In vitro selection of an RNA sequence that interacts with high affinity with thymidylate synthase.
Previous studies have shown that the repressive effect of thymidylate synthase (TS) mRNA translation is mediated by direct binding of TS itself to two cis-acting elements on its cognate mRNA. To identify the optimal RNA nucleotides that interact with TS, we in vitro synthesized a completely degenerate, linear RNA pool of 25 nt and employed in vitro selection to isolate high affinity RNA ligands that bind human TS protein. After 10 rounds of selection and amplification, a single RNA molecule was selected that bound TS protein with nearly 20-fold greater affinity than native, wild-type TS RNA sequences. Secondary structure analysis of this RNA sequence predicted it to possess a stem-loop structure. Deletion and/or modification of the UGU loop element within the RNA sequence decreased binding to TS by up to 1000-fold. In vivo transfection experiments revealed that the presence of the selected RNA sequence resulted in a significant increase in the expression of a heterologous luciferase reporter construct in human colon cancer H630 and TS-overexpressing HCT-C:His-TS+ cells, but not in HCT-C18 cells expressing a functionally inactive TS. In addition, the presence of this element in H630 cells leads to induced expression of TS protein. An immunoprecipitation method using RT-PCR confirmed a direct interaction between human TS protein and the selected RNA sequence in transfected human cancer H630 cells. This study identified a novel RNA sequence from a degenerate RNA library that specifically interacts with TS.
Lin
X
X
Department of Medicine and Pharmacology, Yale Cancer Center, Yale University School of Medicine and VA Connecticut Healthcare System, New Haven, CT 06520, USA.
Mizunuma
N
N
Chen
T
T
Copur
S M
SM
Maley
G F
GF
Liu
J
J
Maley
F
F
Chu
E
E
D001483:Base Sequence; D015153:Blotting, Western; D003110:Colonic Neoplasms; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D009154:Mutation / Q000235:genetics; D023282:Nuclease Protection Assays; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D011233:Precipitin Tests; D011485:Protein Binding; D012313:RNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D013940:Thymidylate Synthase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012313:RNA; D013940:Thymidylate Synthase