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）最初被分离得到，作为胸苷酸合酶（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（exoribonuclease）EXOSC10/Rrp6——这是RNA外切体（RNA exosome）的催化亚基，负责降解并加工编码蛋白的转录本与非编码转录本。以往针对5-FU细胞毒性的研究多聚焦于信使RNA（messenger RNA，mRNA）以及核糖体RNA（ribosomal RNA，rRNA）、转运RNA（transfer RNA，tRNA）、小核仁RNA（small nucleolar RNA，snoRNA）等非编码转录本。然而，5-FU对长链非编码RNA（long non-coding RNAs，lncRNAs）的调控作用仍有待阐明——这类RNA包含了对细胞生长与分化具有关键调控功能的转录本。对经5-FU处理的同步化酵母细胞开展RNA表达谱分析与蛋白质实验检测后发现，该药物可通过降低旁系同源的Swi5与Ace2转录激活因子的表达水平，特异性抑制一系列参与有丝分裂过程的细胞周期调控基因。此外，我们在经药物处理的细胞中观察到多种类型的lncRNAs广泛积累，而这类lncRNAs通常在EXOSC10/Rrp6基因缺失的酵母菌株中呈高表达状态。对5-FU具有响应性的lncRNAs包含潜在的调控性反义转录本，这类转录本可与存在重叠区域的有义mRNA形成双链RNA（double-stranded RNAs，dsRNAs）。其中部分转录本可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。除了揭示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作用外，本研究结果还为5-FU介导的细胞毒性中推定调控性lncRNAs的功能提供了新的研究视角。本研究数据提出了一个引人关注的可能性：该药物可扰乱参与真核细胞分裂调控的lncRNAs与dsRNAs的表达稳态，由此揭示了一类极具潜力的新型治疗靶点。