Telomere Length Determines TERRA and R-Loop Regulation through the Cell Cycle

Abstract

Maintenance of a minimal telomere length is essential to prevent cellular senescence. When critically short telomeres arise in the absence of telomerase, they can be repaired by homology-directed repair (HDR) to prevent premature senescence onset. It is unclear why specifically the shortest telomeres are targeted for HDR. We demonstrate that the non-coding RNA TERRA accumulates as HDR-promoting RNA-DNA hybrids (R-loops) preferentially at very short telomeres. The increased level of TERRA and R-loops, exclusively at short telomeres, is due to a local defect in RNA degradation by the Rat1 and RNase H2 nucleases, respectively. Consequently, the coordination of TERRA degradation with telomere replication is altered at shortened telomeres. R-loop persistence at short telomeres contributes to activation of the DNA damage response (DDR) and promotes recruitment of the Rad51 recombinase. Thus, the telomere length-dependent regulation of TERRA and TERRA R-loops is a critical determinant of the rate of replicative senescence.

Yra1-bound RNA–DNA hybrids cause orientation-independent transcription–replication collisions and telomere instability

“…TERRA hybridization at telomeres promote the recruitment of Rad51 (Graf et al 2017). Collectively, these data support our model that Yra1 binding to R loops causes replication impairment and hyperrecombination all over the genome.…”

“…As predicted, RNA-DNA hybrids were increased in est2Δ cells (Fig. 6E) as shown in the single TelVI-R telomere and consistent with previous results indicating that shortening of telomeres in est2Δ cells triggers TERRA transcription and RNA-DNA hybrid accumulation (Graf et al 2017). Importantly, such hybrids were significantly increased under Yra1 overexpression, consistent with a putative role of Yra1 overexpression in RNA-DNA hybrid stabilization (Fig.…”

“…6), favoring the idea that Yra1 excess would be shortening the telomere through R-loop stabilization. Telomere repeats and subtelomeric regions are transcribed in lncRNAs known as TERRA, which form unstable R loops (Maicher et al 2014;Azzalin and Lingner 2015) that need to be removed before chromosomal ends are replicated (Graf et al 2017). Therefore, it is likely that, by stabilizing TERRA R loops, Yra1 overexpression impairs telomere replication progression.…”

“…This is restrained by the actions of RNase H and the THO complex (Balk et al 2013;Pfeiffer et al 2013;Arora et al 2014;Yu et al 2014) and favored by telomere shortening. TERRA RNA-DNA hybrids promote homologous recombination (Graf et al 2017) between telomeric repeats, enabling alternative lengthening of telomeres (ALT), a mechanism used by telomerase-deficient tumor cells to prevent telomere shortening (Balk et al 2013;Arora et al 2014;Yu et al 2014).…”

Molecular and physiological consequences of faulty eukaryotic ribonucleotide excision repair

“…In any case, the significance of the PIP-box within the RNase H2 complex requires clarification. While RNase H2 did not localize to particular genomic regions in chromatin immunoprecipitations from asynchronously growing yeast cells (Zimmer & Koshland, 2016), it could be crosslinked to telomeres in cells synchronized late in S phase, at times when the bulk of genomic DNA has been replicated (Graf et al, 2017). All other, non-catalytic RNase H2 subunit proteins are constitutively expressed throughout the cell cycle, and the complex resides exclusively within the nuclear compartment (Reijns et al, 2012;Lockhart et al, 2019).…”