Global Impact of Arginine Methylation on Splicing in Arabidopsis

精氨酸（R）甲基化引起的翻译后蛋白质修饰在细胞中起关键作用。R甲基化是许多RNA结合蛋白的常见修饰，但其对高等植物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（PRMT）从植物到人类都是保守的，并且与许多生理和病理过程（包括癌症）相关，至少部分地通过它们对参与选择性剪接的RNA结合蛋白的作用而起作用。我们的目的是评估如何翻译后修饰剪接因子的R甲基化影响其功能的前mRNA剪接，从而影响植物的生理过程。作为范例，我们选择拟南芥PRMT5。PRMT5突变体在选择性剪接中显示出全局缺陷，并且具有异常的昼夜节律钟。特别是，时钟基因假反应调节因子9（PRR9）的选择性剪接被改变，表明PRR9的异常剪接有助于减慢时钟。我们将重点关注PRMT 5目标LSM 4、GRP7和GRP8。LSM 4是小核核糖核蛋白复合物的组分，其是剪接体的一部分。Lsm4突变体在一系列AS事件中受到影响，并显示出异常的昼夜节律，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、先天免疫和开花中发挥调节作用。具有改变的PRMT5或GRP7水平的植物在剪接中显示出共同和不同的变化。因此，我们假设PRMT5可能会影响其靶转录物的剪接，并部分通过调节GRP7和GRP8的活性来影响生物钟和开花。我们将通过PRMT5确定R甲基化与LSM4、GRP7和GRP8体内功能的相关性。我们将使可甲基化的R残基突变，使得修饰的蛋白质不能再被甲基化或模拟组成型甲基化变体。在grp7、lsm 4和prmt 5突变体全基因组范围内受影响的剪接事件的比较将揭示PRMT 5对剪接的控制是否涉及GRP7和LSM 4以及在何种程度上涉及GRP7和LSM 4。这些数据将被用来构建一个网络，说明PRMT5和它的目标之间的层次相互作用。 此外，目前还不清楚PRMT作者沉积在RNA结合蛋白上的精氨酸甲基化标记如何在拟南芥中被识别。我们将测试TUDOR蛋白作为GRP 7、GRP 8和LSM 4中甲基化标记读取器的作用。剪接相关的PRMT5靶标与帝舵结构域蛋白的相互作用将通过解释PRMT5活性的输出来提供调节SF活性的提示。这将为植物中的选择性剪接增加一个很大程度上未探索的控制水平，并有助于定义植物中Tudor结构域的功能输出，这是一个很大程度上未探索的主题。

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.