Data_Sheet_1_A Role of U12 Intron in Proper Pre-mRNA Splicing of Plant Cap Binding Protein 20 Genes.docx

The nuclear cap-binding complex (CBC) is composed of two cap-binding proteins: CBP20 and CBP80. The CBP20 gene structure is highly conserved across land plant species. All studied CBP20 genes contain eight exons and seven introns, with the fourth intron belonging to the U12 class. This highly conserved U12 intron always divides the plant CBP20 gene into two parts: one part encodes the core domain containing the RNA binding domain (RBD), and the second part encodes the tail domain with a nuclear localization signal (NLS). In this study, we investigate the importance of the U12 intron in the Arabidopsis thaliana CBP20 gene by moving it to different intron locations of the gene. Relocation of the U12 intron resulted in a significant decrease in the U12 intron splicing efficiency and the accumulation of wrongly processed transcripts. These results suggest that moving the U12 intron to any other position of the A. thaliana CBP20 gene disturbs splicing, leading to substantial downregulation of the level of properly spliced mRNA and CBP20 protein. Moreover, the replacement of the U12 intron with a U2 intron leads to undesired alternative splicing events, indicating that the proper localization of the U12 intron in the CBP20 gene secures correct CBP20 pre-mRNA maturation and CBP20 protein levels in a plant. Surprisingly, our results also show that the efficiency of U12 splicing depends on intron length. In conclusion, our study emphasizes the importance of proper U12 intron localization in plant CBP20 genes for correct pre-mRNA processing.

细胞核帽结合复合物（nuclear cap-binding complex, CBC）由两种帽结合蛋白组成：CBP20与CBP80。陆生植物物种间的CBP20基因结构高度保守。所有已研究的CBP20基因均包含8个外显子与7个内含子，其中第4个内含子属于U12型内含子。这一高度保守的U12型内含子始终将植物CBP20基因划分为两个区段：一段编码包含RNA结合结构域（RNA binding domain, RBD）的核心结构域，另一段编码携带核定位信号（nuclear localization signal, NLS）的尾部结构域。本研究通过将拟南芥（Arabidopsis thaliana）CBP20基因中的U12型内含子迁移至该基因的不同内含子位置，探究其功能重要性。将U12型内含子异位后，其剪接效率显著下降，且错误加工的转录本积累量增加。上述结果表明，将U12型内含子迁移至拟南芥CBP20基因的任意其他位置均会干扰剪接过程，进而导致正确剪接的mRNA与CBP20蛋白水平显著下调。此外，将U12型内含子替换为U2型内含子会引发异常的可变剪接事件，这提示CBP20基因中U12型内含子的正确定位可保障植物体内CBP20前体mRNA的正确成熟与蛋白水平。令人意外的是，本研究结果还显示U12型内含子的剪接效率取决于内含子自身长度。综上，本研究强调了植物CBP20基因中U12型内含子的正确定位对前体mRNA正确加工的关键意义。