Cloning, characterization, and steroid-dependent posttranscriptional processing of RUSH-1 alpha and beta, two uteroglobin promoter-binding proteins
Cloning, characterization, and steroid-dependent posttranscriptional processing of RUSH-1 alpha and beta, two uteroglobin promoter-binding proteins
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DOI：
10.1210/me.10.11.1335
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发表时间：
1996-11-01
影响因子：
--
通讯作者：
Chilton, BS
中科院分区：
文献类型：
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作者：
HaywardLester, A;Hewetson, A;Chilton, BS
我们先前使用凝胶迁移分析、西南印迹和UV交联来鉴定与兔子宫珠蛋白基因的203-bp 5 '侧翼区（-194/+9）结合的四种蛋白质。在这里，我们报告克隆，识别位点筛选，两个子宫珠蛋白启动子结合蛋白（95 kDa和113 kDa）的cDNA。它们假定的核苷酸结合基序与SWI 2/SNF 2解旋酶超家族共享61%的同一性，并且每个蛋白质在其C末端附近具有新的C3 HC 4（RING）锌指签名。RUSH-1 α，113-kDa的蛋白质，是人HIP 116的兔同源物，HIP 116是一种与人免疫缺陷病毒-1启动子结合的蛋白质。RUSH-1 β是RUSH-1 α的95-kDa截短版本，其由57-bp外显子的选择性剪接产生，如通过基因组克隆所证实的。北方分析显示，孕酮+/- PRL诱导mRNA表达（5.2kb），雌激素拮抗该表达。然而，由于这两种蛋白质是由一个57 bp外显子的选择性剪接产生的，因此它们的mRNA大小的微小差异不能通过北方分析检测到。因此，使用竞争性RT-PCR和HPLC来定量它们的mRNA比率的差异。与RUSH-1 β相比，孕酮+/- PRL治疗增加了RUSH-1 α的信息比率（P < 0.005）。Western分析显示，RUSH-1 α蛋白响应于孕酮+/- PRL而增加，响应于雌激素而减少。用于免疫印迹的抗血清在凝胶迁移实验中特异性超迁移子宫珠蛋白启动子-蛋白复合物。由于在肺、肝和HRE-H9细胞中检测到RUSH-1 α和β信息，这些蛋白质可能调节许多细胞类型中的基因。
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 smalt 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.