Alternative splicing gives rise to two isoforms of Zfhep, a zinc finger/homeodomain protein that binds T3-response elements
Alternative splicing gives rise to two isoforms of Zfhep, a zinc finger/homeodomain protein that binds T3-response elements
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DOI：
10.1089/dna.1996.15.643
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发表时间：
1996-08-01
影响因子：
3.1
通讯作者：
Darling, DS
中科院分区：
文献类型：
--
作者：
Cabanillas, AM;Darling, DS
我们先前已经分离了一种称为Zfhep（锌指同源结构域增强子结合蛋白）的转录因子的cDNA，其含有两个单独的锌指结构域ZD 1和ZD 2，每个锌指结构域结合DNA，以及一个同源结构域。大鼠Zfhep cDNA缺乏5 '-甲硫氨酸密码子，存在于其他物种的一些同源物中。因此，本工作的目的是分离大鼠Zfhep cDNA的5 '端。Zfhep-2cDNA被分离，具有2.5kbp的总长度，包括超过1.1kbp的新序列，随后是与Zfhep-1克隆相同的1.4kbp。1.1 kbp的新序列在所有阅读框中含有多个终止密码子，表明它代表大鼠Zfhep-2 mRNA的5 '-非翻译（5'-UT）区。然而，Zfhep-2克隆不包含Zfhep-1编码序列的末端5 '外显子，这可能是由于Zfhep RNA的选择性剪接。为了区分剪接点与内含子-外显子连接，使用大鼠基因组DNA和来自Zfhep-2序列的连接侧翼引物进行聚合酶链反应（PCR）。通过两对不同的引物从基因组DNA中没有扩增出条带，表明Zfhep-2特异性序列不是内含子。进行核糖核酸酶保护测定以研究多种Zfhep mRNA的表达。在大鼠卵巢、后脑、前脑、心脏、肾脏、小肠和GH4 C1细胞的总RNA或mRNA中检测到两条受保护的条带。Zfhep-2占每个组织中Zfhep RNA的约20%。因此，两种mRNA在这些组织中表达，证实了选择性剪接。为了独立地确认Zfhep-2和Zfhep-1 mRNA两者的存在，使用跨越Zfhep剪接位点的引物进行逆转录酶（RT）-PCR。获得代表两种RNA的特异性条带。将Zfhep-2 PCR产物亚克隆，DNA序列分析证实在开放阅读框的5 '端附近不存在ATG密码子。Zfhep-2克隆的理论翻译预测了比Zfhep-1更小的蛋白质。网织红细胞裂解物中的体外翻译显示Zfhep-2比Zfhep-1小约40 kD。因此，Zfhep-2显然缺乏Zfhep-1的大部分第一锌指结构域（ZD 1）。因为两个锌指结构域结合不同的DNA序列，Zfhep-2被预测为仅结合Zfhep-1识别的基因的子集。
We have previously isolated a cDNA for a transcription factor referred to as Zfhep (Zinc finger Homeodomain Enhancer-binding protein) containing two separate zinc finger domains, ZD1 and ZD2, each of which binds DNA, and a homeodomain. The rat Zfhep cDNA lacks a 5'-methionine codon, present in some homologs from other species. Hence, the aim of this work was to isolate the 5'-end of the rat Zfhep cDNA. Zfhep-2 cDNA was isolated, having a total length of 2.5 kbp, including more than 1.1 kbp of novel sequence followed by 1.4 kbp identical to the Zfhep-1 clone. The 1.1 kbp of novel sequence contains multiple stop codons in all reading frames, suggesting that it represents the 5'-untranslated (5'-UT) region of the rat Zfhep-2 mRNA. However, the Zfhep-2 clone does not contain the extreme 5'-exon(s) of the Zfhep-1 coding sequence, possibly due to alternative splicing of Zfhep RNA. To distinguish between a splice junction versus an intron-exon junction, the polymerase chain reaction (PCR) with rat genomic DNA and junction-flanking primers from the Zfhep-2 sequence was conducted. No bands were amplified from the genomic DNA by two different pairs of primers, indicating that the Zfhep-2-specific sequence is not intronic. Ribonuclease protection assays were performed to investigate the expression of multiple Zfhep mRNAs. Two protected bands were detected, and both were identified in total RNA or mRNA of rat ovary, hindbrain, forebrain, heart, kidney, small intestine, and GH4C1 cells. Zfhep-2 represents about 20% of the Zfhep RNA in each tissue. Hence, two mRNAs are expressed in these tissues, confirming the alternative splicing. To confirm independently the presence of both Zfhep-2 and Zfhep-1 mRNAs, reverse transcriptase (RT)-PCR was done using primers that span the Zfhep splice site. Specific bands representing both RNAs were obtained. The Zfhep-2 PCR product was subcloned and DNA sequence analysis confirmed the absence of ATG codons near the 5'-end of the open reading frame. The theoretical translation of the Zfhep-2 clone predicts a smaller protein than Zfhep-1. In vitro translation in reticulocyte lysates showed that Zfhep-2 is about 40 kD smaller than Zfhep-1. Hence, Zfhep-2 apparently lacks most of the first zinc finger domain (ZD1) of Zfhep-1. Because the two zinc finger domains bind different DNA sequences, Zfhep-2 is predicted to bind to only a subset of genes recognized by Zfhep-1.