Cloning, characterization, and steroid-dependent posttranscriptional processing of RUSH-1 alpha and beta, two uteroglobin promoter-binding proteins.
We previously used gel shift assays, Southwestern blots, and UV cross-linking to identify four proteins that bind to the 203-bp 5'-flanking region (-194/ +9) of the rabbit uteroglobin gene. Here we report cloning, by recognition site screening, the cDNAs for two of the uteroglobin promoter-binding proteins (95 kDa and 113 kDa). Their presumptive nucleotide-binding motifs share 61% identity with the SWI2/SNF2 helicase superfamily, and each protein has the novel C3HC4 (RING) zinc-finger signature near its C terminus. RUSH-1 alpha, the 113-kDa protein, is the rabbit homolog of human HIP116, a protein that binds to the human immunodeficiency virus-1 promoter. RUSH-1 beta is a 95-kDa truncated version of RUSH-1 alpha that results from alternative splicing of a 57-bp exon as confirmed by genomic cloning. Northern analysis showed mRNA expression (5.2 kb) was induced by progesterone +/- PRL and antagonized by estrogen. However, because the two proteins result from alternative splicing of a 57-bp exon, the small difference in their mRNA sizes could not be detected by Northern analysis. Therefore, competitive RT-PCR and HPLC were used to quantify differences in the ratios of their mRNAs. Progesterone +/- PRL treatment increased (P < 0.005) the ratio of message for RUSH-1 alpha compared with RUSH-1 beta. Western analysis showed the RUSH-1 alpha protein is increased in response to progesterone +/- PRL and decreased in response to estrogen. The antiserum used for immunoblotting specifically supershifts uteroglobin promoter-protein complexes in gel shift experiments. Because RUSH-1 alpha and beta messages were detected in lung, liver, and HRE-H9 cells, these proteins may regulate genes in numerous cell types.
Hayward-Lester
A
A
Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, Lubbock 79430, USA.
Hewetson
A
A
Beale
E G
EG
Oefner
P J
PJ
Doris
P A
PA
Chilton
B S
BS
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003001:Cloning, Molecular; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D004717:Endometrium / Q000378:metabolism; D004971:Estrus / Q000378:metabolism; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D016133:Polymerase Chain Reaction / Q000379:methods; D011374:Progesterone / Q000378:metabolism / Q000494:pharmacology; D011388:Prolactin / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013256:Steroids / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics; D014158:Transcription, Genetic; D014598:Uteroglobin / Q000235:genetics* / Q000378:metabolism
D004268:DNA-Binding Proteins; C100023:HLTF protein, human; D009687:Nuclear Proteins; D012333:RNA, Messenger; C499336:SMARCA1 protein, human; C084011:SMARCA2 protein, human; C104270:SMARCA3 protein, Oryctolagus cuniculus; D013256:Steroids; D014157:Transcription Factors; D011374:Progesterone; D011388:Prolactin; D014598:Uteroglobin; C084108:SMARCA4 protein, human; D004265:DNA Helicases