Lou H, McCullough A J, Schuler M A
Department of Plant Biology, University of Illinois, Urbana 61801.
Plant J. 1993 Mar;3(3):393-403. doi: 10.1046/j.1365-313x.1993.t01-22-00999.x.
In vivo and in vitro gene transfer experiments have suggested that the elements mediating intron recognition differ in mammalian, yeast and plant nuclei. Differences in the sequence dependencies, which also exist between dicotyledonous and monocotyledonous nuclei, have prevented some monocot introns from being spliced in dicot nuclei. To locate elements which modulate efficient recognition of introns in dicot nuclei, the maize Adh1 gene has been expressed in full-length and single intron constructs in Nicotiana benthamiana nuclei using an autonomously replicating plant expression vector. Quantitative PCR-Southern analyses indicate that the inefficient splicing of the maize Adh1 intron 1 (57% AU) in these dicot nuclei can be dramatically enhanced by increasing the degree of U1 snRNA complementarity at the 5' splice site. This indicates that the 5' splice site plays a significant role in defining the splicing efficiency of an intron in dicot nuclei and that, most importantly, the remainder of this monocot intron contains no elements which inhibit its accurate recognition in dicot nuclei. Deletions in intron 3 (66% AU) which effectively move the 3' boundary between AU-rich intron and GC-rich exon sequences strongly activate a cryptic upstream splice site; those which do not reposition this boundary activate a downstream cryptic splice site. This suggests that 3' splice site selection in dicot nuclei is extremely flexible and not dependent on strict sequence requirements but rather on the transition points between introns and exons. Our results are consistent with a model in which potential splice sites are selected if they are located upstream (5' splice site) or downstream (3' splice site) of AU transition points and not if they are embedded within AU-rich sequences.
体内和体外基因转移实验表明，介导内含子识别的元件在哺乳动物、酵母和植物细胞核中存在差异。双子叶植物和单子叶植物细胞核之间也存在序列依赖性差异，这使得一些单子叶植物内含子无法在双子叶植物细胞核中进行剪接。为了定位调节双子叶植物细胞核中内含子有效识别的元件，利用自主复制的植物表达载体，在本氏烟草细胞核中以全长和单内含子构建体表达了玉米Adh1基因。定量PCR- Southern分析表明，通过增加5'剪接位点处U1 snRNA的互补程度，可显著提高这些双子叶植物细胞核中玉米Adh1内含子1（57% AU）的低效剪接效率。这表明5'剪接位点在确定双子叶植物细胞核中内含子的剪接效率方面起着重要作用，最重要的是，该单子叶植物内含子的其余部分不包含抑制其在双子叶植物细胞核中准确识别的元件。内含子3（66% AU）中的缺失有效地移动了富含AU的内含子和富含GC的外显子序列之间的3'边界，强烈激活了一个隐蔽的上游剪接位点；而那些没有重新定位该边界的缺失则激活了一个下游隐蔽剪接位点。这表明双子叶植物细胞核中3'剪接位点的选择极具灵活性，不依赖于严格的序列要求，而是取决于内含子和外显子之间的过渡点。我们的结果与一个模型一致，即如果潜在的剪接位点位于AU过渡点的上游（5'剪接位点）或下游（3'剪接位点），则会被选择，而如果它们嵌入富含AU的序列中则不会被选择。
