Polypyrimidine tract-binding protein-associated splicing factor is a negative regulator of transcriptional activity of the porcine p450scc insulin-like growth factor response element.
The porcine P-450 cholesterol side-chain cleavage enzyme gene (P450scc) contains a 30-bp region [insulin-like growth factor response element (IGFRE)] that mediates insulin-like growth factor I (IGF-I)-stimulated gene expression and binds Sp1. In this study, we showed that polypyrimidine tract-binding protein (PTB)-associated splicing factor (PSF), an RNA-binding component of spliceosomes, binds to the IGFRE. Southwestern analysis with an IGFRE oligonucleotide showed that a protein (from Sp1-immunodepleted HeLa extract) fractionated on SDS-PAGE at 100 kDa. Microsequence analysis of 100-kDa band HeLa proteins detected PSF. DNA affinity chromatography, using an IGFRE mutant oligonucleotide that does not bind Sp1, isolated a protein that immunoreacted with PSF antibody. Deoxyribonuclease I (DNase I) footprint analysis showed recombinant PSF binds 5' of the Sp1-binding GC box of the IGFRE, and mutant oligonucleotides further delineated this region to a palindrome, CTGAGTC. Functional analysis of these mutants by transfection experiments in a cell line overexpressing the IGF-I receptor (NWTb3) found that an inability to bind PSF significantly increased the IGFRE transcriptional activity, while retaining responsiveness to IGF-I. Moreover, transfection of expression vectors for Sp1 and PSF in porcine granulosa cells found that Sp1 expression stimulated IGFRE transcriptional activity while PSF inhibited activity even with coexpression of Sp1. In conclusion, we identified PSF as an independent, inhibitory regulator of the transcriptional activity of the porcine P450scc IGFRE.
Urban
R J
RJ
Department of Internal Medicine, University of Texas Medical Branch, Galveston 77555-1060, USA.
Bodenburg
Y
Y
Kurosky
A
A
Wood
T G
TG
Gasic
S
S
D000818:Animals; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics*; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism; D018983:DNA Footprinting; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006107:Granulosa Cells / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D009705:Nucleosides / Q000378:metabolism; D000072670:PTB-Associated Splicing Factor; D012326:RNA Splicing; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000378:metabolism; D020218:Response Elements; D017422:Sequence Analysis, DNA; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013552:Swine; D014162:Transfection
D009705:Nucleosides; D000072670:PTB-Associated Splicing Factor; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D016329:Sp1 Transcription Factor; D007334:Insulin-Like Growth Factor I; D004247:DNA; D002786:Cholesterol Side-Chain Cleavage Enzyme