Temperature-dependent phase separation of RNA binding proteins in chloroplasts

植物作为无根生物，在受到环境压力（包括温度压力）的挑战时无法运行。相反，它们已经发展出大量复杂的生理和分子反应来维持体内平衡。相分离在植物逆境响应中的重要性才刚刚显现。我们发现叶绿体RNA结合蛋白CP29A在体内和体外具有液-液相分离（LLPS）的能力。相分离是由低温诱导的，在返回环境温度后完全可逆，依赖于CP29A的朊病毒样结构域（PLD）的存在。CP29A的破坏或PLD的缺失会导致叶片白化和光合活性降低。eCLIP和RNA binding -and-seq实验鉴定出CP29A的多个叶绿体RNA靶点，包括rbcL mRNA。CP29A突变体在寒冷条件下表现出rbcL mRNA的强烈减少。重组CP29A的核磁共振和生物物理分析证实了低温诱导的LLPS，并在残留物水平上详细了解了凝聚相中的分子相互作用。此外，对CP29As两个单独的RRM基序的核磁共振分析发现了它们RNA结合特性的惊人差异。受CP29A研究结果的鼓舞，我们询问其他叶绿体rbp是否也具有LLPS能力。利用计算机预测、体内瞬时表达分析和体外LLPS分析的结合，我们提出了两种参与叶绿体RNA编辑的蛋白质和三种叶绿体翻译所需的蛋白质能够进行LLPS的证据。现在，我们建议在对叶绿体rbp的功能LLPS的第一次描述之后，通过(i)对CP29A LLPS的生化和生物物理机制进行详细分析，以及（ii）对在这里发现的颗粒中存在的其他叶绿体RNA结合蛋白的LLPS功能进行一般表征。为了实现这一目标，我们将我们在植物遗传学和植物RNA生物学方面的互补专业知识（Schmitz-Linneweber实验室）与在大分子结构、动力学和相互作用分析方面的专业知识（Sattler实验室）结合在一起。我们相信我们的方法的优势在于将LLPS的详细生物物理和生化探索与植物的分子，特别是有机表型联系起来的协同作用。此外，我们将确定在响应冷移时允许LLPS发生的细节。最后，LLPS很少在植物中被分析，尽管事实上植物作为无根生物将极大地受益于LLPS是适应环境变化的一种调节选择。我们有能力填补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.