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 <i>TLC1,</i> 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 <i>tlc1Δ</i> 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 <i>npl3Δ</i> 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.

缺乏端粒酶（telomerase）的真核细胞，每经历一轮细胞分裂，端粒（telomere）便会逐渐缩短，直至触发细胞周期阻滞，进而引发复制性衰老。当编码端粒酶RNA模板的酵母TLC1基因被敲除时，衰老进程会伴随TERRA（端粒重复序列非编码RNA，non-coding telomere repeat-containing RNA）表达水平的升高。Npl3是一种具备端粒维持功能的RNA加工蛋白，敲除Npl3会加速tlc1Δ细胞的衰老进程，并显著提升TERRA水平。本研究采用遗传学方法，旨在解析Npl3调控TERRA表达、维持端粒稳态的具体机制。尽管Npl3可调控过度重组，但我们发现，其无法像核糖核酸酶H1（RNase H1）与H2那样，消解TERRA合成过程中形成的RNA:DNA杂交双链。此外，在缺失Npl3的细胞中，细胞仍需依赖Rad52通过端粒重组以逃离衰老。Npl3的功能亦独立于用于加工新生RNA并介导其核输出的THO/TREX通路。然而，敲除Dot1——一种负责将端粒锚定至核周边区域的组蛋白甲基转移酶（histone methyltransferase）——可挽救npl3Δ细胞的加速衰老表型。综上，本研究表明，Npl3在RNA:DNA杂交双链消解与共转录加工之外，还承担着调控细胞衰老的额外功能。