Heme is involved in microRNA processing

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  • An Addendum to this article was published on 01 October 2008

Abstract

MicroRNAs (miRNAs) regulate the expression of a large number of protein-coding genes. Their primary transcripts (pri-miRNAs) have to undergo multiple processing steps to reach the functional form. Little is known about how the processing of miRNAs is modulated. Here we show that the RNA-binding protein DiGeorge critical region-8 (DGCR8), which is essential for the first processing step, is a heme-binding protein. The association with heme promotes dimerization of DGCR8. The heme-bound DGCR8 dimer seems to trimerize upon binding pri-miRNAs and is active in triggering pri-miRNA cleavage, whereas the heme-free monomer is much less active. A heme-binding region of DGCR8 inhibits the pri-miRNA–processing activity of the monomer. This putative autoinhibition is overcome by heme. Our finding that heme is involved in pri-miRNA processing suggests that the gene-regulation network of miRNAs and signal-transduction pathways involving heme might be connected.

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Figure 1: DGCR8 contains a heme cofactor.
Figure 2: Heme promotes the dimerization of DGCR8.
Figure 3: A conserved cysteine probably serves as an axial ligand of heme.
Figure 4: Dimer and monomer of NC1 form different higher-order structures upon binding pri-miRNA.
Figure 5: The DGCR8 dimer is active in pri-miRNAs processing in vitro.
Figure 6: The dsRBD domains (NC9) contain the core pri-miRNA–binding and pri-miRNA–processing activities.

Change history

  • 06 October 2008

    Here we report that the calibration peaks of size-exclusion chromatography (SEC) were misassigned in our paper. The estimated molecular weights based on the new calibration curve are larger than those reported (see PDF for details). However, our conclusions about oligomerization states of the heme-bound and heme-free NC1 and the NC9 proteins are supported by additional experimental evidence, and are unchanged. The heme-bound NC1 is a dimer, as was directly confirmed using ESI MS. NC9 and the heme-free NC1 are largely monomeric, as indicated by our unpublished ESI mass spectra. In addition, NC9 is similar to the monomeric DGCR8 core, as demonstrated by its crystal structure (Nat. Struct. Mol. Biol. 14, 847–853, 2007). In our paper, we proposed a cooperative trimer model in which the heme-free NC1 dimer, as well as NC9, further trimerizes upon association with the pri-miR-30a RNA. This model is supported by the new SEC estimates. Therefore, SEC seems to systematically overestimate the molecular weights of DGCR8 proteins and protein–RNA complexes, probably due to their elongated shapes. The new estimated molecular weight of the heme-free NC1 in complex with pri-miR-30a is consistent with a model in which three heme-free NC1 molecules associate with one pri-miRNA, but this estimate is not conclusive. Further corroboration on the oligomerization state of the heme-free NC1 upon binding to pri-miRNAs is needed.

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Acknowledgements

A clone containing human Drosha sequence was kindly provided by V.N. Kim (Seoul National University). We thank T. Cech, D. Eisenberg, J. Fukuto, C.W. Goulding and S. Kurtistani for comments of the manuscript, R. Landgraf, V. Kickhoefer and L. Simpson for assistance and advice on insect-cell protein expression, and D. Black, S. Clarke, A. Fire, A. Fulco, O. Hankinson, S. Merchant and J.S. Valentine for discussion. This work was supported in part by seed grants from the Stein Oppenheimer Endowment Fund and the Jonsson Comprehensive Cancer Center at UCLA to F.G. The UCLA Functional Proteomics Center was established and equipped by a grant from the W.M. Keck Foundation. J.A.L. acknowledges support from the UCLA–US Department of Energy Institute for Genomics and Proteomics, and the US National Institutes of Health (RR 20004).

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Correspondence to Feng Guo.

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Supplementary information

Supplementary Fig. 1

Fluorescence excitation and emission spectra of NC1. (PDF 531 kb)

Supplementary Fig. 2

ESI mass spectrum of NC1 dimer. (PDF 642 kb)

Supplementary Fig. 3

Standard curves for SEC experiments. (PDF 783 kb)

Supplementary Fig. 4

Quantification of the heme cofactor bound to DGCR8. (PDF 802 kb)

Supplementary Fig. 5

SEC experiments of C352H and C430A. (PDF 714 kb)

Supplementary Methods (PDF 49 kb)

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