Primary acute lymphoblastic leukemia cells use a novel promoter and 5'noncoding exon for the human reduced folate carrier that encodes a modified carrier translated from an upstream translational start.
The human reduced folate carrier (hRFC) is reported to be regulated by up to seven alternatively spliced noncoding exons (A1, A2, A, B, C, D, and E). Noncoding exon and promoter usage was analyzed in RNAs from 27 childhood acute lymphoblastic leukemia (ALL) specimens by real-time PCR and/or 5' rapid amplification of cDNA ends (5' RACE) assay. By real-time PCR, total hRFC transcripts in ALL spanned a 289-fold range. Over 90% of hRFC transcripts were transcribed with A1, A2, and B 5' untranslated regions (UTRs). Analysis of 5' RACE clones showed that the A1 + A2 5'UTRs contained A1 sequence alone or a fusion of A1 and A2, implying the existence of a single, alternatively spliced 1021-bp A1/A2 noncoding region. High frequency sequence polymorphisms (AGG deletion, C/T transition) identified in the A1/A2 region by 5'RACE were confirmed in normal DNAs. By reporter assays in HepG2 hepatoma and Jurkat leukemia cells, A1/A2 promoter activity was localized to a 134-bp minimal region. Translation from an upstream AUG in the A1/A2 noncoding region in-frame with the normal translation start resulted in synthesis of a larger ( approximately 7 kDa) hRFC protein with transport properties altered from those for wild-type hRFC. Although there was no effect on transcript or protein stabilities, in vitro translation from A1/A2 transcripts was decreased compared with those with the B 5'UTR. Our results document the importance of the hRFC A1/A2 upstream region in childhood ALL and an intricate transcriptional and posttranscriptional regulation of hRFC-A1/A2 mRNAs. Furthermore, they suggest that use of the A1/A2 5'UTR may confer a transport phenotype distinct from the other 5'UTRs due to altered translation efficiency and transport properties.
Flatley
Robin M
RM
Experimental and Clinical Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Detroit, Michigan 48201, USA.
Payton
Scott G
SG
Taub
Jeffrey W
JW
Matherly
Larry H
LH
D020121:5' Untranslated Regions; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000378:metabolism; D005091:Exons; D017353:Gene Deletion; D017930:Genes, Reporter; D005838:Genotype; D006801:Humans; D019169:Jurkat Cells; D020014:K562 Cells; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D008727:Methotrexate / Q000494:pharmacology; D018613:Microscopy, Confocal; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011110:Polymorphism, Genetic; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism; D058978:Reduced Folate Carrier Protein; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014158:Transcription, Genetic
D020121:5' Untranslated Regions; D018076:DNA, Complementary; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; D008156:Luciferases; D008727:Methotrexate