Characterization of a cis-acting regulatory element in the protein coding region of thymidylate synthase mRNA.
Thymidylate synthase (TS) functions as an RNA-binding protein by interacting with two different sequences on its own mRNA. One site is located in the 5'-upstream region of human TS mRNA while the second site is located within the protein coding region corresponding to nt 434-634. In this paper, a 70 nt RNA sequence, corresponding to nt 480-550, was identified that binds TS protein with an affinity similar to that of full-length TS mRNA and TS434-634 RNA. In vitro translation studies confirmed that this sequence is critical for the translational autoregulatory effects of TS. To document in vivo biological significance, TS sequences contained within this region were cloned onto the 5'-end of a luciferase reporter plasmid and transient transfection experiments were performed using H630 human colon cancer cells. In cells transfected with p644/TS434-634 or p644/TS480-550, luciferase activity was decreased 2.5-fold when compared to cells transfected with p644 plasmid alone. Luciferase mRNA levels were identical for each of these conditions as determined by RNase protection and RT-PCR analysis. Immunoprecipitation of TS ribonucleoprotein complexes revealed a direct interaction between TS protein and TS480-550 RNA in transfected H630 cells. Treatment with 5-fluorouridine resulted in a nearly 2-fold increase in luciferase activity only in cells transfected with p644/TS434-634 and p644/TS480-550. This study identifies a 70 nt TS response element in the protein coding region of TS mRNA with in vitro and in vivo translational regulatory activity.
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.
Parsels
L A
LA
Voeller
D M
DM
Allegra
C J
CJ
Maley
G F
GF
Maley
F
F
Chu
E
E
D001665:Binding Sites; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism; D005472:Fluorouracil / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D020128:Inhibitory Concentration 50; D009154:Mutation / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics*; D011233:Precipitin Tests; D014176:Protein Biosynthesis / Q000187:drug effects / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics*; D013379:Substrate Specificity; D013940:Thymidylate Synthase / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D013940:Thymidylate Synthase; D005472:Fluorouracil