title: Three different rearrangements in a single intron truncate sterol regulatory element binding protein-2 and produce sterol-resistant phenotype in three cell lines. Role of introns in protein evolution.
abstract: The cholesterol analogue 25-hydroxycholesterol kills animal cells by blocking the proteolytic activation of two sterol-regulated transcription factors designated sterol regulatory element binding protein-1 and -2 (SREBP-1 and SREBP-2). These proteins, each approximately 1150 amino acids in length, are embedded in the membranes of the nucleus and endoplasmic reticulum by virtue of hydrophobic COOH-terminal segments. In cholesterol-depleted cells the proteins are cleaved to release soluble NH2-terminal fragments of approximately 480 amino acids that enter the nucleus and activate genes encoding the low density lipoprotein receptor and enzymes of cholesterol synthesis. 25-Hydroxycholesterol blocks this cleavage, and cells die of cholesterol deprivation. We previously described a mutant 25-hydroxycholesterol-resistant hamster cell line (SRD-1 cells) in which the SREBP-2 gene had undergone a recombination between the intron following codon 460 and an intron in an unrelated gene. The SREBP-2 sequence terminated at residue 460, eliminating the membrane attachment domain and producing a constitutively active factor that no longer required proteolysis and thus was not inhibited by 25-hydroxycholesterol. Here, we report that two additional sterol-resistant cell lines (SRD-2 and SRD-3) have also undergone genomic rearrangements in the intron following codon 460 of the SREBP-2 gene. Although the molecular rearrangements differ in the three mutant lines, each leads to the production of a constitutively active transcription factor whose SREBP-2 sequence terminates at residue 460. These findings provide a dramatic illustration of the advantage that introns provide in allowing proteins to gain new functions in response to new environmental challenges.
authors.affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235, USA.
authors.forename: J
authors.initials: J
authors.lastname: Yang
authors.forename: M S
authors.initials: MS
authors.lastname: Brown
authors.forename: Y K
authors.initials: YK
authors.lastname: Ho
authors.forename: J L
authors.initials: JL
authors.lastname: Goldstein
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D034961:Antigens, Nuclear; D001483:Base Sequence; D001692:Biological Transport; D022762:CCAAT-Enhancer-Binding Proteins; D016466:CHO Cells; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000235:genetics*; D004351:Drug Resistance; D005347:Fibroblasts; D005796:Genes; D018257:Helix-Loop-Helix Motifs; D006888:Hydroxycholesterols / Q000633:toxicity*; D007438:Introns / Q000235:genetics; D000072200:Ku Autoantigen; D008168:Lung; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D051780:Sterol Regulatory Element Binding Protein 1; D051782:Sterol Regulatory Element Binding Protein 2; D014157:Transcription Factors / Q000235:genetics*; D015533:Transcriptional Activation
chemical_list: D034961:Antigens, Nuclear; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D006888:Hydroxycholesterols; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D051780:Sterol Regulatory Element Binding Protein 1; D051782:Sterol Regulatory Element Binding Protein 2; D014157:Transcription Factors; C007997:25-hydroxycholesterol; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen