Functional and structural characterization of a novel heme- and micro RNA-binding human protein

人类基因组序列的确定是过去十年中最伟大的科学成就之一，其通过例如鉴定与疾病状态有关的基因和蛋白质来改善人类状况的前景受到了广泛的宣传。人类（和复杂的真核生物）的基因组比细菌（原核生物）的基因组大得多。人类基因组包含大量功能不确定的DNA（脱氧核糖核酸），以及可识别为编码蛋白质或参与基因表达调节的区域。然而，人类基因组的大部分不参与蛋白质生产的事实并不意味着它们是多余的。最近，已经清楚的是，约3%的人类基因组DNA用于编码RNA（核糖核酸）分子，最终用于通过“基因沉默”调节其他基因。最终的基因调控产物是微小RNA（或miRNAs）。它们的产生起始于初级RNA转录物（pri-miRNAs）的转录，其可以非常长（高达数千个核糖核苷酸单位）。这些在细胞核中被称为“微处理器”的分子机器切割，该分子机器可能包含两种蛋白质的多个拷贝/一种称为DGCR 8或“Pasha”的RNA结合蛋白和一种称为“Drosha”的RNA切割（RNase）酶。微处理器反应的缩短产物是前体miRNA（pre-miRNA），并且这些前体miRNA从细胞核转运到细胞质中，在细胞质中它们被另一种称为“Dicer”的RNA酶进一步加工/最终形成执行基因调控作用的成熟miRNA。现在很明显，miRNA在控制重要的人类过程中起关键作用，包括器官和组织的分化、细胞的程序性死亡（细胞凋亡）和癌症发展。关于核微处理器的结构和催化性质知之甚少，但最近的研究表明DGCR 8结合血红素辅因子/与血红蛋白中的血红素相同。我们表达和纯化DGCR 8蛋白的初步工作证实了这一发现，我们还做了其他几项研究，表明血红素中心的铁原子被两个配体结合，可能是DGCR 8中的氨基酸。我们还表明，血红素铁处于还原（亚铁）状态，在这种状态下，血红素能够与氧气，一氧化氮（NO）和一氧化碳（CO）等气体相互作用。已知这些气体中的每一种都对细胞过程如呼吸和血液流动产生深远的影响。在这项研究中，我们将利用我们在血红素蛋白质和RNA代谢研究的专业知识，进行详细的表征的微处理器复合体及其组件。这项工作将确切地确定血红素是如何与DGCR 8蛋白结合的，以及血红素对DGCR 8结构及其聚集趋势的影响。我们还将研究pri-miRNA结合对DGCR 8构象和聚集的影响，并研究NO和CO对血红素及其蛋白连接状态的影响，因为这些配体可能影响DGCR 8的结构和反应性。我们将使用现代结构方法来定义DGCR 8/Drosha蛋白的寡聚化状态，然后分析它们在微处理器复合物中的相互作用和寡聚状态的性质。我们将使用先进的动力学方法来研究血红素与DGCR 8的结合及其与蛋白质配位的中间状态，并检查pri-miRNA的加工速率。我们还将进行晶体学研究，以解析DGCR 8/Drosha蛋白质（或其部分，“结构域”）的原子结构，并合理化这些蛋白质结合血红素和pri-miRNA并进行反应的机制。总的来说，这项工作将使我们在理解人类健康和发展所涉及的一个关键系统的结构和机制方面向前迈出一大步。

The determination of the human genome sequence has been one of the great scientific achievements of the last decade, receiving enormous publicity with respect to its prospects for improving the human condition through e.g. identification of genes and proteins implicated in disease states. The genomes of humans (and complex eukaryotes) are vastly larger than those of bacteria (prokaryotes). The human genome contains swathes of DNA (deoxyribonucleic acid) of uncertain function, alongside regions recognizable as encoding proteins or involved in regulation of gene expression. The fact that large sections of the human genome are not involved in protein production does not, however, mean they are redundant. Recently, it has become clear that ~3 % of human genomic DNA is used to encode RNA (ribonucleic acid) molecules ultimately used for regulation of other genes by 'gene silencing'. The ultimate gene regulatory products are micro RNAs (or miRNAs). Their production initiates with transcription of primary RNA transcripts (pri-miRNAs), which can be very long (up to thousands of ribonucleotide units). These are cleaved in the nucleus by a molecular machine called the 'microprocessor', which likely contains multiple copies of two proteins / an RNA-binding protein called DGCR8 or 'Pasha' and a RNA-cleaving (RNase) enzyme called 'Drosha'. The shortened products of the microprocessor reaction are precursor miRNAs (pre-miRNAs) and these are transported from the nucleus into the cell cytoplasm, where they are further processed by another RNase called 'Dicer' / ultimately forming mature miRNAs that perform gene regulatory roles. It is now evident that miRNAs play critical roles in control of important human processes / including differentiation of organs and tissues, programmed death of cells (apoptosis) and cancer development. Relatively little is known about structures and catalytic properties of the nuclear microprocessor, but recent studies revealed that DGCR8 binds a heme cofactor / identical to the heme in hemoglobin. Our preliminary work to express and purify DGCR8 protein have confirmed this finding, and we have done several other studies that indicate that the iron atom at the centre of the heme is bound by two ligands, likely to be amino acids within DGCR8. We have also showed that the heme iron is in a reduced (ferrous) state, and in this state hemes are able to interact with gases such as oxygen, nitric oxide (NO) and carbon monoxide (CO). Each of these gases is known to exert profound effects over cellular processes such as respiration and blood flow. In this study, we will exploit our expertise in study of heme proteins and RNA metabolism to perform a detailed characterization of the microprocessor complex and its components. This work will establish exactly how heme is bound to the DGCR8 protein, and the influence of heme on the DGCR8 structure and its tendency to aggregate. We will also investigate the effect of pri-miRNA binding on conformation and aggregation of DGCR8, and examine influence of NO and CO on the state of the heme and its protein ligation, since these ligands may influence DGCR8 structure and reactivity. We will use modern structural methods to define the oligimerization state of both DGCR8/Drosha proteins, and then analyse the nature of their interactions and their oligomeric state in the microprocessor complex. We will use advanced kinetic methods to study binding of heme to DGCR8 and intermediate states in its coordination to the protein, and to examine the rate of processing of pri-miRNA. We will also undertake crystallographic studies to resolve the atomic structures of the DGCR8/Drosha proteins (or sections, 'domains', thereof) and to rationalise the mechanism by which these proteins bind heme and pri-miRNA and perform their reaction. Collectively, this work will lead to a large step forward in our understanding of structure and mechanism of a crucial system involved in human health and development.