Identification of in vivo target RNA sequences bound by thymidylate synthase.
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.
Chu
E
E
NCI-Navy Medical Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20889-5105, USA.
Cogliati
T
T
Copur
S M
SM
Borre
A
A
Voeller
D M
DM
Allegra
C J
CJ
Segal
S
S
D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011485:Protein Binding; D018075:RNA, Complementary; D012333:RNA, Messenger / Q000378:metabolism; D012334:RNA, Neoplasm / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D013940:Thymidylate Synthase / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D018075:RNA, Complementary; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D016601:RNA-Binding Proteins; D013940:Thymidylate Synthase