XRN2 interactome reveals its synthetic lethal relationship with PARP1 inhibition

Praveen L. Patidar,Megha Khandelwal,Farjana J. Fattah,Edward A. Motea,Talysa Viera,Julio C. Morales,Naveen Singh

doi:10.1038/s41598-020-71203-7

Abstract

Persistent R-loops (RNA–DNA hybrids with a displaced single-stranded DNA) create DNA damage and lead to genomic instability. The 5′-3′-exoribonuclease 2 (XRN2) degrades RNA to resolve R-loops and promotes transcription termination. Previously, XRN2 was implicated in DNA double strand break (DSB) repair and in resolving replication stress. Here, using tandem affinity purification-mass spectrometry, bioinformatics, and biochemical approaches, we found that XRN2 associates with proteins involved in DNA repair/replication (Ku70-Ku80, DNA-PKcs, PARP1, MCM2-7, PCNA, RPA1) and RNA metabolism (RNA helicases, PRP19, p54(nrb), splicing factors). Novel major pathways linked to XRN2 include cell cycle control of chromosomal replication and DSB repair by non-homologous end joining. Investigating the biological implications of these interactions led us to discover that XRN2 depletion compromised cell survival after additional knockdown of specific DNA repair proteins, including PARP1. XRN2-deficient cells also showed enhanced PARP1 activity. Consistent with concurrent depletion of XRN2 and PARP1 promoting cell death, XRN2-deficient fibroblast and lung cancer cells also demonstrated sensitivity to PARP1 inhibition. XRN2 alterations (mutations, copy number/expression changes) are frequent in cancers. Thus, PARP1 inhibition could target cancers exhibiting XRN2 functional loss. Collectively, our data suggest XRN2’s association with novel protein partners and unravel synthetic lethality between XRN2 depletion and PARP1 inhibition.

Highlights

Persistent R-loops (RNA–DNA hybrids with a displaced single-stranded DNA) create DNA damage and lead to genomic instability
The role of XRN2 is well understood in RNA metabolic processes including transcription termination and RNA p rocessing[25]; little is known about its involvement in other cellular processes
We devised a strategy that combines proteomics, bioinformatics, biochemical, and biological approaches to systematically investigate the cellular roles of XRN2 in processes beyond RNA metabolism

Summary

Introduction

Persistent R-loops (RNA–DNA hybrids with a displaced single-stranded DNA) create DNA damage and lead to genomic instability. The 5′-3′-exoribonuclease 2 (XRN2) degrades RNA to resolve R-loops and promotes transcription termination. Abbreviations BER Base excision repair Co-IP Co-immunoprecipitation DAVID Database for Annotation, Visualization and Integrated Discovery DSBs DNA double strand breaks FDR False discovery rate HR Homologous recombination IPA Ingenuity Pathway Analysis MS Mass spectrometry NHEJ Non-homologous end joining PAR Poly(ADP-ribose) PMSF Phenylmethylsulfonyl fluoride STRING Search Tool for the Retrieval of INteracting Genes/proteins TAP Tandem affinity purification. The nucleic acid structures containing RNA–DNA hybrids with a displaced single-stranded DNA (R-loops) are generated in cells during transcription and their persistence leads to DNA damage and genomic. The XRN2 protein processively degrades downstream cleaved RNAPII-associated RNA to promote termination, and its ribonuclease activity is essential for genome-wide poly(A) site-dependent RNAPII termination[9,10]. XRN2 is involved in general RNA degradation, gene silencing, and rRNA m aturation[17,18,19]

Methods

Results

Conclusion