Network of interactions of a novel plant-specific Arg/Ser-rich protein, atRSZ33, with atSC35-like splicing factors
Network of interactions of a novel plant-specific Arg/Ser-rich protein, atRSZ33, with atSC35-like splicing factors
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DOI：
10.1074/jbc.m206455200
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发表时间：
2002-10-18
影响因子：
4.8
通讯作者：
Barta, A
中科院分区：
文献类型：
--
作者：
Lopato, S;Forstner, C;Barta, A
精氨酸/丝氨酸丰富（RS）蛋白质在剪接中起着至关重要的作用，并参与后生动物剪接位点的选择。在植物中，由于特定的因子要求，内含子识别似乎与动物中的内含子识别不同。在这里，我们描述了一种新的植物特异性RS丰富的蛋白，atRSZ 33，具有独特的结构域结构组成的RNA识别基序（RRM），两个锌关节嵌入在一个基本的RS区域，和一个酸性的C-末端结构域。atRSZ 33被发现是一种磷蛋白，集中在核斑点中，主要存在于根和花中。在酵母双杂交筛选中，atRSZ 33与剪接因子atSRp 34/SR 1相互作用，atSRp 34/SR 1是人SF 2/ASF的拟南芥直向同源物; atRSZp 21和atRSZp 22与人9 G8相似;以及三种新的SC 35样剪接因子atSCL 28，atSCL 30和atSCL 33/SR 33。SCL家族的另外两个成员，即SCL 30 a和哺乳动物SC 35的直系同源物atSC 35，也被发现与atRSZ 33相互作用。通过体外结合试验验证了这些相互作用;此外，发现atRSZ 33的转录活性与其相互作用伴侣的转录活性重叠。这些特异性相互作用加上atRSZ 33与SR蛋白的许多相似性表明其主要活性是剪接体组装。atRSZ 33和atSCL 33/SR 33之间蛋白质-蛋白质相互作用所必需的区域的作图揭示了两个锌关节以及RS和RRM结构域的一小部分是有效结合所必需的。然而，相互作用结构域相对较小，允许结合额外的蛋白质，这一特征与atRSZ 33在剪接体组装中的拟议作用一致。
Arg/Ser-rich (RS) proteins play a crucial role in splicing and are implicated in splice site selection in metazoa. In plants, intron recognition seems to differ from the one in animals due to specific factor requirements. Here we describe a new plant-specific RS-rich protein, atRSZ33, with a unique domain structure consisting of an RNA recognition motif (RRM), two zinc knuckles embedded in a basic RS region, and an acidic C-terminal domain. atRSZ33 was found to be a phosphoprotein that concentrates in nuclear speckles and is predominantly present in roots and flowers. In a yeast two-hybrid screen, atRSZ33 interacted with splicing factors atSRp34/SR1, an Arabidopsis ortholog of human SF2/ASF; atRSZp21 and atRSZp22, which are similar to the human 9G8; and three novel SC35-like splicing factors termed atSCL28, atSCL30, and atSCL33/SR33. Two further members of the SCL family, namely SCL30a and the ortholog of mammalian SC35, atSC35, were also found to interact with atRSZ33. These interactions were verified by in vitro binding assays; furthermore, the transcriptional activity of atRSZ33 was found to overlap with the ones of its interacting partners. These specific interactions coupled with the many similarities of atRSZ33 to SR proteins suggest that its main activity is in spliceosome assembly. Mapping of regions necessary for protein-protein interaction between atRSZ33 and atSCL33/SR33 revealed that both zinc knuckles together with a small part of the RS and the RRM domain are required for efficient binding. However, the interacting domain is relatively small, allowing binding of additional proteins, a feature that is consistent with the proposed role of atRSZ33 in spliceosome assembly.