TERRA G-quadruplex RNA interaction with TRF2 GAR domain is required for telomere integrity

Yang Mei,Olga Vladimirova,Zhong Deng,William C Drosopoulos,F Brad Johnson,Carl L Schildkraut,Nitish Gulve,Paul M Lieberman

doi:10.1038/s41598-021-82406-x

Abstract

Telomere dysfunction causes chromosomal instability which is associated with many cancers and age-related diseases. The non-coding telomeric repeat-containing RNA (TERRA) forms a structural and regulatory component of the telomere that is implicated in telomere maintenance and chromosomal end protection. The basic N-terminal Gly/Arg-rich (GAR) domain of telomeric repeat-binding factor 2 (TRF2) can bind TERRA but the structural basis and significance of this interaction remains poorly understood. Here, we show that TRF2 GAR recognizes G-quadruplex features of TERRA. We show that small molecules that disrupt the TERRA-TRF2 GAR complex, such as N-methyl mesoporphyrin IX (NMM) or genetic deletion of TRF2 GAR domain, result in the loss of TERRA, and the induction of γH2AX-associated telomeric DNA damage associated with decreased telomere length, and increased telomere aberrations, including telomere fragility. Taken together, our data indicates that the G-quadruplex structure of TERRA is an important recognition element for TRF2 GAR domain and this interaction between TRF2 GAR and TERRA is essential to maintain telomere stability.

Highlights

Telomeres are nucleoprotein structures that protect and maintain the ends of linear chromosomes and are necessary for genomic stability[1,2,3,4]
Several different small molecules, including the acridine derivative BRACO-19 and porphyrin N-methyl mesoporphyrin IX (NMM) (Fig. 1B) have been shown to interact with DNA and/or RNA G4 structures in vitro[69] and to regulate telomere functions in vivo[46,50,70]. To determine if these compounds bound with selectivity to telomeric repeat-containing RNAs (TERRA) or telomeric G-rich strand DNA (TeloDNA), we generated fluorescent oligonucleotides for G4 TeloDNA, TERRA, and their antisense oligonucleotides by attaching a 5′ fluorescein label that could be used for fluorescence polarization (FP) assays to measure ligand binding (Fig. 1C,D)
We found EBNA1 G4 RNA bound with PDS (199 nM) and BRACO-19 (227 nM), it did not display any binding with NMM nor with negative control protoporphyrin IX (PP) (Fig. S4)

Summary

Introduction

Telomeres are nucleoprotein structures that protect and maintain the ends of linear chromosomes and are necessary for genomic stability[1,2,3,4]. TERRA has been shown to be involved in numerous functions including regulation of telomerase activity, inhibition of histone methyltransferase LSD1, competition with single-stranded DNA-binding proteins, and modulation of telomeric c hromatin[19,20,21,22,23,24,25]. The TRF2 GAR domain can bind to telomere DNA structures, including 4-way junctions formed at telomere T-loops[60,66]. We set out to investigate the ability of G4 interacting molecules, such as BRACO-19 and NMM, to bind TERRA and disrupt its interaction with the TRF2 GAR domain We assayed their effects on TERRA expression and telomeric DNA integrity in living cells, and whether these effects resemble the effects of genetic disruption of the TRF2 GAR domain

Methods

Results

Conclusion