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

我们将研究南瓜在感知和/或改变嘧啶库以及在调控转录后过程中的作用。南瓜是唯一的质体UMP激酶。南瓜是一种同源多聚体蛋白，它与未知成分形成含有多达百万吨大小的RNA的复合体。其他一些复合体可能不含有RNA，因为它们是抗核糖核酸酶的。值得注意的是，根据叶绿体转录组广谱关联研究(RIP-Seq)，南瓜在体内与5个叶绿体转录本NDHA、PetB、PETD、trnG-UCC和trnV-UAC的内含子特异相关。南瓜还在体外以高特异性和亲和力结合PetB内含子RNA，并稳定前体转录，最可能的是通过掩盖内切酶敏感部位。南瓜的零等位基因和敲除基因是可行的，但在生长、叶绿体转换和光合作用表现方面明显受到影响。在南瓜突变体中，由叶绿体编码的聚合酶产生的转录本的水平降低，而由核编码的聚合酶产生的转录本的水平甚至增加，这表明UTP对叶绿体转录的供应通常不受限制。因此，问题是南瓜的催化功能是否已经被传感功能部分取代。然而，南瓜在体外和体内都具有酶活性，并催化依赖于ATP的UMP转化为UDP，具有已知的必要的真细菌UMP激酶的特性。我们将在假设南瓜的酶和RNA稳定功能不一定是互斥的假设的基础上，调查在何种程度和条件下，南瓜的酶和RNA稳定功能的缺失对突变表型有贡献。因此，我们将剖析南瓜作为UMP激酶在嘧啶代谢中的个体作用以及作为兼职RNA结合蛋白在转录后过程中的作用。将探索目标RNA对南瓜酶特性的影响以及核苷酸在与其目标RNA结合方面的作用。南瓜突变体与一个可能与南瓜靶RNA无关的真细菌UMP激酶以及与不具有酶或RNA结合活性的重组南瓜形式的互补将被执行。此外，南瓜突变的代谢后果，如尿嘧啶核苷酸依赖的代谢物产生的水平，将通过质谱学进行定量检测。将确定相互作用的伙伴和精确的目标，并解决南瓜-RNA复合体的结构。将分析另一种未知的真细菌UMP激酶(NUMPKIN)对细胞嘧啶代谢的贡献，该蛋白可能存在于细胞核中，也具有兼职功能。最后，我们的数据将在多大程度上突出两个UMP激酶作为整合传感器的作用，将叶绿体和/或核基因表达与尿嘧啶核苷酸池中的变化联系起来。

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.