Interaction of JMJD6 with single-stranded RNA
Xia Honga,1, Jianye Zangb,1, Janice Whitec,1, Chao Wanga, Cheol-Ho Pand, Rui Zhaoe, Robert C. Murphyf,
Shaodong Daia, Peter Hensona,g, John W. Kapplera,c,2, James Hagmana,e, and Gongyi Zhanga,2
aIntegrated Department of Immunology, National Jewish Health, Denver, CO 80206;
China, Hefei, China;
Science and Technology, Gangneung Institute, Gangneung, Korea;
of Colorado, Aurora, CO 80045;
Pediatrics, National Jewish Heath, Denver, CO 80206
bSchool of Life Science, University of Science and Technology of
dNatural Products Research Center, Korea Institute of
eDepartment of Biochemistry and Molecular Genetics, School of Medicine, University
fDepartment of Pharmacology, School of Medicine, University of Colorado, Aurora, CO 80045; and
cHoward Hughes Medical Institute, National Jewish Health, Denver, CO 80206;
Contributed by John W. Kappler, June 28, 2010 (sent for review May 10, 2010)
JMJD6 is a Jumonji C domain-containing hydroxylase. JMJD6 binds
α-ketoglutarate and iron and has been characterized as either a
histone arginine demethylase or U2AF65 lysyl hydroxylase. Here,
we describe the structures of JMJD6 with and without α-keto-
glutarate, which revealed a novel substrate binding groove and
two positively charged surfaces. The structures also contain a stack
of aromatic residues located near the active center. The side chain
of one residue within this stack assumed different conformations
in the two structures. Interestingly, JMJD6 bound efficiently to
single-stranded RNA, but not to single-stranded DNA, double-
stranded RNA, or double-stranded DNA. These structural features
and truncation analysis of JMJD6 suggest that JMJD6 may bind and
modify single-stand RNA rather than the previously reported
RNA binding proteins ∣ RNA modification ∣ RNA splicing
dying cells by macrophages and fibroblasts (1). Targeted deletion
of gene encoding PSR in mice and morpholino knock-downs of
PSR in zebrafish resulted in embryonic lethality, with severe
defects in hematopoiesis and aberrant development of eye, brain,
and heart (2–5). In contrast, knock-down of PSR expression in
Caenorhabditis elegans produced only a mild phenotype (5).
Somewhat surprisingly, sequence analysis suggested that JMJD6
contains a Jumonji C (JMJC) domain, which places it within a
highly conserved, cupin fold-containing enzyme family (6–8).
Further analysis demonstrated that the protein is localized spe-
cifically in the nucleus (7–9). Despite the significant effects of
JMJD6 deficiency, knockout mice engulfed apoptotic cells
normally (9). Based on these studies and additional sequence
analysis, the protein was recategorized as an α-ketoglutarate-
and Fe2þ-dependent hydroxylase and was named JMJD6 (10).
Recent studies demonstrated that most JMJC domain-contain-
ing proteins function as histone demethylases by specifically act-
ing on lysine residues in histone tails (11–14). For example, the
specific interactions between enzymes from the JMJD2 subfamily
and methylated peptides have been structurally characterized
(15–18). Interestingly, JMJD6 was reported to demethylate argi-
nine residues in histone tails (10). Several laboratories including
ours, however, have been unable to reproduce these results. In
other studies, JMJD6 was identified as a lysine hydroxylase that
specifically recognizes the protein tail of U2AF65, a mediator of
RNA splicing (19).
To resolve the disparate results and further elucidate the
structure and functions of JMJD6, we determined X-ray crystal-
lographic structures of the protein with and without α-keto-
glutarate. To obtain these structures, JMJD6 was cocrystallized
with a Fab fragment derived from a JMJD6-specific hamster
monoclonal antibody. Intriguingly, the structure of JMJD6 is
dramatically different from known structures of other JMJC do-
main superfamily proteins including FIH (20, 21), JMJD2A (16),
and AlkB (22). Our structural and biochemical analyses suggest
MJD6 was first characterized as a receptor for phosphatidyl-
serine (PSR), which facilitates the phagocytosis of dead and
that JMJD6 may recognize substrates including nucleic acids in
addition to the known peptide tails.
Overall Structure. As described in Methods, full-length human
JMJD6 was crystallized in the presence of Fab fragments ob-
tained from a JMJD6-specific monoclonal antibody. Due to the
flexibility of the C terminus of JMJD6, the Fab fragments are
essential to obtain crystals of the entire JMJD6 protein. Briefly,
the initial phases and structure were determined using the single
wavelength anomalous dispersion (SAD) method and a mercury
derivative. For refinement, data from multiple additional crystals
with or without α-KG were used to obtain structures both at
2.7-Å resolution. In the final models, residues 1 to 334 of JMJD6
are well defined; however, the C-terminal, serine-rich region
(residues 335 to 403) is completely disordered (Fig. 1 and
Fig. S1 A and B). The structure contains a total of 15 α-helices,
with α2, α3, α5, α6, α10, and α12 containing only one-turn helix.
These one-turn helices distribute all over the surface of the mo-
lecule and are connected by a variety of coil regions, a unique
feature for JMJD6 with unknown function (Fig. 1). With the ex-
ceptions of β3 and β4, 11 of the 13 β-strands in JMJD6 contribute
to the cupin fold, a hallmark of this enzyme family (Fig. 1) (6).
The structure can be divided into an N-terminal domain and
C-terminal domain, which associate via β13 and α9 of the N-term-
inal domain and α13 of the C-terminal domain. Several hydro-
phobic residues are involved in these interactions, including
Leu160, Phe161, and Tyr163 of the N-terminal domain and resi-
dues Trp298, Phe294, Leu308, Trp312, Leu316, and Leu323 from
the C-terminal domain (Fig. S1C). Two consecutive proline
residues between α9 and β6 and the hydrophobic core assembled
between α9 and the C-terminal domain suggest a relatively rigid
association between the N-terminal and C-terminal domains. An
Fe2þion is chelated by three residues that are highly conserved
among cupin domains: His187, Asp189, and His 273. The cofac-
tor α-ketoglutarate is bound to the Fe2þion and the side chains of
residues Lysine 204 and Asn277, as well as the main chain amide
of Ser184. The Fab fragment was found binding at the back tail
of the C-terminal domain (Fig. S1A); conformational changes
Author contributions: J.W.K. and G.Z. designed research; X.H., J.Z., J.W., C.W., C.-H.P., R.Z.,
J.H., R.C.M., S.D., and P.H. performed research; C.W., C.-H.P., R.Z., J.H., R.C.M., S.D., and P.H.
contributed new reagents/analytic tools; X.H., J.Z., J.W., J.W.K., and G.Z. analyzed data;
and J.W.K. and G.Z. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank, www.pdb.org (PDB ID code 3LD8 for JMJD6 plus Fab and PDB ID code
3LDB for JMJD6 plus Fab plus alpha-keto glutarate).
1X.H., J.Z., and J.W. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or zhangg@
This article contains supporting information online at www.pnas.org/lookup/suppl/
14568–14572 ∣ PNAS ∣ August 17, 2010 ∣ vol. 107 ∣ no. 33www.pnas.org/cgi/doi/10.1073/pnas.1008832107
caused by Fab fragments could be limited due to the rigid con-
nection between the N-terminal domain and C-terminal domain.
Structural Comparisons Among JMJD6, FIH, JMJD2, and AlkB. JMJD6
has been characterized as an arginine demethylase and U2AF65
tail lysine hydroxylase (10, 19). Therefore, we compared the
structure of JMJD6 with representative structures from these two
protein families. Overlapping features were observed between
the catalytic cores of the lysine demethylase JMJD2A (16) and
FIH (20, 21), a well-characterized asparaginyl hydroxylase (Fig. 2
and Fig. S2). Nevertheless, with the exception of the cupin fold,
we did not detect much similarity among the three proteins. This
suggests that each protein may represent a distinct subfamily.
Comparisons with the structure of AlkB—a known DNA/RNA
demethylase (22–24)—also revealed fairly limited structural
homology (Fig. 2 and Fig. S3). However, if only the cupin folds
are involved, high similarity can be found. The rmsds between the
cupin fold of JMJD6 and those of JMJD2A, FIH, as well as AlkB,
are 1.14 Å, 1.11 Å, and 1.20 Å, respectively (Fig. 2A).
Unique Structural Features of JMJD6. Our analysis revealed a num-
ber of interesting structural features in JMJD6. As described
above, JMJD6 contains an N-terminal cupin fold and a C-termi-
nal helix-turn-helix-like motif (Fig. 1). The two domains are con-
nected by an inflexible region containing two proline residues and
the hydrophobic core, which likely limit the relative movement of
the two domains (Fig. 3). These features suggest that substrate
binding may not cause significant conformational changes in
JMJD6. Furthermore, the two domains of JMJD6 create a large
groove, characterized by a diameter of ∼20 Å at its narrowest and
∼30 Å at its widest (Fig. 1). Of note, the helix-turn-helix-like
motif within the C-terminal domain and the β-hairpin comprising
β3 and β4 from the N-terminal domain are exposed along the
open groove (Fig. 1).
JMJD6 contains three aromatic residues close to the catalytic
center, which are not found in other family members. Interest-
ingly, the three side chains from Tyr131, Phe133, and Tyr174
are stacked against each other in the α-ketoglutarate- and iron
(II)-bound complex (Fig. 3). The side chain of Phe assumes
two conformations: the stacked arrangement and a conformation
that is rotated by 90° (Fig. 3).
RNA and DNA-Binding Properties of JMJD6. A surface potential map
was built based on the distribution of charged residues (Fig. 1B).
The map showed positively charged areas on the flat side of the
molecule and within the groove containing the helix-turn-helix-
like motif (Fig. 1B). These positively charged structural features
suggest JMJD6 could bind RNA or DNA.
The full-length JMJD6 protein was used in DNA and RNA
EMSAs as describe in Methods. JMJD6 strongly bound a 27-nt
single-stranded RNA (ssRNA) probe with an approximate affi-
nity of ∼40 nM (Fig. 4A), but failed to bind to the equivalent
ssDNA (Fig. S4A), double-stranded RNA (dsRNA) (Fig. S4B),
or dsDNA (Fig. S4C) probe. To identify requirements for the
binding of JMJD6 to ssRNA, we compared its binding to progres-
sively shorter probes (Fig. 4B). JMJD6 bound similarly to ssRNA
secondary structures are colored as follows: alpha-Helix, red; beta-sheet, yellow; coil, green. (B) The electrostatic surface potentials of JMJD6, positive charged
surface is colored red, negative charged surface is colored blue, and neutral surface is colored gray.
The overall structure of JMJD6, a new structure of JMJC family. (A) The stereo view of the structure of JMJD6 at present of alpha-KG and Fe2þ. The
AlkB. JMJD6 is colored magenta, Jmjd2a is colored blue, FIH is colored cyan, and AlkB is colored green. (B) The comparison of JMJD6 on the catalytic core of
Jmjd2a. JMJD6 is colored magenta, and Jmjd2a is colored blue.
The similarity and difference of JMJD6 among the cupin fold family members. (A) The cupin fold of JMJD6 is compared to those of Jmjd2a, FIH, and
Hong et al.PNAS
August 17, 2010
probes of 27, 24, and 21 nt. Binding decreased dramatically to an
18-nt probe and was not detected using probes of 15 or 13 nt. To
identify regions of JMJD6 that are necessary for ssRNA binding,
truncated proteins were generated. Only full-length JMJD6 in-
cluding the unstructured serine-rich C terminal domain (residues
and Fig. S1B). The structured fragment containing only residues
1–337 exhibited weak binding to ssRNA, whereas fragments
containing residues 290–403 or 334–403 did not bind ssRNA
Members of the α-ketoglutarate-dependent hydroxylase protein
family participate in different reactions, including antibiotic bio-
synthesis, detection of hypoxia, and metabolite processing (25).
Moreover, these enzymes include DNA, RNA, and histone tail
demethylase functions, which contribute to nucleobase, nucleo-
side, nucleotide, and chromatin metabolism (25). Each of these
subfamilies is characterized by unique structural features that
allow the enzymes to recognize and process their cognate
Sequence similarities among the members of the JMJC
domain-containing protein family and the overall structure of
JMJD6 were used to identify the catalytic core domain of the
JMJD2 histone demethylase subfamily (15, 26); the structure
of JMJD6 was determined before these activities of JMJD2 were
identified in the laboratory. Although proteins from this family
are thought to mostly function as histone demethylases, so far
we have been unable to demethylate various histone tails using
JMJD6 in vitro. Also, we have not detected DNA demethylase
or deaminase activities. It has been reported that JMJD6 hydro-
xylates lysine residues specifically in the tail of U2AF65, suggest-
ing a potential role of JMJD6 in the regulation of mRNA splicing
(19). Hydroxylase activities were also identified during the char-
acterization of JMJD6 as an arginine demethylase (10), suggest-
ing that JMJD6 hydroxylates protein tails nonspecifically. In this
study, we have shown that JMJD6 contains a large groove around
the catalytic center, which should be accessible to elongated
Phe133, and Trp174 are aligned but not stacking on each other. (B) The overlay of the active center of JMJD6 with and without alpha-KG. In the presence of
alpha-KG, the side chain of PHe133 stacks with those of Tyr131 and Trp174. This may represent a mode of substrate recognition.
Unique structural features around the active center of JMJD6. (A) The active center without binding of alpha-KG. Three aromatic residues Tyr131,
ssRNA probe prior to fractionation in an EMSA. (B) Progressively shorter ssRNA probes were incubated with (+) or without (−) full-length JMJD6 prior to
fractionation in an EMSA. (C) Binding of truncated JMJD6 proteins to the 27-nt ssRNA probe. 1. JMJD6 (1–403), 2. JMJD6 (1–337), 3. JMJD6 (290–403)
and 4. JMJD6 (334–403). All protein concentrations are about 200 nM. (D) The potential model of JMJD6 binding to ssRNA.
JMJD6 binds ssRNA. (A) Binding of full-length JMJD6 to ssRNA. Increasing concentrations of JMJD6 were incubated with a constant amount of 27-nt
www.pnas.org/cgi/doi/10.1073/pnas.1008832107 Hong et al.
peptides. This may facilitate the access of lysine side chains of any
flexible protein tails to the catalytic center.
A question remains as to whether JMJD6 acts on other sub-
strates in addition to proteins, or what is the real cognate
substrate? Several lines of evidence led us to hypothesize that
ssRNA may serve as substrates. The groove around the JMJD6
catalytic center would accommodate RNAor DNA. The C-termi-
nal groove region includes a helix-turn-helix-like motif, which is
typical for DNA or RNA binding proteins (Fig. 3). The antipar-
allel β3- and β4-sheet, which forms a β-hairpin, is also located
close to the groove (Fig. 1). β-hairpins, including acidic residues
at the apex, often make contacts with nucleotide bases. Mean-
while, the positively charged surface within the groove is consis-
tent with the property required for a RNA- or DNA-binding
domain. Moreover, a striking feature is the stack of aromatic
side chains (Fig. 3), a feature similar to that found in several cap-
binding proteins such as the m7G-cap dimethyltransferase TGS1,
the eukaryotic initiation factor 4E, and the cap-binding complex,
etc. (27). The flexibility of the side chain of the middle Phe within
the stack may allow for interactions with bases, which may be
important for binding to nucleic acid substrates during the enzy-
matic reaction. Furthermore, direct experimental data demon-
strated the binding of JMJD6 to ssRNA. Finally, further domain
characterization demonstrated that the N-terminal domain,
C-terminal domain, and unstructured serine-rich region in the
C-terminal of the protein were required for this binding (Fig. 4C).
None of these regions alone produced detectable binding to
ssRNA; however, the structured core containing residues from
1 to 337 (including the N-terminal domain and C-terminal do-
main) binds to ssRNA weakly (Fig. 4C). This result suggests that
the groove region within the structured core is essential for the
ssRNA binding; the unstructured C-terminal serine-rich motif is
also required to enhance the interaction. Here, we hypothesize
that ssRNAs are cognate substrates of JMJD6. As a potential me-
chanism of action, bases within the ssRNA substrates could insert
into the stack for enzymatic modifications similar to the intera-
tion of mRNA with cap-binding proteins, although the enzymatic
consequences are likely tobe different. Based on our data, a mod-
el of JMJD6 binding to ssRNA can be built (Fig. 4D). It should be
noted that the ssRNA sequence used in this study was selected
randomly, suggesting a lack of base sequence specificity in bind-
ing to JMJD6. However, how promiscuous this specificity is will
require additional studies.
A key issue that still remains to be resolved is the relationship
All these features, the severe phenotype of JMJD6 knockout,
the special structural features, and the ssRNA binding activity,
differentiate JMJD6 from other members of the JMJC protein
family, which work mainly as histone demethylases and protein
hydroxylases. The nature of the catalytic activity, however, is still
unclear. Based on the structural information, biochemistry data
generated so far, and the enzymatic activity nature for JMJC pro-
teins, methyl groups on nucleic acids are likely candidates for the
enzymatic target moiety. More specifically, JMJD6 may function
as a component of the splicesome (including U2AF65) at the
branch site to affect the alternative splicing of pre-mRNA. Future
studies will address the identities of these chemical targets and
their regulatory roles in cells.
Protein Expression, Purification, and Crystallization. The cDNA clone encoding
wild-type human JMJD6 was described previously (1). DNA fragments encod-
ing wild-type human JMJD6 (amino acids 1–403) or the mutant variants were
amplified in PCRs and ligated into pET23b (Novagen) with a C-terminal 6X His
tag or without a tag. The final clones were verified by restriction enzyme
digestion and DNA sequencing. Rosetta (DE3) cells (Novagen) were trans-
formed with the recombinant plasmid containing the JMJD6 gene. JMJD6
expression was induced by adding IPTG to an 8-L growing culture (37 °C)
at an OD600of 0.8. After 4 h of additional growth, cells were harvested
and resuspended in buffer A [20 mM Tris-HCl (pH 8.0), 500 mM NaCl, and
5 mM imidazole] supplemented with protease inhibitors (Invitrogen). After
cell lysis using a continuous-flow French press and a low-speed spin, the
soluble fractionwas loaded onto His affinitybeads (Novagen). After intensive
washing with buffer A, JMJD6 was eluted from the beads with buffer A con-
taining 1 M imidazole. The JMJD6 sample was loaded onto a MonoS column
(Pharmacia). After elution with a NaCl gradient, the homogeneity of the pro-
tein sample was evaluated using Coomassie blue-stained SDS-polyacrylamide
gels. JMJD6 was further purified using a Superdex 200 column (Amersham).
All other truncated versions of JMJD6 were produced similarly as the entire
version of JMJD6. Purified anti-JMJD6 hamster monoclonal antibody
(http://www.scbt.com/datasheet-32740-psr-apsr-14-4-antibody.html) was di-
gested into Fab fragments using papain (p4406, Sigma) at 37 °C for 4 h in
buffer B [20 mM phosphate buffer (pH 7.0), 10 mM EDTA, and 25 mM cy-
steine]. Fab fragments were loaded onto a MonoS column. Purified JMJD6
and the Fab fragments were mixed at 4°C for 2 h and loaded on a Superdex
200 column. Purified JMJD6-Fab complexes (6 mg∕mL) were crystallized
at 4 °C using vapor diffusion against 2.1 M (ðNH4Þ2SO4and 100 mM Bis-Tris
(pH 5.6). For data collection, crystals were gradually transferred into
cryobuffer (reservoir buffer supplemented with 20% glycerol) and flash
cooled in liquid N2.
Structure Determination and Refinement. Native crystals of the JMJD6–Fab
complexes diffracted poorly (∼3.2 Å) when examined in a synchrotron.
The crystal diffraction quality dramatically improved to 2.6 Å after the crys-
tals were incubated for one week in crystallization solution saturated with
methylmercury chloride, although the crystals remained sensitive to the
X-ray beam. Because the crystal was highly symmetric, a complete dataset
could be collected from a single crystal. Data were processed using
HKL2000 (28). The initial phases were derived from a native dataset and
anomalous derivative data using SOLVES (29) (Table S1). The phases were ex-
tended from 4.5 Å to 3.2 Å using SOLOMON (30). From the initial calculated
map, β-strands and α-helixes were identified. The Fab fragment was quickly
built into the model. The map of JMJD6 improved after adding the Fab frag-
ments via the Sigma program (30). The final model, which contained residues
1 to 334of JMJD6, was further refined using higher resolution datacombined
from data of two crystals by the Crystallography and NMR System program
(CNS) (31) (Table S1). Cocrystals of α-ketoglutarate and JMJD6 were grown at
a similar condition as the native form. The complex structure of α-ketoglu-
tarate and JMJD6 was determined using a Fourier transformation and
refined using a dataset from three crystals by CNS (31) (Table S1). All struc-
tural models were built and adjusted in the O program (32). All structural
figures were made using the PyMOL program (http://pymol.sourceforge.
net). Crystallographic data are briefly summarized in Table S1.
EMSA for ssRNA and Others. All RNA probes were purchased from Integrated
DNA Technologies. Single-stranded RNA (5′-rAUACGAUGCUUUACGGUG-
CUAUUUUGU-3′; 27 nt) was 5′ end labeled using T4 polynucleotide kinase
(New England Biolabs) and γ32P-ATP (6;000 Ci∕mmol; PerkinElmer Life and
Analytical Sciences) according to the manufacturers’ instructions. Following
extraction with phenol:chloroform, unincorporated nucleotides were re-
moved using illustra™ MicroSpin™ G-25 columns. Progressively shorter
RNA probes included 5′-AUACGAUGCUUUACGGUGCUAUUU-3′ (24 nt),
5′-AUACGAUGCUUUACGGUGCUA-3′ (21 nt), 5′-AUACGAUGCUUUACGGUG-
3′ (18 nt), 5′-AUACGAUGCUUUACG-3′ (15 nt), and 5′-GCUUUACGUGCU-3′
(13 nt). For EMSA, 15,000 cpm of each probe were incubated with increasing
amounts of recombinant JMJD6 protein in 20 μL of 10 mM Hepes pH7.6,
100 mM NaCl, 1 mM MgCl2, 1 mM dithiothreitol, 5% glycerol, 100 μg∕mL
BSA, and 0.5 μL RNasin® (Promega Corporation). Binding reactions were in-
cubated at 4 °C for 30 min; 8 μL of each reaction were fractioned by electro-
phoresis on a 5% polyacrylamide/1X Tris/glycine/EDTA gel (4°C, 240 V for
45 min) and autoradiographed as described (33). Data were quantitated
using a Typhoon 9200 PhosphorImager system (Molecular Dynamics/
Amersham/GE Healthcare). DsRNA and dsDNA binding assays follow the
ACKNOWLEDGMENTS. We thank Dr. James Kappler for editing; the Howard
Hughes Medical Institute, the Zuckerman/Canyon Ranch, and Alan Lapporte
for supporting our x-ray and computing facilities; and Dr. Philippa C. Marrack,
Dr. James D. Crapo, and other researchers at National Jewish for their kind
support. All datasets were collected from Howard Hughes Medical Institute
Beamlines 8.2.1 and 8.2.2 at the Advanced Light Source (Berkeley, CA).
J.H. was supported by National Institutes of Health (NIH) Grants AI54661
and AI22295. G.Z. was supported by NIH Grants GM80719 and AI22295
(to P.M.) and an intramural grant (2Z03300) from the Korean Institute of
Hong et al.PNAS
August 17, 2010
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