CELF family RNA-binding protein UNC-75 regulates two sets of mutually exclusive exons of the unc-32 gene in neuron-specific manners in Caenorhabditis elegans.
CELF family RNA-binding protein UNC-75 regulates two sets of mutually exclusive exons of the unc-32 gene in neuron-specific manners in Caenorhabditis elegans.
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DOI：
10.1371/journal.pgen.1003337
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发表时间：
2013
期刊：
影响因子：
4.5
通讯作者：
Hagiwara M
中科院分区：
文献类型：
--
作者：
Kuroyanagi H;Watanabe Y;Hagiwara M
在多细胞生物体中，大量的可选择的前mRNA剪接模式由有限数量的调节蛋白和顺式元件协调地定义。互斥选择性剪接应该受到严格的监管，是一个具有挑战性的模型，阐明调控机制。在这里，我们提供了两套相互排斥的外显子，4a-4c和7a-7 b的秀丽隐杆线虫不协调（unc）-32基因，编码的一个亚基的V0复合物的液泡型H+-ATP酶的调节模型。我们可视化的选择模式的外显子4和外显子7在体内通过利用一个三人组和一对对称的荧光剪接报告基因，分别，以证明它们在组织特异性的方式进行调节。遗传分析表明，RBFOX家族RNA结合蛋白ASD-1和FOX-1以及内含子7 b中的UGCAUG延伸参与外显子7a的神经元特异性选择。通过进一步的正向遗传筛选，我们确定了一个神经元特异性的CELF家族RNA结合蛋白，其功能未知，是外显子7a选择的重要调节因子。电泳迁移率变动分析指定内含子7a中的一个短片段作为识别位点，并证明了ESTA-75通过其三个RNA识别基序特异性地结合到包括UUGUUGUUGU延伸的元件。报告基因小基因中的UUGUUGUUGU片段实际上是神经系统中选择外显子7a所必需的。我们比较了野生型和unc-75突变体背景中部分剪接RNA的数量，并提出了一个RBFOX家族和UNC-75对unc-32外显子7进行互斥选择的模型。unc-32外显子4 b的神经元特异性选择也受nc-75的调节，并且unc-75突变抑制unc-32突变体的外显子4 b特异性等位基因的Unc表型。两者合计，nc-75是神经元特异性剪接因子，并调节unc-32基因的两组互斥外显子。组织特异性和相互排斥的替代性前体mRNA剪接是一个具有挑战性的模型，阐明调控机制。我们先前证明了进化上保守的RBFOX家族RNA结合蛋白ASD-1和FOX-1以及肌肉特异性RNA结合蛋白E12协同指导C.线虫egl-15基因。在这里，我们证明了两组相互排斥的外显子，4a-4c和7a-7 b，unc-32基因的组织特异性的方式进行调节，ASD-1和FOX-1，在各种组织中表达，可以调节神经元特异性选择unc-32外显子7a与神经元特异性CELF家族RNA结合蛋白结合。我们确定了RBFOX家族的顺式元件和RBFOX-75，它们分别位于内含子7 b和内含子7a。通过分析部分剪接的RNA种类，我们提出了内含子去除的顺序和作用位点的RBFOX家族和ESTA-75在互斥选择外显子7a和外显子7 b。我们还证明，ESTA-75调节外显子4 b的神经元特异性选择，并提出了模型的互斥选择外显子4a，4 b和4c。因此，这些研究提供了组织特异性和相互排斥的选择性剪接在体内的新的调节模式。
An enormous number of alternative pre–mRNA splicing patterns in multicellular organisms are coordinately defined by a limited number of regulatory proteins and cis elements. Mutually exclusive alternative splicing should be strictly regulated and is a challenging model for elucidating regulation mechanisms. Here we provide models of the regulation of two sets of mutually exclusive exons, 4a–4c and 7a–7b, of the Caenorhabditis elegans uncoordinated (unc)-32 gene, encoding the a subunit of V0 complex of vacuolar-type H+-ATPases. We visualize selection patterns of exon 4 and exon 7 in vivo by utilizing a trio and a pair of symmetric fluorescence splicing reporter minigenes, respectively, to demonstrate that they are regulated in tissue-specific manners. Genetic analyses reveal that RBFOX family RNA–binding proteins ASD-1 and FOX-1 and a UGCAUG stretch in intron 7b are involved in the neuron-specific selection of exon 7a. Through further forward genetic screening, we identify UNC-75, a neuron-specific CELF family RNA–binding protein of unknown function, as an essential regulator for the exon 7a selection. Electrophoretic mobility shift assays specify a short fragment in intron 7a as the recognition site for UNC-75 and demonstrate that UNC-75 specifically binds via its three RNA recognition motifs to the element including a UUGUUGUGUUGU stretch. The UUGUUGUGUUGU stretch in the reporter minigenes is actually required for the selection of exon 7a in the nervous system. We compare the amounts of partially spliced RNAs in the wild-type and unc-75 mutant backgrounds and raise a model for the mutually exclusive selection of unc-32 exon 7 by the RBFOX family and UNC-75. The neuron-specific selection of unc-32 exon 4b is also regulated by UNC-75 and the unc-75 mutation suppresses the Unc phenotype of the exon-4b-specific allele of unc-32 mutants. Taken together, UNC-75 is the neuron-specific splicing factor and regulates both sets of the mutually exclusive exons of the unc-32 gene. Tissue-specific and mutually exclusive alternative pre–mRNA splicing is a challenging model for elucidating regulation mechanisms. We previously demonstrated that evolutionarily conserved RBFOX family RNA–binding proteins ASD-1 and FOX-1 and a muscle-specific RNA–binding protein SUP-12 cooperatively direct muscle-specific selection of exon 5B of the C. elegans egl-15 gene. Here we demonstrate that two sets of mutually exclusive exons, 4a–4c and 7a–7b, of the unc-32 gene are regulated in tissue-specific manners and that ASD-1 and FOX-1, expressed in a variety of tissues, can regulate the neuron-specific selection of unc-32 exon 7a in combination with the neuron-specific CELF family RNA–binding protein UNC-75. We determine the cis-elements for the RBFOX family and UNC-75, which separately reside in intron 7b and intron 7a, respectively. By analyzing the partially spliced RNA species, we propose the orders of intron removal and the sites of action for the RBFOX family and UNC-75 in the mutually exclusive selection of exon 7a and exon 7b. We also demonstrate that UNC-75 regulates the neuron-specific selection of exon 4b and propose the models of the mutually exclusive selection of exons 4a, 4b, and 4c. These studies thus provide novel modes of regulation for tissue-specific and mutually exclusive alternative splicing in vivo.
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