Toward understanding the structural basis of DGCR8 RNA-binding heme domain

Abstract

MicroRNAs (miRNAs) play an important role in the regulation of protein expression in eukaryotes. The initial step of producing pre-miRNAs from primary transcripts (pri-miRNAs) is processed by a Microprocessor consisting of a nuclease Drosha and its coactivator – a dimer of DiGeorge Critical Region 8 (DGCR8). The efficiency and accuracy of Microprocessor activity depend on heme-binding with DGCR8. The DGCR8 RNA heme-binding domain (Rhed) binds to an apical junction of pri-miRNA with a specific UGU motif. Previous studies showed that ferric [Fe(III)] heme is essential for processing pri-miRNAs in a precise way. Besides, the reduction of ferric [Fe(III)] heme to ferrous [Fe(II)] inhibits the Microprocessor activity. In the dimer structure of DGCR8, the ferric [Fe(III)] heme binds with two cysteines 352 as axial ligands. However, the mechanism of how heme enhances the binding of DGCR8 with pri-miRNA is still unclear. Here, in this study, we attempted to obtain a crystal structure of Rhed of DGCR8 with RNAs to understand its mechanism. We found that the protein was not stable during crystallization and only dimerization domain was crystalized, suggesting that the Rhed instability is a common problem. The structure we obtained is similar to the previously published structure, which is also very similar to the structure in Xenopus laevis. In additions, we discovered that adding reducing reagents such as dithiothreitol or 2-mercaptoethanol increases the dissociation of heme from Rhed. We also provide the optimal buffer to keep the protein more stable for further study to determine full-length structure of DGCR8 Rhed.

Date of Award	2019
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology