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

Lead Research Organisation: University of Manchester
Department Name: Life Sciences


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.

Technical Summary

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.


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Description MicroRNAs are important regulatory molecules that participate in RNAi (RNA interference) pathways to regulate expression of protein-encoding genes. The microRNAs are produced from longer RNA (ribonucleic acid) molecules through the action of an enzyme complex referred to as the Microprocessor. This has two major components - an RNA cleaving enzyme (an RNase) and a heme-binding protein called DGCR8 that is required for the function of the Microprocessor. The major purposes of this proposal was to express and purify different forms of human DGCR8 and to characterize this protein in terms of its heme binding properties (including exactly how the heme is ligated and which amino acids in the protein are required to coordinate to the heme iron to stabilize its binding). Other biochemical studies involved analysis of the aggregation state of the DGCR8 protein and its ability to interact with the RNA substrate (the primary microRNA) of the microprocessor. Key findings included the ability of DGCR8 to aggregate and dimerize. This was consistent with structural studies done by another group (who identified a dimerization domain responsible for dimer formation). Analysis of the DGCR8 heme protein's spectroscopic features was done using a variety of methods, including UV-visible, electron paramagnetic resonance (EPR), resonance Raman, electron nuclear double resonance (ENDOR) spectroscopy and magnetic circular dichroism (MCD). The UV-vis characteristics of DGCR8 are typical of proteins in which the heme is coordinated by two cysteine amino acid residues, and in which sulfur atoms (as negatively charged thiolates) bind the iron above and below the plane of the heme. The combined spectroscopic approaches supported this assignment and effectively ruled out the possibility of a nitrogen or an oxygen ligand to the heme iron (e.g. from a histidine amino acid or from a water molecule). Other spectroscopic studies showed that the DGCR8 heme iron is highly selective for other small chemicals that might displace one or other of the cysteines, and it was found that carbon monoxide (CO) was the only gaseous ligand that effectively displaced a cysteine, forming a distinctive spectral complex. This raises the possibility that CO might be a regulator of DGCR8 function, since this gas is known to regulate a number of biological processes in both higher and lower organisms. Our work revealed several novel features of this enzyme, and we overcame problems with expressing this enzyme (using different length forms of the protein and different types of protein production systems). Binding of RNA substrate to the DGCR8 protein was also achieved, and the heme protein was prepared for structural studies, but proved difficult to crystallize in light of a propensity for aggregation and precipitation. However, recent studies from a Korean group have produced a structure for DGCR8's partner protein Drosha. We have recently submitted a manuscript reporting the detailed spectroscopic characterization of the heme binding site in DGCR8, revealing that the heme undergoes a change in heme iron ligation state on reduction, consistent with an observed loss of activity in the reduced state and with an important role of the heme in regulating DGCR8 function. Overall, our studies provide fundamental new data on an important hemoprotein, and work is continuing to provide more detailed characterization of this enzyme.
Exploitation Route DGCR8 is a crucial enzyme in regulation of protein expression. The data provided in our study (particularly with respect to understanding the heme binding site in the protein) are crucial for studies done by groups worldwide on this important enzyme, and for developing our knowledge of its function and how changes to heme ligation regulate DGCR8 and its microRNA processing function. These findings include a potential key role for carbon monoxide (CO) as a regulator of DGCR8 and Microprocessor complex activity.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

Description Our work in this project was aimed at furthering our knowledge of the structure and function of a novel enzyme system now recognized as crucial in regulation of protein expression and intensively studied worldwide. The data we have produced have provided, in particular, key insights into the essential heme binding site in the protein; including how the heme is ligated and retained, and the nature of other ligands (particularly carbon monoxide) that can displace one of the heme ligands and that may be important in the regulation of DGCR8 function. We have also developed improved methods for the isolation of the enzyme, demonstrated that the heme and RNA binding sites on DGCR8 are distinct, and characterized the mode of heme binding in oxidized and reduced forms of the enzyme using a range of spectroscopic approaches. These key data have been published in February, and demonstrate an important change in heme coordination state on reduction of the enzyme, underpinning a loss of activity in the reduced form of DGCR8. These data have important ramifications in understanding the structure and mechanism of this important enzyme in microRNA biosynthesis, which impacts on human health and disease.
First Year Of Impact 2014
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

Description Manchester Institute of Biotechnology Open Day - annual event from 2012 onwards 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Scientific demonstrations to senior secondary school students to enthuse them about a scientific career and to provide advice on career development and the courses on offer at the University of Manchester.

Annual event - such that lessons are learned from one year's activity and are carried forward to the following year's presentations.
Year(s) Of Engagement Activity 2012,2013,2014,2015
Description Schools visit (Wilmslow) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Presentation to primary school children in final year on general science/genetics - talk sparked questions and general discussion

Students registered interests in scientific career. Invite for further talk in following year obtained.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011,2014