Dimerization and Heme Binding Are Conserved in Amphibian and Starfish Homologues of the microRNA Processing Protein DGCR8

Rachel Senturia,Brooke D Scheidemantle,Ian Barr,Arthur Laganowsky,Feng Guo,Vladimir N Uversky

doi:10.1371/journal.pone.0039688

Rachel Senturia, Brooke D Scheidemantle + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0039688

Copy DOI

Journal: PloS one	Publication Date: Jul 2, 2012
Citations: 56	License type: CC BY 4.0

Affiliation: University of California, Los Angeles

Abstract

Human DiGeorge Critical Region 8 (DGCR8) is an essential microRNA (miRNA) processing factor that is activated via direct interaction with Fe(III) heme. In order for DGCR8 to bind heme, it must dimerize using a dimerization domain embedded within its heme-binding domain (HBD). We previously reported a crystal structure of the dimerization domain from human DGCR8, which demonstrated how dimerization results in the formation of a surface important for association with heme. Here, in an attempt to crystallize the HBD, we search for DGCR8 homologues and show that DGCR8 from Patiria miniata (bat star) also binds heme. The extinction coefficients (ε) of DGCR8-heme complexes are determined; these values are useful for biochemical analyses and allow us to estimate the heme occupancy of DGCR8 proteins. Additionally, we present the crystal structure of the Xenopus laevis dimerization domain. The structure is very similar to that of human DGCR8. Our results indicate that dimerization and heme binding are evolutionarily conserved properties of DGCR8 homologues not only in vertebrates, but also in at least some invertebrates.

Highlights

MicroRNAs are a class of,22 nucleotide noncoding RNAs that negatively regulate gene expression by destabilizing target mRNAs or inhibiting their translation [1,2]
In the first step of miRNA processing, a pri-miRNA is cleaved in the nucleus to produce an intermediate called the precursor miRNA, by a protein complex called the Microprocessor, which is minimally composed of the ribonuclease III enzyme Drosha, and the RNA-binding partner DiGeorge Critical Region 8 (DGCR8) [4,5,6,7,8]. pre-miRNAs are exported to the cytoplasm where they undergo additional cleavages by another ribonuclease III enzyme Dicer to produce miRNA duplexes. miRNA duplexes are incorporated into the miRNA-induced silencing complex and unwound into the mature singlestranded form
Heme Binding is Conserved in a Starfish DGCR8 In our pursuit to crystallize the DGCR8 heme-binding domain (HBD), we searched for homologues within other organisms

Summary

Introduction

MicroRNAs (miRNAs) are a class of ,22 nucleotide (nt) noncoding RNAs that negatively regulate gene expression by destabilizing target mRNAs or inhibiting their translation [1,2]. Mature miRNAs originate from primary transcripts (pri-miRNAs) that may be transcribed as introns of mRNAs or as independent transcripts [3]. In the first step of miRNA processing, a pri-miRNA is cleaved in the nucleus to produce an intermediate called the precursor miRNA (premiRNA), by a protein complex called the Microprocessor, which is minimally composed of the ribonuclease III enzyme Drosha, and the RNA-binding partner DGCR8 [4,5,6,7,8]. Pre-miRNAs are exported to the cytoplasm where they undergo additional cleavages by another ribonuclease III enzyme Dicer to produce miRNA duplexes. Dgcr8+/2 mouse models indicated pri-miRNA processing defects in the brain and neurological defects and symptoms similar to those observed in DiGeorge syndrome [12,13,14]

Methods

Results

Conclusion