Alternative splicing landscape in Arabidopsis

Alternative splicing (AS) is a key regulatory mechanism that contributes to transcriptome and proteome diversity. As very few genome-wide studies analyzing AS in plants are available, we have performed high-throughput sequencing of a normalized cDNA library which resulted in a high coverage transcriptome map of Arabidopsis. We detect ~150,000 splice junctions derived mostly from typical plant introns, including a large number of U12 introns (2,069). Around 61% of multi-exonic genes are alternatively spliced under normal growth conditions. These values are much higher than previously reported and imply a significant role of AS in Arabidopsis transcriptome complexity. Moreover we provide experimental validation of 540 AS transcripts (from 256 genes coding for important regulatory factors) using high resolution RT-PCR and Sanger sequencing. Intron retention (IR) is the most frequent AS event (~40%) but many IRs have relatively low read coverage and are less well represented in assembled transcripts. Additionally ~51% of Arabidopsis genes produce AS transcripts which do not involve IR. Therefore the significance of IR in generating transcript diversity is in general overestimated. IR analysis allowed the identification of a large set of cryptic introns inside annotated exons. Importantly 29% of these cryptic introns are in frame indicating a role in protein diversity. Furthermore we show extensive AS coupled to nonsense-mediated decay in AFC2, encoding a highly conserved LAMMER kinase which phosphorylates splicing factors, thus establishing a complex loop in AS regulation. We provide the most comprehensive analysis of AS to date which will serve as a valuable resource for the plant community to study transcriptome complexity and gene regulation.

可变剪接（Alternative splicing, AS）是促成转录组与蛋白质组多样性的关键调控机制。鉴于目前已发表的植物全基因组水平可变剪接研究寥寥无几，本研究对归一化互补DNA（complementary DNA, cDNA）文库开展高通量测序，构建了覆盖度极高的拟南芥（Arabidopsis）转录组图谱。本研究共检测到约15万个剪接位点，其来源多为典型的植物内含子，其中包含2069个U12型内含子。正常生长条件下，约61%的多外显子基因发生可变剪接。该数值远高于此前报道结果，提示可变剪接在拟南芥转录组复杂性调控中发挥重要作用。此外，本研究通过高分辨率逆转录聚合酶链反应（reverse transcription polymerase chain reaction, RT-PCR）与桑格测序（Sanger sequencing），对来自256个编码重要调控因子的基因的540条可变剪接转录本进行了实验验证。内含子滞留（Intron retention, IR）是最为常见的可变剪接事件（占比约40%），但多数内含子滞留事件的测序读段覆盖度相对较低，在组装得到的转录本中占比也偏低。此外，约51%的拟南芥基因所产生的可变剪接转录本并不涉及内含子滞留事件，由此可见，学界对于内含子滞留在转录组多样性生成中的作用普遍存在高估。对内含子滞留的分析还鉴定出一大批注释外显子内部的隐蔽内含子。值得注意的是，其中29%的隐蔽内含子处于开放阅读框内，提示其可参与蛋白质多样性的调控。进一步研究发现，在编码高度保守的LAMMER激酶（可磷酸化剪接因子）的AFC2基因中，存在大量与无义介导的mRNA降解（nonsense-mediated decay, NMD）相关联的可变剪接事件，由此构建了可变剪接调控的复杂反馈环路。本研究提供了截至目前最为全面的可变剪接分析数据，将为植物学界开展转录组复杂性与基因调控相关研究提供宝贵的参考资源。