Yeast Npl3 regulates replicative senescence outside of TERRA R-loop resolution and co-transcriptional processing.

Eukaryotic cells without telomerase experience progressively shorter telomeres with each round of cell division until cell cycle arrest is initiated, leading to replicative senescence. When yeast TLC1, which encodes the RNA template of telomerase, is deleted, senescence is accompanied by increased expression of TERRA (non-coding telomere repeat-containing RNA). Deletion of Npl3, an RNA-processing protein with telomere maintenance functions, accelerates senescence in tlc1Δ cells and significantly increases TERRA levels. Using genetic approaches, we set out to determine how Npl3 is involved in regulating TERRA expression and maintaining telomere homeostasis. Even though Npl3 regulates hyperrecombination, we found that Npl3 does not help resolve RNA:DNA hybrids formed during TERRA synthesis in the same way as RNase H1 and H2. Furthermore, Rad52 is still required for cells to escape senescence by telomere recombination in the absence of Npl3. Npl3 also works separately from the THO/TREX pathway for processing nascent RNA for nuclear export. However, deleting Dot1, a histone methyltransferase involved in tethering telomeres to the nuclear periphery, rescued the accelerated senescence phenotype of npl3Δ cells. Thus, our study suggests that Npl3 plays an additional role in regulating cellular senescence outside of RNA:DNA hybrid resolution and co-transcriptional processing.