The anti-cancer drug 5-fluorouracil affects cell cycle regulators and potential regulatory long non-coding RNAs in yeast

5-fluorouracil (5-FU) was isolated as an inhibitor of thymidylate synthase, which is important for DNA synthesis. The drug was later found to also affect the conserved 3ʹ-5ʹ exoribonuclease EXOSC10/Rrp6, a catalytic subunit of the RNA exosome that degrades and processes protein-coding and non-coding transcripts. Work on 5-FU’s cytotoxicity has been focused on mRNAs and non-coding transcripts such as rRNAs, tRNAs and snoRNAs. However, the effect of 5-FU on long non-coding RNAs (lncRNAs), which include regulatory transcripts important for cell growth and differentiation, is poorly understood. RNA profiling of synchronized 5-FU treated yeast cells and protein assays reveal that the drug specifically inhibits a set of cell cycle regulated genes involved in mitotic division, by decreasing levels of the paralogous Swi5 and Ace2 transcriptional activators. We also observe widespread accumulation of different lncRNA types in treated cells, which are typically present at high levels in a strain lacking EXOSC10/Rrp6. 5-FU responsive lncRNAs include potential regulatory antisense transcripts that form double-stranded RNAs (dsRNAs) with overlapping sense mRNAs. Some of these transcripts encode proteins important for cell growth and division, such as the transcription factor Ace2, and the RNA exosome subunit EXOSC6/Mtr3. In addition to revealing a transcriptional effect of 5-FU action via DNA binding regulators involved in cell cycle progression, our results have implications for the function of putative regulatory lncRNAs in 5-FU mediated cytotoxicity. The data raise the intriguing possibility that the drug deregulates lncRNAs/dsRNAs involved in controlling eukaryotic cell division, thereby highlighting a new class of promising therapeutical targets.

5-氟尿嘧啶（5-fluorouracil，5-FU）最初被分离为胸苷酸合酶（thymidylate synthase）的抑制剂，而该酶对DNA合成至关重要。后续研究发现，该药物还可作用于保守的3'-5'核糖核酸外切酶EXOSC10/Rrp6——这是RNA外切体（RNA exosome）的一个催化亚基，负责降解并加工蛋白质编码转录本与非编码转录本。既往针对5-FU细胞毒性的研究多聚焦于信使RNA（mRNA）以及核糖体RNA（rRNA）、转运RNA（tRNA）、核仁小RNA（snoRNA）等非编码转录本。但目前人们对5-FU作用于长非编码RNA（long non-coding RNAs，lncRNAs）的效应仍知之甚少，而lncRNAs涵盖了对细胞生长与分化至关重要的调控性转录本。对经5-FU处理的同步化酵母细胞开展RNA谱分析与蛋白质实验后发现，该药物可通过降低同源Swi5与Ace2转录激活因子的表达水平，特异性抑制一组参与有丝分裂的细胞周期调控基因。我们还观察到，经药物处理的细胞中出现了多种lncRNAs的广泛积累，而这类积累模式通常在缺失EXOSC10/Rrp6的菌株中呈现高丰度状态。对5-FU响应的lncRNAs包含潜在的调控性反义转录本，这类转录本可与重叠的有义mRNA形成双链RNA（double-stranded RNAs，dsRNAs）。其中部分转录本编码对细胞生长与分裂至关重要的蛋白质，例如转录因子Ace2，以及RNA外切体亚基EXOSC6/Mtr3。本研究不仅揭示了5-FU可通过参与细胞周期进程的DNA结合调控因子发挥转录调控效应，其研究结果还为5-FU介导的细胞毒性中潜在调控性lncRNAs的功能提供了新的启示。本研究数据提出了一个极具吸引力的可能性：该药物可扰乱参与真核细胞分裂调控的lncRNAs/dsRNAs的稳态，由此凸显了一类极具潜力的新型治疗靶点。