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

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.

The microprocessor is a nuclear molecular machine involved in the first step of processing of primary microRNAs (pri-miRNAs), leading to their scission. Products are exported to the cytoplasm, further processed by another RNase (Dicer), and ultimately produce mature miRNAs involved in e.g. regulation of tissue and organ development, hematopoietic differentiation and cellular proliferation. The microprocessor is a large molecular assembly, activity of which is reconstituted by the proteins DGCR8 and Drosha. DGCR8 is a RNA-binding protein; Drosha is a RNaseIII. DGCR8 binds heme and has an unusual spectrum suggesting a cysteine ligand to the iron. Heme is implicated in dimerization of DGCR8 and in promoting microprocessor activity. We will exploit expertise of the applicants in hemopotein and RNA enzyme structure/mechanism to perform an integrated study of structural/catalytic properties of the DGCR8 and Drosha proteins. We will use spectroscopy (MCD and EPR) to determine heme ligands in DGCR8, and examine influence of potential gaseous regulators (O2, CO, NO) on heme coordination and DGCR8 function. Oligomeric states of DGCR8 and Drosha will be analysed by light scattering and AUC methods, and influence of binding of heme/pri-miRNA on DGCR8 states will be determined. The Drosha/DGCR8 complex will also be analysed to define quaternary structure. Mass spectrometry will be used to further define its composition and to assess stoichiometry of miRNA/heme binding. ITC will be used to determine miRNA affinity for DGCR8. Transient kinetic studies of binding of heme will be used to probe for intermediates in heme coordination. Steady-state assays will establish kinetic parameters of the microprocessor. Crystallography will be done on intact DGCR8 and smaller heme- and RNA-binding domains, and using Drosha and/or its domains. Collectively these studies will resolve key issues with respect to structure and catalytic properties of this crucial human enzyme system.