Incorporation of 5-fluorouracil into U2 snRNA blocks pseudouridylation and pre-mRNA splicing in vivo.
Incorporation of 5-fluorouracil into U2 snRNA blocks pseudouridylation and pre-mRNA splicing in vivo.
复制标题
将 5-氟尿嘧啶掺入 U2 snRNA 中可阻断体内假尿苷化和前 mRNA 剪接。
DOI：
10.1093/nar/gkl1084
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发表时间：
2007
影响因子：
14.9
通讯作者：
Yu,Yi-Tao
中科院分区：
文献类型：
--
作者：
Zhao,Xinliang;Yu,Yi-Tao
5-氟尿嘧啶（5FU）是一种有效的抗癌药物，但其作用机制尚不清楚。在这里，我们检查了 5FU 对体内前 mRNA 剪接的影响。使用 RT-PCR，我们发现在暴露于低剂量 5FU 的 HeLa 细胞中，许多前体 mRNA 的剪接受到抑制。看来这种抑制作用并不是由于其掺入前体mRNA中，因为部分或完全被5FU取代的前体mRNA在注射到爪蟾细胞中时，与未取代的前体mRNA一样被剪接。对 5FU 处理的细胞的详细分析表明，5FU 并入 U2 snRNA 中重要的天然存在的假尿苷化位点。值得注意的是，5FU 的掺入有效阻止了 U2 snRNA 中重要假尿苷的形成，因为当细胞暴露于低剂量 5FU 5 天时，仅检测到痕量假尿苷。将低假尿苷化的 HeLa U2 snRNA 注射到 U2 耗尽的爪蟾细胞中无法重建前 mRNA 剪接，而从未处理或尿嘧啶处理的 HeLa 细胞中分离的对照 U2 则完全重建剪接。我们的结果首次证明5FU在体内天然假尿苷化位点处掺入剪接体snRNA，从而抑制snRNA假尿苷化和剪接。这种机制可能对 5FU 介导的细胞死亡有重大贡献。
5-fluorouracil (5FU) is an effective anti-cancer drug, yet its mechanism of action remains unclear. Here, we examine the effect of 5FU on pre-mRNA splicingin vivo. Using RT–PCR, we show that the splicing of a number of pre-mRNAs is inhibited in HeLa cells that have been exposed to a low dose of 5FU. It appears that this inhibitory effect is not due to its incorporation into pre-mRNA, because partially or fully 5FU-substituted pre-mRNA, when injected intoXenopusoocytes, is spliced just as well as is the unsubstituted pre-mRNA. Detailed analyses of 5FU-treated cells indicate that 5FU is incorporated into U2 snRNA at important naturally occurring pseudouridylation sites. Remarkably, 5FU incorporation effectively blocks the formation of important pseudouridines in U2 snRNA, as only a trace of pseudouridine is detected when cells are exposed to a low dose of 5FU for 5 days. Injection of the hypopseudouridylated HeLa U2 snRNA into U2-depletedXenopusoocytes fails to reconstitute pre-mRNA splicing, whereas control U2 isolated from untreated or uracil-treated HeLa cells completely reconstitutes the splicing. Our results demonstrate for the first time that 5FU incorporates into a spliceosomal snRNA at natural pseudouridylation sitesin vivo, thereby inhibiting snRNA pseudouridylation and splicing. This mechanism may contribute substantially to 5FU-mediated cell death.