Identification of a cis-acting element of human dihydrofolate reductase mRNA.
Human dihydrofolate reductase (DHFR) is a critical target in cancer chemotherapy. Previous studies showed that an 82-nt RNA fragment within the DHFR mRNA protein-coding region functions as a DHFR cis-acting response element. In this study, we further investigated the key elements contained within this sequence that are required for the DHFR mRNA-DHFR protein interaction. Using enzymatic foot-printing assays and RNA-binding experiments, we isolated a 27-nt sequence (DHFR27, corresponding to nts 407-433), which bound with high affinity and specificity to human DHFR to form a ribonucleoprotein complex. In vivo transient transfection experiments using a luciferase reporter system revealed that DHFR27 RNA could repress the luciferase expression in a DHFR-dependent manner when placed upstream of luciferase mRNA. This work provides new insights into the essential molecular elements that mediate RNA-protein interactions.
Department of Medicine and Pharmacology, Developmental Therapeutic Program, Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06520, USA.
Ningwen
N
Tai
John C
JC
Schmitz
Tian-Min
TM
Chen
Michelle B
MB
O'Neill
Edward
E
Chu
D001483:Base Sequence; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D049410:Luciferases, Renilla / Q000235:genetics; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D038621:Regulatory Sequences, Ribonucleic Acid; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D012333:RNA, Messenger; D038621:Regulatory Sequences, Ribonucleic Acid; D049410:Luciferases, Renilla; D013762:Tetrahydrofolate Dehydrogenase