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

我们将调查的作用，pAPKIN，唯一的质体UMP激酶，无论是在传感和/或改变嘧啶池，以及在管理转录后过程。PKIN是一种同源多聚体蛋白，可与未知组分形成含RNA的高达兆道尔顿大小的复合物。一些其他复合物可能不含RNA，因为它们是RNA酶抗性的。值得注意的是，如质体转录组全关联研究（RIP-Seq）所揭示的，在体内，PKIN与五种质体转录物ndhA、petB、petD、trnG-UCC和trnV-UAC的内含子特异性相关。PKIN还在体外以高特异性和亲和力结合petB内含子RNA，并最可能通过掩蔽核酸内切酶敏感位点来稳定前体转录物。PKIN的等位基因和敲低是可行的，但明显影响生长，质体翻译和光合性能。在南瓜突变体中，由质体编码的聚合酶产生的转录物的水平降低，而由核编码的聚合酶产生的转录物的水平甚至增加，这表明用于质体转录的UTP供应通常不受限制。因此，出现了一个问题，即PKIN的催化功能是否部分被传感功能所取代。然而，PKIN在体外和体内都具有酶活性，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。我们将研究在何种程度上和在何种条件下缺乏酶和RNA稳定功能的PKIN有助于突变表型的基础上的假设，这两种功能不一定是相互排斥的。因此，我们将剖析在嘧啶代谢中作为UMP激酶和在转录后过程中作为兼职RNA结合蛋白的PKIN的个体作用。将探索靶RNA对PKIN的酶性质的影响以及核苷酸对其靶RNA结合的作用。将进行南瓜突变体与真细菌UMP激酶的互补，所述真细菌UMP激酶推测不与PKIN靶RNA相关，并且与缺乏酶或RNA结合活性的重组PKIN形式互补。此外，南瓜突变的代谢结果，如尿嘧啶核苷酸依赖性代谢产物的产生水平，将通过质谱法定量检测。相互作用的伙伴和精确的目标将被确定，并将解决PKIN-RNA复合物的结构。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，也具有兼职功能。最后，我们的数据将突出在何种程度上的两个UMP激酶作为整合传感器，连接质体和/或核基因表达与尿嘧啶核苷酸池的变化。

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.