Role of the Plastid UMP Kinase PUMPKIN in Coupling the Primary Pyrimidine Metabolism with the Stabilization of Intron-Containing RNAs

质体 UMP 激酶 PUMPKIN 在初级嘧啶代谢与含内含子 RNA 稳定耦合中的作用

• 批准号: 422702898
• 负责人: Privatdozent Dr. Jörg Meurer
• 金额: --
• 依托单位: Lehrstuhl für Molekularbiologie der Pflanzen
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/422702898?language=en
• 关键词: Role; Plastid; UMP; Kinase; PUMPKIN

We will investigate the roles of PUMPKIN, the sole plastid UMP kinase, both in sensing and/or changing the pyrimidine pool as well as in governing posttranscriptional processes. PUMPKIN is a homomultimeric protein that forms RNA-containing up to megadalton-sized complexes with unknown components. Some other complexes presumably do not contain RNA since they are RNase resistant. Remarkably, PUMPKIN is associated specifically with the introns of the five plastid transcripts ndhA, petB, petD, trnG-UCC and trnV-UAC in vivo as revealed by plastid transcriptome wide association studies (RIP-Seq). PUMPKIN also binds the petB intron RNA with high specificity and affinity in vitro and stabilizes the precursor transcripts most likely by masking endonuclease-sensitive sites. Null alleles and knockdowns of PUMPKIN are viable but clearly affected in growth, plastid translation and photosynthetic performance. In pumpkin mutants, levels of transcripts generated by the plastid-encoded polymerase are reduced, while those produced by the nuclear-encoded polymerase are even increased, indicating that UTP supply for plastid transcription is generally not limiting. Thus, the question arises whether the catalytic function of PUMPKIN had been replaced partially by a sensing function. Nevertheless, PUMPKIN is enzymatically active in vitro and in vivo and catalyses the ATP-dependent conversion of UMP to UDP with properties characteristic for known essential eubacterial UMP kinases. We will investigate to which extent and under which conditions lack of the enzymatic and of the RNA stabilizing functions of PUMPKIN contribute to the mutant phenotype based on the hypothesis that both functions are not necessarily mutually exclusive. Thus, we will dissect the individual roles of PUMPKIN as UMP kinase in the pyrimidine metabolism and as moonlighting RNA binding protein in posttranscriptional processes. The impact of the target RNAs on the enzymatic properties of PUMPKIN and the role of nucleotides on binding to its target RNAs will be explored. Complementation of pumpkin mutants with a eubacterial UMP kinase presumably not associated with the PUMPKIN target RNAs and with recombinant PUMPKIN forms devoid of the enzymatic or the RNA binding activity will be performed. Furthermore, the metabolic consequences of the pumpkin mutation, such as levels of the uracil nucleotide-dependent production of metabolites will be examined quantitatively by mass spectrometry. Interacting partners and the precise targets will be identified and the structure of the PUMPKIN-RNA complex will be solved. The contribution to the cellular pyrimidine metabolism of a further uncharacterized eubacterial UMP kinase (NUMPKIN), presumably present in the nucleus and also harbouring a moonlighting function, will be analysed. Finally, our data will highlight to which extent the two UMP kinases act as integrative sensors that link plastid and/or nuclear gene expression with changes in the uracil nucleotide pool.

我们将研究 PUMPKIN（唯一的质体 UMP 激酶）在感知和/或改变嘧啶池以及控制转录后过程中的作用。 PUMPKIN 是一种同源多聚体蛋白质，可形成包含 RNA 的高达兆道尔顿大小且成分未知的复合物。其他一些复合物可能不含 RNA，因为它们具有 RNase 抗性。值得注意的是，正如质体转录组广泛关联研究 (RIP-Seq) 所揭示的，PUMPKIN 在体内与五个质体转录本 ndhA、petB、petD、trnG-UCC 和 trnV-UAC 的内含子特异性相关。 PUMPKIN 还在体外以高特异性和亲和力结合 petB 内含子 RNA，并很可能通过掩蔽核酸内切酶敏感位点来稳定前体转录本。 PUMPKIN 的无效等位基因和敲低是可行的，但对生长、质体翻译和光合作用性能有明显影响。在南瓜突变体中，质体编码聚合酶产生的转录物水平降低，而核编码聚合酶产生的转录物水平甚至增加，表明质体转录的UTP供应通常不受限制。因此，问题出现了：PUMPKIN 的催化功能是否已部分被传感功能取代。尽管如此，PUMPKIN 在体外和体内均具有酶活性，并催化 UMP 向 UDP 的 ATP 依赖性转化，具有已知的必需真细菌 UMP 激酶的特性。我们将基于两种功能不一定相互排斥的假设，研究 PUMPKIN 酶促和 RNA 稳定功能的缺乏在何种程度上以及在何种条件下导致突变表型。因此，我们将剖析 PUMPKIN 在嘧啶代谢中作为 UMP 激酶以及在转录后过程中作为兼职 RNA 结合蛋白的各自作用。将探讨靶标 RNA 对 PUMPKIN 酶学特性的影响以及核苷酸在与其靶标 RNA 结合方面的作用。将进行南瓜突变体与推测与PUMPKIN靶RNA不相关的真细菌UMP激酶和缺乏酶促或RNA结合活性的重组PUMPKIN形式的互补。此外，南瓜突变的代谢后果，例如尿嘧啶核苷酸依赖性代谢物的产生水平，将通过质谱法进行定量检查。相互作用的伙伴和精确的靶点将被识别，PUMPKIN-RNA 复合物的结构也将得到解决。将分析进一步未表征的真细菌 UMP 激酶 (NUMPKIN) 对细胞嘧啶代谢的贡献，该激酶可能存在于细胞核中，并且还具有兼职功能。最后，我们的数据将强调这两种 UMP 激酶在多大程度上充当将质体和/或核基因表达与尿嘧啶核苷酸库的变化联系起来的整合传感器。