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.

我们将研究南瓜（唯一的质体UMP激酶）在感测和/或更改嘧啶池以及管理后的后期过程中的作用。南瓜是一种同源蛋白，形成含RNA的含有RNA的蛋白质，直至具有未知成分的Megadalton大小的复合物。其他一些复合物大概不含RNA，因为它们具有RNase耐药性。值得注意的是，南瓜与体内五个质体转录本，PETB，PETD，TRNG-UCC和TRNV-UAC的内含子特别相关，如质体转录组广泛的关联研究（RIP-SEQ）所示。南瓜还可以在体外具有高特异性和亲和力结合PETB内含子RNA，并稳定前体转录本最有可能通过掩盖核酸内切酶敏感的位点。南瓜的无效等位基因和敲低是可行的，但在生长，质体翻译和光合作用性能方面明显影响。在南瓜突变体中，由塑料编码的聚合酶产生的转录本水平降低，而核编码聚合酶产生的转录本甚至增加了，这表明质体转录的UTP供应通常不是限制。因此，出现了一个问题，即南瓜的催化功能是否已被传感函数部分取代。然而，南瓜在体外和体内都具有酶活性，并催化了UMP转化为UMP的UDP转化，其特征是已知必需的Eubacterial UMP激酶的特征。我们将研究南瓜缺乏酶促和RNA稳定功能的条件的程度和在哪些条件下，基于两个功能不一定互斥的假设有助于突变表型。因此，我们将在嘧啶代谢中剖析南瓜作为UMP激酶的个体作用，并在转录后过程中剖析月亮的RNA结合蛋白。将探索靶RNA对南瓜酶特性的影响以及核苷酸在与其靶RNA结合的作用。南瓜突变体与细菌UMP激酶的互补可能与南瓜靶RNA无关，并且将与没有酶促或RNA结合活性的重组南瓜形式有关。此外，南瓜突变的代谢后果（例如尿素核苷酸依赖性产生的代谢产物水平将通过质谱法进行定量检查。将确定相互作用的伙伴和精确的目标，并将解决南瓜-R​​NA复合物的结构。将分析对进一步未表征的Eubacterial UMP激酶（NOMPKIN）的细胞嘧啶代谢的贡献（大概存在于细胞核中并具有月光下的功能）。最后，我们的数据将强调两个UMP激酶在多大程度上充当综合传感器，将塑料和/或核基因表达与尿嘧啶核苷酸池的变化联系起来。