mRNA instability in the nucleus due to a novel open reading frame element is a major determinant of the narrow tissue specificity of folate receptor alpha.
The folate receptor type alpha (FR-alpha) is a promising tumor marker and target. Here, we investigate the mechanistic basis for the tumor specificity and vast overexpression of FR-alpha. Among representative FR-alpha-positive (HeLa and JAR) and FR-alpha-negative (MG63, Caki1, and HT3) cell lines, the transcription rates of the endogenous FR-alpha gene, as well as the FR-alpha promoter activity, were relatively weak and comparable, but the FR-alpha transcript was abundant only in total RNA and nuclear RNA from the FR-alpha-positive cells. Rous sarcoma virus (RSV) promoter-driven expression of the FR-alpha gene was 7 to 30 times greater in the FR-alpha-positive than in FR-alpha-negative cells, both at the protein and mRNA levels, independently of intron sequences. Through the use of chimeric FR-alpha/FR-beta cDNAs, the above pattern of FR-alpha expression was attributed to a 60-bp sequence in the FR-alpha open reading frame. This sequence element, when placed in the 5' untranslated region of RSV promoter-luciferase, decreased the reporter expression approximately 7- to 20-fold in FR-alpha-negative cells (MG63, Caki1, HT3, BG1, and MCF7) relative to FR-alpha-positive cells (HeLa, JAR, and JEG3). Substitution of this FR-alpha element in FR-beta increased the in vivo degradation rate of the transcript in the nuclei of MG63 cells but not in the nuclei of HeLa cells or in the cytosol of MG63 or HeLa cells. The results reveal an efficient mechanism by which a novel sequence element causes differential transcript degradation in the nucleus to ensure narrow tissue specificity for a gene (e.g., that for FR-alpha) whose transcription is weak and relatively nonselective. FR-alpha exhibited constitutive mRNA and protein synthesis during the cell cycle and a slow protein turnover, presumably ensuring a high steady-state level of the receptor in cells that could override the nuclear mRNA instability determinant.
Zheng
Xuan
X
Department of Biochemistry and Molecular Biology, Medical College of Ohio, 3035 Arlington Avenue, Toledo, OH 43614-5804, USA.
Kelley
Karen
K
Elnakat
Hala
H
Yan
Wu
W
Dorn
Ted
T
Ratnam
Manohar
M
D020121:5' Untranslated Regions; D001358:Avian Sarcoma Viruses / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism*; D002822:Choriocarcinoma / Q000473:pathology; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D017930:Genes, Reporter; D005813:Genes, Synthetic; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D016366:Open Reading Frames / Q000235:genetics*; D009928:Organ Specificity; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012334:RNA, Neoplasm / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured / Q000378:metabolism; D014594:Uterine Neoplasms / Q000473:pathology
D020121:5' Untranslated Regions; D002352:Carrier Proteins; D018076:DNA, Complementary; D058973:Folate Receptors, GPI-Anchored; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D005492:Folic Acid; D008156:Luciferases