Characterization of a cis-acting regulatory element in the protein-coding region of human dihydrofolate reductase mRNA.
Previous studies have shown that human DHFR (dihydrofolate reductase), in addition to its critical role in DNA biosynthesis, functions as an RNA-binding protein. The interaction between DHFR and its own mRNA results in translational repression. In this study, we characterized the cis-acting elements on human DHFR mRNA that are required for the DHFR mRNA-DHFR protein interaction. Using a series of gel-shift and nitrocellulose filter-binding assays, a 164 nt RNA sequence, corresponding to nt 401-564, was identified within the coding region that binds to DHFR protein with an affinity similar to that of full-length DHFR mRNA. To document in vivo biological activity, various DHFR sequences contained within the coding region were cloned on to the 5' end of a luciferase reporter plasmid, and transient transfection experiments were performed using human colon cancer RKO cells. In cells transfected with p644/DHFR:401-564, luciferase activity was decreased by 50% when compared with cells transfected with the p644 plasmid alone. Luciferase mRNA levels were identical under each of these conditions, as determined by Northern-blot analysis. In cells transfected with p644/DHFR:401-564, luciferase activity was restored to almost 100% of control when cells were treated with the antifolate analogue methotrexate or with a short-interfering RNA targeting DHFR mRNA. These findings provide evidence that the DHFR 401-564 sequence is a DHFR-response element. In vitro and in vivo studies further localized this cis-element to an 82 nt sequence corresponding to nt 401-482. This work provides new insights into critical elements that mediate RNA-protein interactions.
Tai
Ningwen
N
Department of Medicine and Pharmacology, Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06520, USA.
Schmitz
John C
JC
Chen
Tian-min
TM
Chu
Edward
E
D001665:Binding Sites; D045744:Cell Line, Tumor; D005091:Exons; D005786:Gene Expression Regulation; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D038621:Regulatory Sequences, Ribonucleic Acid; D020218:Response Elements; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics*
D012333:RNA, Messenger; D038621:Regulatory Sequences, Ribonucleic Acid; D008156:Luciferases; D013762:Tetrahydrofolate Dehydrogenase