Muscle-Specific Splicing Factors ASD-2 and SUP-12 Cooperatively Switch Alternative Pre-mRNA Processing Patterns of the ADF/Cofilin Gene in Caenorhabditis elegans

Pre–mRNAs are often processed in complex patterns in tissue-specific manners to produce a variety of protein isoforms from single genes. However, mechanisms orchestrating the processing of the entire transcript are not well understood. Muscle-specific alternative pre–mRNA processing of the unc-60 gene in Caenorhabditis elegans, encoding two tissue-specific isoforms of ADF/cofilin with distinct biochemical properties in regulating actin organization, provides an excellent in vivo model of complex and tissue-specific pre–mRNA processing; it consists of a single first exon and two separate series of downstream exons. Here we visualize the complex muscle-specific processing pattern of the unc-60 pre–mRNA with asymmetric fluorescence reporter minigenes. By disrupting juxtaposed CUAAC repeats and UGUGUG stretch in intron 1A, we demonstrate that these elements are required for retaining intron 1A, as well as for switching the processing patterns of the entire pre–mRNA from non-muscle-type to muscle-type. Mutations in genes encoding muscle-specific RNA–binding proteins ASD-2 and SUP-12 turned the colour of the unc-60 reporter worms. ASD-2 and SUP-12 proteins specifically and cooperatively bind to CUAAC repeats and UGUGUG stretch in intron 1A, respectively, to form a ternary complex in vitro. Immunohistochemical staining and RT–PCR analyses demonstrate that ASD-2 and SUP-12 are also required for switching the processing patterns of the endogenous unc-60 pre-mRNA from UNC-60A to UNC-60B in muscles. Furthermore, systematic analyses of partially spliced RNAs reveal the actual orders of intron removal for distinct mRNA isoforms. Taken together, our results demonstrate that muscle-specific splicing factors ASD-2 and SUP-12 cooperatively promote muscle-specific processing of the unc-60 gene, and provide insight into the mechanisms of complex pre-mRNA processing; combinatorial regulation of a single splice site by two tissue-specific splicing regulators determines the binary fate of the entire transcript.前体mRNA（pre-mRNA）通常以复杂且组织特异性的模式进行加工，从而从单个基因产生多种蛋白质异构体。然而，调控整条转录本加工的分子机制尚未被充分阐释。秀丽隐杆线虫（Caenorhabditis elegans）中unc-60基因的肌肉特异性可变前体mRNA加工，可编码两种在调控肌动蛋白组织过程中具备不同生化特性的组织特异性肌动蛋白解聚因子/丝切蛋白（ADF/cofilin）异构体，为复杂且组织特异性的前体mRNA加工提供了极佳的体内研究模型；该加工路径包含单个第一外显子与两组独立的下游外显子序列。 本研究借助不对称荧光报告基因迷你基因，可视化了unc-60前体mRNA复杂的肌肉特异性加工模式。通过破坏内含子1A中相邻的CUAAC重复序列与UGUGUG延伸序列，我们证实这些元件对于保留内含子1A、以及将整条前体mRNA的加工模式从非肌肉型转换为肌肉型均是必需的。 编码肌肉特异性RNA结合蛋白ASD-2与SUP-12的基因突变，会使携带unc-60报告基因的线虫的荧光颜色发生改变。ASD-2与SUP-12蛋白可分别特异性结合内含子1A中的CUAAC重复序列与UGUGUG延伸序列，并在体外形成三元复合物。免疫组织化学染色与逆转录聚合酶链反应（RT-PCR）分析显示，ASD-2与SUP-12对于肌肉内源性unc-60前体mRNA的加工模式从UNC-60A转换为UNC-60B同样不可或缺。 此外，对部分剪接RNA的系统性分析揭示了不同mRNA异构体的内含子实际切除顺序。综合来看，本研究结果证实肌肉特异性剪接因子ASD-2与SUP-12可协同促进unc-60基因的肌肉特异性加工，并为复杂前体mRNA加工机制提供了新见解：两种组织特异性剪接调控因子对单个剪接位点的组合调控，决定了整条转录本的二元加工命运。