Lopato S, Mayeda A, Krainer A R, Barta A
Institute of Biochemistry, Vienna Biocenter, Austria.
Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):3074-9. doi: 10.1073/pnas.93.7.3074.
The fact that animal introns are not spliced out in plants suggests that recognition of pre-mRNA splice sites differs between the two kingdoms. In plants, little is known about proteins required for splicing, as no plant in vitro splicing system is available. Several essential splicing factors from animals, such as SF2/ASF and SC-35, belong to a family of highly conserved proteins consisting of one or two RNA binding domain(s) (RRM) and a C-terminal Ser/Arg-rich (SR or RS) domain. These animal SR proteins are required for splice site recognition and spliceosome assembly. We have screened for similar proteins in plants by using monoclonal antibodies specific for a phosphoserine epitope of the SR proteins (mAb1O4) or for SF2/ASF. These experiments demonstrate that plants do possess SR proteins, including SF2/ASF-like proteins. Similar to the animal SR proteins, this group of proteins can be isolated by two salt precipitations. However, compared to the animal SR proteins, which are highly conserved in size and number, SR proteins from Arabidopsis, carrot, and tobacco exhibit a complex pattern of intra- and interspecific variants. These plant SR proteins are able to complement inactive HeLa cell cytoplasmic S1OO extracts that are deficient in SR proteins, yielding functional splicing extracts. In addition, plant SR proteins were active in a heterologous alternative splicing assay. Thus, these plant SR proteins are authentic plant splicing factors.
动物内含子在植物中不能被剪接出去，这一事实表明两个王国中前体mRNA剪接位点的识别存在差异。在植物中，由于没有可用的植物体外剪接系统，关于剪接所需蛋白质的了解很少。来自动物的几种必需剪接因子，如SF2/ASF和SC-35，属于一类高度保守的蛋白质家族，由一个或两个RNA结合结构域（RRM）和一个C末端富含丝氨酸/精氨酸（SR或RS）的结构域组成。这些动物SR蛋白是剪接位点识别和剪接体组装所必需的。我们通过使用针对SR蛋白的磷酸丝氨酸表位（单克隆抗体1O4）或针对SF2/ASF的单克隆抗体，在植物中筛选了类似的蛋白质。这些实验表明植物确实拥有SR蛋白，包括类似SF2/ASF的蛋白。与动物SR蛋白相似，这组蛋白可以通过两次盐沉淀分离出来。然而，与大小和数量高度保守的动物SR蛋白相比，来自拟南芥、胡萝卜和烟草的SR蛋白表现出种内和种间变体的复杂模式。这些植物SR蛋白能够补充缺乏SR蛋白的无活性HeLa细胞胞质S100提取物，产生功能性剪接提取物。此外，植物SR蛋白在异源可变剪接试验中具有活性。因此，这些植物SR蛋白是真正的植物剪接因子。
