JBIC Journal of Biological Inorganic Chemistry

, Volume 21, Issue 8, pp 1021–1035 | Cite as

CO and NO bind to Fe(II) DiGeorge critical region 8 heme but do not restore primary microRNA processing activity

  • Judy P. Hines
  • Aaron T. Smith
  • Jose P. Jacob
  • Gudrun S. Lukat-Rodgers
  • Ian Barr
  • Kenton R. Rodgers
  • Feng Guo
  • Judith N. Burstyn
Original Paper
  • 316 Downloads

Abstract

The RNA-binding heme protein DiGeorge critical region 8 (DGCR8) and its ribonuclease partner Drosha cleave primary transcripts of microRNA (pri-miRNA) as part of the canonical microRNA (miRNA) processing pathway. Previous studies show that bis-cysteine thiolate-coordinated Fe(III) DGCR8 supports pri-miRNA processing activity, while Fe(II) DGCR8 does not. In this study, we further characterized Fe(II) DGCR8 and tested whether CO or NO might bind and restore pri-miRNA processing activity to the reduced protein. Fe(II) DGCR8 RNA-binding heme domain (Rhed) undergoes a pH-dependent transition from 6-coordinate to 5-coordinate, due to protonation and loss of a lysine ligand; the ligand bound throughout the pH change is a histidine. Fe(II) Rhed binds CO and NO from 6- and 5-coordinate states, forming common CO and NO adducts at all pHs. Fe(II)–CO Rhed is 6-coordinate, low-spin, and pH insensitive with the histidine ligand retained, suggesting that the protonatable lysine ligand has been replaced by CO. Fe(II)–NO Rhed is 5-coordinate and pH insensitive. Fe(II)–NO also forms slowly upon reaction of Fe(III) Rhed with excess NO via a stepwise process. Heme reduction by NO is rate-limiting, and the rate would be negligible at physiological NO concentrations. Importantly, in vitro pri-miRNA processing assays show that both CO- and NO-bound DGCR8 species are inactive. Fe(II), Fe(II)–CO, and Fe(II)–NO Rhed do not bear either of the cysteine ligands found in the Fe(III) state. These data support a model in which the bis-cysteine thiolate ligand environment of Fe(III) DGCR8 is necessary for establishing proper pri-miRNA binding and enabling processing activity.

Keywords

Heme microRNA RNA processing Carbon monoxide Nitric oxide 

Abbreviations

5cHS

5-Coordinate high spin

6cLS

6-Coordinate low spin

CAPS

N-cyclohexyl-3-aminopropanesulfonic acid

CHES

N-cyclohexyl-2-aminoethanesulfonic acid

CTT

C-terminal tail

DaCld

Dechloromonas aromatica chlorite dismutase

DEANO

2-(N,N-diethylamino)-diazenolate-2-oxide

DGCR8

DiGeorge critical region 8

DSD

Dimerization subdomains

dsRBD1

Double-stranded RNA-binding domain 1

dsRBD2

Double-stranded RNA-binding domain 2

DTT

Dithiothreitol

EPPS

4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid

HEPES

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

HBD

Heme-binding domain

MCD

Magnetic circular dichroism

MES

2-(N-morpholino)ethanesulfonic acid

miRNA

mircoRNA

MOPS

3-(N-morpholino)propanesulfonic acid

NLS

Nuclear localization signal

Ns

Nostdoc sp.

pre-miRNA

Precursor microRNA

pri-miRNA

Primary microRNA

PPIX

Protoporphyrin IX

Rhed

RNA-binding heme domain

RMSD

Root-mean-square deviation

rR

Resonance Raman

SEC

Size exclusion chromatography

WW

WW-motif

Supplementary material

775_2016_1398_MOESM1_ESM.pdf (1.7 mb)
Supplementary material 1 (PDF 1716 kb)

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Copyright information

© SBIC 2016

Authors and Affiliations

  • Judy P. Hines
    • 1
  • Aaron T. Smith
    • 2
  • Jose P. Jacob
    • 3
  • Gudrun S. Lukat-Rodgers
    • 4
  • Ian Barr
    • 3
  • Kenton R. Rodgers
    • 4
  • Feng Guo
    • 3
  • Judith N. Burstyn
    • 1
  1. 1.Department of ChemistryUniversity of Wisconsin-MadisonMadisonUSA
  2. 2.Department of Chemistry and BiochemistryUniversity of Maryland, Baltimore CountyBaltimoreUSA
  3. 3.Department of Biological ChemistryDavid Geffen School of Medicine, Molecular Biology Institute, University of California Los AngelesLos AngelesUSA
  4. 4.Department of Chemistry and Molecular BiologyNorth Dakota State UniversityFargoUSA

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