Molecular and functional characterization of telomeric repeat-containing RNAs in Chinese hamster ovary cells

Mammalian telomeric DNA comprises long tracts of tandem TTAGGG repeats. The same repeats are also found at internal chromosomal regions called interstitial telomeric sequences (ITSs). Telomeres are transcribed into UUAGGG-containing transcripts, named TERRA, which serve multiple functions in maintaining telomere integrity. Complementary RNAs containing C-rich telomeric repeats, named ARIA, have also been identified in few yeast mutants and mammalian cells with dysfunctional telomeres. The molecular features and functions of ARIA remain understudied, mainly due to its low abundance and the lack of suitable cellular systems. Here, we show that Chinese hamster ovary (CHO) cells produce abundant TERRA and ARIA transcripts, predominantly originating from ITSs. Both RNAs are polyadenylated, exhibit relatively short half-lives and form large cellular foci. We also show that ARIA depletion leads to exposure of single-stranded (ss) DNA at ITSs and that ssDNA exposure increases when ITS DNA is damaged. SsDNA formation does not require the DNA damage signaling kinases ATM and ATR, nor the exonucleases DNA2 and EXO1; however, ATM prevents excessive ssDNA accumulation when ARIA function is inhibited. These findings establish CHO cells as a powerful model to dissect telomeric RNA functions and reveal ARIA as a key regulator of telomeric repeat DNA integrity.