Minigene reporter for identification and analysis of cis elements and trans factors affecting pre-mRNA splicing
Minigene reporter for identification and analysis of cis elements and trans factors affecting pre-mRNA splicing
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DOI：
10.2144/000112208
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发表时间：
2006-08-01
期刊：
影响因子：
2.7
通讯作者：
Cooper, Thomas A.
中科院分区：
文献类型：
--
作者：
Singh, Gopal;Cooper, Thomas A.
所有的人类基因都含有一系列的顺式作用元件，这些元件位于内含子和外显子内，是正确和有效的前体信使RNA（pre-mRNA）剪接所必需的。最近的计算分析预测，大多数人类外显子含有剪接所需的元素，这与破坏前mRNA剪接的突变导致疾病的高频率一致。微基因提供了一种直接确定致病突变或单核苷酸多态性（SNP）是否影响剪接效率的方法。小基因也有助于研究选择性剪接，以确定细胞特异性剪接事件所需的顺式元件，证明特定RNA结合蛋白对个体剪接事件的调节，并将这些剪接调节因子与剪接调节的结合相关联。在这里，我们提出了一个通用的小基因质粒载体设计的快速克隆和分析的顺式元件和反式作用因子，影响剪接效率或调节细胞特异性剪接。无处不在的表达和独特的限制性位点允许直接替换各种基因片段，以分析核苷酸取代对剪接的影响，鉴定组织特异性调控元件，或确定对共表达蛋白质或小分子的响应性。
All human genes contain a diverse array of cis-acting elements within introns and exons that are required for correct and efficient precursor messenger RNA (pre-MRNA) splicing. Recent computational analyses predict that most human exons contain elements required for splicing coinciding with an appreciation for the high frequency with which mutations that disruption pre-mRNA splicing cause disease. Minigenes provide a means to directly determine whether disease-causing mutations or single nucleotide polymorphisms (SNPs) affect splicing efficiency. Minigenes have also been instrumental in investigations of alternative splicing to identify cis elements required for cell-specific splicing events, demonstrating regulation of individual splicing events by specific RNA binding proteins, and correlating binding of these splicing regulators with splicing regulation. Here we present a versatile minigene plasmid vector designed for rapid cloning and analysis of cis elements and trans-acting factors that influence splicing efficiency or regulate cell-specific splicing. Ubiquitous expression and unique restriction sites allow for straightforward replacement of a variety of gene segments to analyze the effects of nucleotide substitutions on splicing, to identify tissue-specific regulatory elements, or to determine responsiveness to coexpressed proteins or small molecules.