterogeneous nuclear ribonucleoprotein E1 binds polycytosine DNA and monitors genome integrity.

Bidyut K Mohanty,William S Streitfeld,Annamarie C Dalton,Je-Hyun Yoon,Lata Balakrishnan,Simon Grelet,David T Long,Walter J Chazin,Breege V Howley,Joseph Aq Karam,Toros Dincman,Philip H Howe

doi:10.26508/lsa.202000995

Abstract

Heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) is a tumor suppressor protein that binds site- and structure-specifically to RNA sequences to regulate mRNA stability, facilitate alternative splicing, and suppress protein translation on several metastasis-associated mRNAs. Here, we show that hnRNP E1 binds polycytosine-rich DNA tracts present throughout the genome, including those at promoters of several oncogenes and telomeres and monitors genome integrity. It binds DNA in a site- and structure-specific manner. hnRNP E1-knockdown cells displayed increased DNA damage signals including γ-H2AX at its binding sites and also showed increased mutations. UV and hydroxyurea treatment of hnRNP E1-knockdown cells exacerbated the basal DNA damage signals with increased cell cycle arrest, activation of checkpoint proteins, and monoubiquitination of proliferating cell nuclear antigen despite no changes in deubiquitinating enzymes. DNA damage caused by genotoxin treatment localized to hnRNP E1 binding sites. Our work suggests that hnRNP E1 facilitates functions of DNA integrity proteins at polycytosine tracts and monitors DNA integrity at these sites.

Highlights

Genome instability is a hallmark of cancer (Negrini et al, 2010)
We examined chromosomal localization of hnRNP E1 in A549 cells by chromatin immunoprecipitation (ChIP) and observed that hnRNP E1 was enriched at promoter-proximal regions of c-MYC, h-RAS, and EGFR (Fig 1C)
In a hypoxanthine phosphorybosyl transferase (HPRT) gene assay that detects mutations (Johnson, 2012) (See the Materials and Methods section for details), E1KD cells showed an ~fivefold increase in mutation frequency over control cells (Fig 4J), indicating that hnRNP E1 plays a critical role in maintaining genome integrity. This is the first report on genome-wide binding of hnRNP E1 to polycytosine-repeats and its global role in maintenance of DNA secondary structures such as i-motifs and suppression of G4s, and protection of cells from DNA damage/replication stress. hnRNP E1 knockdown resulted in increased accumulation of γ-H2AX, G4s, replication protein A (RPA), proliferating cell nuclear antigen (PCNA) monoubiquitination, and increase in mutations

Summary

Introduction

Genome instability is a hallmark of cancer (Negrini et al, 2010). Cells are constantly exposed to various exogenous agents such as UV, X-rays, and chemicals, and endogenous agents such as reactive oxygen species that can damage DNA and cause genome instability (Friedberg, 2008; Chatterjee & Walker, 2017). Various cellular processes that involve breaks in DNA or DNA-free ends, including replication, repair, recombination, transcription, and related cell cycle progression, have the potential to cause genome instability (Aguilera & Garcıa-Muse, 2013; Tubbs & Nussenzweig, 2017). Upon DNA damage or replication blockage, a battery of checkpoint proteins including sensors, adaptors, and effectors are activated and halt cell cycle progression (Harrison & Haber, 2006). Hydroxyurea (HU) treatment reduces the nucleotide pool in the cell, which uncouples replicative helicase and DNA polymerase thereby generating stretches of ssDNA; ssDNA binding proteins such as RPA play important role in protecting the ssDNA (Balajee & Geard, 2004; Alvino et al, 2007; Papadopoulou et al, 2015; Singh & Xu, 2016)

Methods

Results

Conclusion