Temperature-dependent phase separation of RNA binding proteins in chloroplasts

植物作为固着生物，在受到包括温度胁迫在内的环境胁迫时不能奔跑。相反，它们已经发展出了大量复杂的生理和分子反应来维持体内平衡。相分离对植物逆境响应的重要性才刚刚显现。我们发现叶绿体RNA结合蛋白CP 29 A能够在体内和体外进行液-液相分离（LLPS）。相分离由低温诱导，在返回到环境温度后完全可逆，并且取决于CP 29 A的朊病毒样结构域（PLD）的存在。CP 29 A的破坏或PLD的缺失导致叶片漂白和受损的光合活性在寒冷中。eCLIP和RNA-bind-and-seq实验鉴定了CP 29 A的多个叶绿体RNA靶标，包括rbcL mRNA。CP 29 A的突变体在寒冷中显示rbcL mRNA的强烈减少。重组CP 29 A的NMR和生物物理分析证实了低温诱导的LLPS，并提供了在残留物水平详细了解凝聚相中的分子相互作用。此外，CP 29 As两个单独的RRM基序的NMR分析鉴定了它们的RNA结合性质的惊人差异。受到我们对CP 29 A的发现的鼓舞，我们询问其他叶绿体RBP是否也能够LLPS。使用计算机预测，在体内瞬时表达分析和体外LLPS测定的组合，我们提出的证据参与叶绿体RNA编辑和叶绿体翻译所需的三种蛋白质的LLPS能力的两个蛋白质。我们现在建议跟进这些功能LLPS叶绿体RBP的第一个描述与（i）的生化和生物物理机制的详细分析CP 29 A LLPS，和（ii）通过LLPS的功能的一般表征的额外的叶绿体RNA结合蛋白居住在这里发现的颗粒。为了实现这一目标，我们将植物遗传学和植物RNA生物学（Schmitz-Linneweber实验室）的互补专业知识与大分子结构，动力学和相互作用分析（Sattler实验室）的专业知识结合起来。我们认为，我们的方法的优势是将LLPS的详细生物物理和生物化学探索与植物的分子，特别是器官表型联系起来的协同作用。此外，我们成立，以确定什么允许LLPS发生在冷移位响应的细节。最后，LLPS很少在植物中进行分析，尽管事实上植物作为固着生物将极大地受益于LLPS是一种适应环境变化的调节选择。我们能够填补一些叶绿体蛋白质的这一空白。

Plants as sessile organisms cannot run when challenged by environmental stress, including temperature stress. Instead, they have developed a plethora of sophisticated physiological and molecular reactions to maintain homeostasis. The importance of phase separation for plant stress response is just emerging. We found that the chloroplast RNA binding protein CP29A is capable of liquid-liquid phase separation (LLPS) in vivo and in vitro. Phase separation is induced by low temperatures, fully reversible after return to ambient temperature and depends on the presence of CP29A’s prion-like domain (PLD). Disruption of CP29A or deletion of the PLD leads to leaf bleaching and compromised photosynthetic activity in the cold. eCLIP and RNA-bind-and-seq experiments identified multiple chloroplast RNA targets of CP29A, including the rbcL mRNA. Mutants of CP29A show a strong reduction of rbcL mRNA in the cold. NMR and biophysical analyses of recombinant CP29A confirm low temperature induced LLPS and provide insight into molecular interactions in the condensed phase at residue level detail. Moreover, NMR analysis of CP29As two individual RRM motifs identified surprising differences in their RNA binding properties. Encouraged by our findings with CP29A, we asked whether other chloroplast RBPs are also capable of LLPS. Using a combination of in silico predictions, in vivo transient expression analysis and in vitro LLPS assays, we present evidence for two proteins involved in chloroplast RNA editing and three proteins required for chloroplast translation capable of LLPS. We now propose to follow up these first descriptions of functional LLPS of chloroplast RBPs with (i) a detailed analysis of the biochemical and biophysical mechanisms underlying CP29A LLPS, and (ii) by a general characterization of the function of LLPS for the additional chloroplast RNA binding proteins residing in granules discovered here. For this goal, we bring together our complementary expertise in plant genetics and plant RNA biology (Schmitz-Linneweber lab) with expertise in the analysis of macromolecule structure, dynamics and interactions (Sattler lab). We believe that a strength of our approach are synergies from linking detailed biophysical and biochemical exploration of LLPS with molecular and in particular organismic phenotypes for plants. Furthermore, we are set up to determine the details of what allows LLPS to occur in response to a cold-shift. Finally, LLPS has rarely been analyzed in plants, despite the fact that plants as sessile organisms would greatly benefit from LLPS is a regulatory option to adjust to environmental changes. We are in a position to fill this gap for a number of chloroplast proteins.