Identification of Functional Exonic Splicing Enhancer Motifs Recognized by Individual Sr Proteins Using an in Vitro Randomization and Functional Selection Procedure, We Have Identified Three Novel Classes of Exonic Splicing Enhancers (eses) Recognized by Human Sf2/asf, Srp40, and Srp55, Respectively-猫眼课题宝

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Hong-Xiang Liu;Michael Q. Zhang;A. Krainer

这三个共有序列在外显子中出现的频率高于内含子中的频率，因此可能有助于定义外显子-内含子边界。它们以簇的形式出现在与自然发生的、映射的 ESE 相对应的区域内。我们得出的结论是，一组非常多样化的序列可以充当 ESE。这些基序的简并性与外显子增强子在极其多样化的蛋白质编码序列中进化并被少数以有限的序列特异性结合RNA的SR蛋白识别的事实相一致。 mRNA 前体剪接由两个酯交换反应组成，这些反应发生在称为剪接体的大型 RNA-蛋白质复合物中。这种高保真过程需要剪接体精确识别内含子-外显子边界。保守性差的后生动物剪接位点和分支位点没有为这种识别提供足够的信息。额外的内含子和外显子序列对于许多高等真核前体 mRNA 的有效和/或准确剪接通常是必需的。积极的外显子顺式作用元件，称为外显子剪接增强子（ESE），通常（但并非总是）在富含嘌呤的环境中发现。一个经过充分研究的例子是小鼠 IgM 基因替代外显子 M2 中的 ESE。该 73 个核苷酸的 ESE 对于外显子 M1 和 M2 之间前面的内含子的剪接至关重要。 M2 ESE 还可以刺激果蝇双性 (dsx) 基因的异源调节内含子的剪接。 IgM 前体 mRNA 中的增强子活性也可以通过插入某些天然或合成的富含嘌呤的序列代替天然 ESE 来获得。然而，删除 M2 ESE 中富含嘌呤的序列并不能完全消除其活性（Watakabe 等人，1993 年；Tanaka 等人，1994 年）。与这一发现一致的是，SELEX 实验表明某些非富含嘌呤的序列也可以充当增强子（Tian 和 Kole 1995；Coulter 等人 1997）。大多数天然 ESE 已在组织特异性或发育调控的外显子中发现，这些外显子通常具有较弱的剪接位点，需要 ESE 来包含外显子。在某些情况下，ESE 被一种或多种 SR 蛋白特异性识别。反过来，SR 蛋白在不同组织中以不同水平表达，并且它们的表达似乎也受到选择性剪接的调节（Jumaa 等人，1997 年；综述参见 Cá-ceres 和 Krainer，1997 年）。 SR 蛋白是一个高度保守的富含丝氨酸/精氨酸的 RNA 结合蛋白家族。它们是重要的剪接因子（Krainer 等人，1990b，1991；Ge 等人，1991；Zahler 等人，1992），并且还调节……

These three consensus sequences occur at higher frequencies in exons than in introns and may thus help define exon–intron boundaries. They occur in clusters within regions corresponding to naturally occurring, mapped ESEs. We conclude that a remarkably diverse set of sequences can function as ESEs. The degeneracy of these motifs is consistent with the fact that exonic enhancers evolved within extremely diverse protein coding sequences and are recognized by a small number of SR proteins that bind RNA with limited sequence specificity. Pre-mRNA splicing consists of two trans-esterification reactions, which occur in a large RNA–protein complex termed the spliceosome. This high-fidelity process requires precise recognition of the intron–exon borders by the spliceosome. The poorly conserved metazoan splice sites and branch site do not provide sufficient information for this recognition. Additional intron and exon sequences are often necessary for efficient and/or accurate splicing of many higher eukaryotic pre-mRNAs. The positive exon cis-acting elements, known as exonic splicing enhancers (ESEs), are often, though not always, found in a purine-rich context. A well-studied example is the ESE in the alternative exon M2 of the mouse IgM gene. This 73-nucleotide ESE is essential for splicing of the preceding intron between exons M1 and M2. The M2 ESE can also stimulate splicing of a heterologous regulated intron of the Drosophila doublesex (dsx) gene. En-hancer activity in the context of the IgM pre-mRNA could also be obtained by insertion of certain natural or synthetic purine-rich sequences in place of the natural ESE. However, deletion of the purine-rich sequences within the M2 ESE did not abolish its activity completely (Watakabe et al. 1993; Tanaka et al. 1994). In agreement with this finding, SELEX experiments revealed that certain nonpurine-rich sequences can also function as enhancers (Tian and Kole 1995; Coulter et al. 1997). Most natural ESEs have been identified in tissue-specific or developmentally regulated exons, which typically have weak splice sites and require the ESE for exon inclusion. In some cases, ESEs are specifically recognized by one or more SR proteins In turn, SR proteins are expressed at different levels in different tissues, and their expression also appears to be regulated by alternative splicing (Jumaa et al. 1997; for review, see Cá-ceres and Krainer 1997). The SR proteins are a family of highly conserved ser-ine/arginine-rich RNA-binding proteins. They are essential splicing factors (Krainer et al. 1990b, 1991; Ge et al. 1991; Zahler et al. 1992) and also regulate …

Identification of Functional Exonic Splicing Enhancer Motifs Recognized by Individual Sr Proteins Using an in Vitro Randomization and Functional Selection Procedure, We Have Identified Three Novel Classes of Exonic Splicing Enhancers (eses) Recognized by Human Sf2/asf, Srp40, and Srp55, Respectively

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