Global Impact of Arginine Methylation on Splicing in Arabidopsis

精氨酸(R)甲基化的翻译后蛋白质修饰在细胞中起着关键作用。R甲基化是许多RNA结合蛋白的常见修饰，但它对高等植物RNA加工的影响在很大程度上是未知的。催化这些修饰的蛋白质精氨酸甲基转移酶(PRMTs)从植物到人类都是保守的，与包括癌症在内的许多生理和病理过程有关，至少部分通过它们对参与选择性剪接的RNA结合蛋白的作用来发挥作用。我们的目的是评估R甲基化对剪接因子的翻译后修饰如何影响它们在前mRNA剪接中的功能，从而影响植物的生理过程。作为一个范例，我们选择了拟南芥PRMT5。PRMT5突变体表现出选择性剪接的全局缺陷，并具有异常的生物钟。特别是，时钟基因PSEUDORESPONSE调节器9(PRR9)的选择性剪接发生了改变，这表明PRR9的异常剪接有助于减慢时钟。我们将重点关注PRMT5目标LSM4、GRP7和GRP8。LSM4是小核核糖核蛋白复合体的组成部分，是剪接体的一部分。Lsm4突变体在一系列AS事件中受到影响，并表现出异常的昼夜节律，表明剪接体的一个组成部分影响时钟功能。富含甘氨酸的RNA结合蛋白GRP7和GRP8调节选择性剪接，并在昼夜节律系统、先天性免疫和开花中发挥调节作用。PRMT5或GRP7水平改变的植物在剪接方面既有共同的也有明显的变化。因此，我们假设PRMT5可能通过调节GRP7和GRP8的活性来影响其一些目标转录本的剪接，以及它对生物钟和开花的影响。我们将在体内确定PRMT5的R甲基化与LSM4、GRP7和GRP8功能的相关性。我们将突变可甲基化的R残基，这样修饰的蛋白质就不能再甲基化或模仿构成甲基化的变体。对grp7、lsm4和prmt5全基因组突变体中影响剪接事件的比较将揭示PRMT5对剪接的控制是否涉及grp7和lsm4，以及在多大程度上涉及grp7和lsm4。这些数据将用于构建一个网络，说明PRMT5与其目标之间的层级互动。此外，目前还不清楚PRMT编写者在RNA结合蛋白上沉积的精氨酸甲基化标记是如何在拟南芥中识别的。我们将测试都铎蛋白作为GRP7、GRP8和LSM4甲基化标记阅读器的作用。剪接相关的PRMT5靶标与Tudor结构域蛋白的相互作用将通过解释PRMT5活性的输出为调节SFS的活性提供线索。这将为植物中的选择性剪接增加一种基本上未被探索的控制水平，并有助于定义植物中都铎结构域的功能输出，这在很大程度上是一个未被探索的话题。

Posttranslational protein modification by arginine (R) methylation plays a key role in the cell. R methylation emerges as a common modification of many RNA-binding proteins, but its impact on RNA processing in higher plants is largely unknown. Protein arginine methyl transferases (PRMTs) that catalyze these modifications are conserved from plants to humans and are connected to many physiological and pathological processes, including cancer, acting at least in part through their effect on RNA-binding proteins involved in alternative splicing. We aim to evaluate how posttranslational modification of splicing factors by R methylation affects their function in pre-mRNA splicing and thus impacts physiological processes in plants. As a paradigm we chose Arabidopsis thaliana PRMT5. PRMT5 mutants show a global defect in alternative splicing and have an aberrant circadian clock. In particular, alternative splicing of the clock gene PSEUDORESPONSE REGULATOR 9 (PRR9) is altered, suggesting that aberrant splicing of PRR9 contributes to slowing down the clock. We will focus on the PRMT5 targets LSM4, GRP7 and GRP8. LSM4 is a component of the small nuclear ribonucleoprotein complexes that are part of the spliceosome. Lsm4 mutants are affected in a suite of AS events and show aberrant circadian rhythms, suggesting that a component of the spliceosome affects clock function. The glycine-rich RNA-binding proteins GRP7 and GRP8 regulate alternative splicing and play a regulatory role in the circadian system, innate immunity, and flowering. Plants with altered PRMT5 or GRP7 levels show both common and distinct changes in splicing. Thus, we hypothesize that PRMT5 may affect splicing of some of its target transcripts and its impact on the circadian clock and flowering partly by modulating GRP7 and GRP8 activity. We will determine the relevance of R methylation by PRMT5 for the LSM4, GRP7, and GRP8 in vivo function. We will mutate the methylable R residues so that the modified proteins can no longer be methylated or mimic a constitutively methylated variant. A comparison of splicing events affected in grp7, lsm4 and prmt5 mutants genome-wide will reveal if and to which extent PRMT5 control of splicing involves GRP7 and LSM4. These data will be used to construct a network illustrating hierarchical interactions between PRMT5 and its targets. Furthermore, it is unknown how the arginine methylation marks deposited on RNA-binding proteins by the PRMT writers are recognized in Arabidopsis. We will test TUDOR proteins for a role as readers of the methylation marks in GRP7, GRP8, and LSM4. An interaction of splicing-relevant PRMT5 targets with a TUDOR domain protein would provide a hint for a regulation of the activity of SFs by interpreting the output of PRMT5 activity. This would add a largely unexplored level of control to alternative splicing in plants and contribute to defining the functional output of Tudor domains in plants, a largely unexplored topic.