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.

我们将研究唯一的质体UMP激酶PUMPKIN在感应和/或改变嘧啶库以及控制转录后过程中的作用。PUMPKIN是一种同源蛋白，可形成含有未知成分的高达兆子大小的rna复合物。其他一些复合物可能不含RNA，因为它们具有RNA抗性。值得注意的是，通过质体转录组全关联研究（RIP-Seq）发现，PUMPKIN在体内与五种质体转录本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，并很可能通过屏蔽内切酶敏感位点来稳定前体转录本。无等位基因和基因敲低对南瓜的生长、质体翻译和光合性能有明显影响。在南瓜突变体中，由质体编码的聚合酶产生的转录本水平降低，而由核编码的聚合酶产生的转录本水平甚至增加，这表明质体转录的UTP供应通常不受限制。因此，南瓜的催化功能是否部分被传感功能所取代的问题就出现了。尽管如此，PUMPKIN在体外和体内都具有酶活性，并催化atp依赖的UMP转化为UDP，具有已知必需真菌性UMP激酶的特性。基于酶和RNA稳定功能并不一定相互排斥的假设，我们将研究南瓜酶和RNA稳定功能的缺乏在多大程度上和在什么条件下导致了突变表型。因此，我们将分析南瓜在嘧啶代谢中的UMP激酶和转录后过程中兼职RNA结合蛋白的个体作用。我们将探讨靶rna对南瓜酶学特性的影响以及核苷酸与靶rna结合的作用。将南瓜突变体与可能与南瓜靶RNA无关的真菌性UMP激酶互补，并与缺乏酶或RNA结合活性的重组南瓜形式互补。此外，南瓜突变的代谢后果，如尿嘧啶核苷酸依赖性代谢产物的水平将通过质谱定量检测。相互作用的伙伴和精确的靶标将被确定，南瓜rna复合物的结构将被解决。我们将分析另一种未被表征的真菌性UMP激酶（NUMPKIN）对细胞嘧啶代谢的贡献，这种激酶可能存在于细胞核中，也具有兼职功能。最后，我们的数据将强调两种UMP激酶在多大程度上作为整合传感器，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。