Abstract
BTEB is a GC-box binding transcription factor that can activate human immunodeficiency virus type 1 long terminal repeat and cellular gene promoters containing multiple GC boxes. The present studies showed that although BTEB mRNA was expressed in various tissues of mammals and cell lines, the expression of BTEB protein was confined to the brain and a neuroblastoma Neuro2A (N2A), suggesting that the BTEB expression was translationally regulated in a cell-specific or tissue-specific manner. The BTEB mRNA was characterized by a long (1.26 kilobases( 5'-untranslated region (5'-UTR) containing 10 upstream AUGs (uAUGs) and a GC-rich tract. To examine whether the 5'-UTR controlled the translation in a cell-specific manner, a fusion plasmid composed of the BTEB 5'-UTR and the chloramphenicol acetyltransferase gene was transfected into HeLa and N2A cells. Translational efficiency of the transcribed mRNA was estimated from the chloramphenicol acetyltransferase activity normalized on the basis of the amount of the mRNA. The 5'-UTR was found to decrease the translational efficiency by 7-fold in HeLa cells; that in N2A was not affected. When one of the uAUGs in the 5'-UTR was mutated to AAG, the inhibition of the translation by the 5'-UTR in HeLa cells was reversed; no effect of the mutation was observed in N2A cells. These results suggest that an uAUG in the 5'-UTR of the BTEB mRNA is, at least in part, responsible for the cell-specific translational control of the BTEB expression.
Highlights
From the mesearch Center for Immunodeficiency, Institute for Virus Research, Shogoin, Sakyo-ku, Kyoto 606 Japan and the Wepartment of Chemistry, Faculty of Science, Aramaki, Aoba-ku, Sendai980 Japan
The present studies showed that BTEB mRNA was expressed in various tissues of mammals and cell lines,the expression of BTEB protein was confinedto the brain and a neuroblastoma Neuro2A (N2A), suggesting that the BTEB expression was translationally regulated in a cell-specific or tissue-specific manner
Theseresults suggest that an upstream AUGs (uAUGs) in the 5‘-UTR otfhe BTEB mRNAisa, t least in part, responsible for the cell-specific translational control of the BTEB expression
Summary
Animals and Cells-MaleWistar rats (9-11 weeks old) werepurchased from Japan SLC, Inc. (Shizuoka,Japan). The abbreviations used are: 5’-UTR, 5”untranslated region; uAUGs, upstream AUGs;PAGE, polyacrylamide gel electrophoresis; CAT,chloramphenicol acetyltransferase;kb, kilobasepairb); ORF, open reading frames. A buffer consisting of 20 mM HEPES-KOH (pH7.8), 0.2 mM EDTA, 0.1 terminal 31 amino acids of the BTEB and the CAT coding region was. Antibodies-Anti-BTEB antiserum was obtained from a rabbit im- was employed for transfection of plasmids into HeLa and N2A.Ten munized with bacterially produced BTEB proteians follows. Western a n d Northern Blotting-For Western blotting, nuclear ex- sense RNA corresponding to the HindIII-EcoRrIegion (240 base pairs) tracts (60 pg of protein) fractionatedby SDS-PAGE were transferred on of the CAT coding region was synthesized in the preseofnc[Ce Y-~~PICTP.
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