Department of Biology, Program in Molecular Plant Biology, Program in Cell and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USADepartment of Computer Science and Program in Molecular Plant Biology, Colorado State University, Fort Collins, CO 80523, USA.
Plant J. 2012 Dec;72(6):935-46. doi: 10.1111/tpj.12004. Epub 2012 Oct 29.
In Arabidopsis, pre-mRNAs of serine/arginine-rich (SR) proteins undergo extensive alternative splicing (AS). However, little is known about the cis-elements and trans-acting proteins involved in regulating AS. Using a splicing reporter (GFP-intron-GFP), consisting of the GFP coding sequence interrupted by an alternatively spliced intron of SCL33, we investigated whether cis-elements within this intron are sufficient for AS, and which SR proteins are necessary for regulated AS. Expression of the splicing reporter in protoplasts faithfully produced all splice variants from the intron, suggesting that cis-elements required for AS reside within the intron. To determine which SR proteins are responsible for AS, the splicing pattern of the GFP-intron-GFP reporter was investigated in protoplasts of three single and three double mutants of SR genes. These analyses revealed that SCL33 and a closely related paralog, SCL30a, are functionally redundant in generating specific splice variants from this intron. Furthermore, SCL33 protein bound to a conserved sequence in this intron, indicating auto-regulation of AS. Mutations in four GAAG repeats within the conserved region impaired generation of the same splice variants that are affected in the scl33 scl30a double mutant. In conclusion, we have identified the first intronic cis-element involved in AS of a plant SR gene, and elucidated a mechanism for auto-regulation of AS of this intron.
在拟南芥中，丝氨酸/精氨酸丰富（SR）蛋白的前 mRNA 经历广泛的选择性剪接（AS）。然而，对于参与调节 AS 的顺式元件和反式作用蛋白知之甚少。我们使用一个剪接报告基因（GFP-intron-GFP），该报告基因由 GFP 编码序列被一个剪接的 SCL33 内含子打断，研究了这个内含子中的顺式元件是否足以进行 AS，以及哪些 SR 蛋白是调节 AS 所必需的。GFP-intron-GFP 报告基因在原生质体中的表达忠实地产生了来自内含子的所有剪接变体，这表明 AS 所需的顺式元件位于内含子内。为了确定哪些 SR 蛋白负责 AS，我们在三个单突变体和三个双突变体的原生质体中研究了 GFP-intron-GFP 报告基因的剪接模式。这些分析表明，SCL33 和一个密切相关的同源物 SCL30a 在从这个内含子产生特定剪接变体方面是功能冗余的。此外，SCL33 蛋白与这个内含子中的保守序列结合，表明 AS 的自我调节。在保守区域内的四个 GAAG 重复突变破坏了同一剪接变体的产生，而这些剪接变体在 scl33 scl30a 双突变体中受到影响。总之，我们已经确定了第一个参与植物 SR 基因 AS 的内含子顺式元件，并阐明了该内含子 AS 自我调节的机制。
