Chu E, Cogliati T, Copur S M, Borre A, Voeller D M, Allegra C J, Segal S
NCI-Navy Medical Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20889-5105, USA.
Nucleic Acids Res. 1996 Aug 15;24(16):3222-8. doi: 10.1093/nar/24.16.3222.
We developed an immunoprecipitation-RNA-random PCR (rPCR) method to isolate cellular RNA sequences that bind to the folate-dependent enzyme thymidylate synthase (TS). Using this approach, nine different cellular RNAs that formed a ribonucleoprotein (RNP) complex with thymidylate synthase (TS) in human colon cancer cells were identified. RNA binding experiments revealed that seven of these RNAs bound TS with relatively high affinity (IC50 values ranging from 1.5 to 6 nM). One of the RNAs was shown to encode the interferon (IFN)-induced 15 kDa protein. Western immunoblot analyses demonstrated that the level of IFN-induced 15 kDa protein was significantly decreased in human colon cancer H630-R10 cells compared with parent H630 cells. While the level of IFN-induced 15 kDa mRNA expression was the same in parent and TS-overexpressing cell lines, the level of IFN-induced 15 kDa RNA bound to TS in the form of a RNP complex was markedly higher in H630-R10 cells relative to parent H630 cells. These studies begin to define a number of cellular target RNA sequences with which TS interacts and suggest that these TS protein-cellular RNA interactions may have a biological role.
我们开发了一种免疫沉淀 - RNA - 随机PCR（rPCR）方法，以分离与叶酸依赖性酶胸苷酸合成酶（TS）结合的细胞RNA序列。使用这种方法，在人结肠癌细胞中鉴定出九种不同的细胞RNA，它们与胸苷酸合成酶（TS）形成核糖核蛋白（RNP）复合物。RNA结合实验表明，其中七种RNA以相对高的亲和力结合TS（IC50值范围为1.5至6 nM）。其中一种RNA被证明编码干扰素（IFN）诱导的15 kDa蛋白。蛋白质免疫印迹分析表明，与亲本H630细胞相比，人结肠癌H630 - R10细胞中IFN诱导的15 kDa蛋白水平显著降低。虽然亲本细胞系和TS过表达细胞系中IFN诱导的15 kDa mRNA表达水平相同，但相对于亲本H630细胞，H630 - R10细胞中以RNP复合物形式与TS结合的IFN诱导的15 kDa RNA水平明显更高。这些研究开始确定TS与之相互作用的一些细胞靶RNA序列，并表明这些TS蛋白 - 细胞RNA相互作用可能具有生物学作用。
