ARTICLE

Backbone 1HN, 13C, and 15N resonance assignments of the tandemRNA-binding domains of human DGCR8

Braden M. Roth • Mirko Hennig

Received: 9 May 2012 / Accepted: 21 June 2012

� Springer Science+Business Media B.V. 2012

Abstract Double-stranded RNA binding domain

(dsRBD) containing proteins are critical components of the

microRNA (miRNA) pathway, with key roles in small

RNA biogenesis, modification, and regulation. DiGeorge

Critical Region 8 (DGCR8) is a 773 amino acid, dsRBD-

containing protein that was originally identified in humans

as a protein encoded in the region of chromosome 22 that is

deleted in patients with DiGeorge syndrome. Now, it is

realized that DGCR8 complements the nuclear RNase III

Drosha to initiate miRNA biogenesis by promoting effi-

cient recognition and cleavage of primary miRNAs (pri-

miRNA). A pair of C-terminal tandem dsRBDs separated

by a flexible linker are required for pri-miRNA substrate

binding and recognition. The crystal structure of the

DGCR8 core region comprising residues 493–720 revealed

that each dsRBD adopts the canonical abbba fold. How-

ever, several residues located in important flexible regions

including the b1-b2-loop implicated in canonical dsRNA

recognition are absent in the crystal structure and no RNA-

bound structure of DGCR8 has been reported. Here we

report the 1HN, 13C, and 15N backbone resonance assign-

ments of the 24 kDa, 214 amino acid human DGCR8core

(residues 493–706) by heteronuclear NMR spectroscopy.

Our assignments lay the foundation for a detailed solution

state characterization of the dynamical and RNA-binding

properties of this protein in solution.

Keywords DiGeorge Critical Region 8 (DGCR8) �Double stranded RNA binding domain (dsRBD) �Microprocessor � microRNA (miRNA)

Biological context

MicroRNAs are non-coding RNAs that regulate gene

expression through directed downregulation of targeted

messenger RNA (mRNA). MiRNA biogenesis involves two

distinct RNase III processing events. Long, noncoding pri-

miRNA transcripts are initially cleaved by the nuclear

RNase III Drosha to release a *70nt precursor (pre)-miR-

NA. Pre-miRNAs undergo a second round of processing by

the cytoplasmic RNase III Dicer before incorporating into

the RISC (RNA-Induced Silencing Complex) where the

mature miRNA serves as a guide to repress translation or

destabilize mRNAs with incomplete complementarity to the

miRNA guide. RNase III-dependent miRNA biogenesis is

facilitated by dsRBD containing protein cofactors. In

humans, the nuclear protein DGCR8 assists Drosha in the

recognition and cleavage of miRNA-containing pri-miRNA

hairpins and is required for embryonic development and

differentiation.

DGCR8 was initially identified as the essential gene

located within a 1.5-Mb chromosomal deletion (del22q11)

commonly associated with DGS. More recently, DGCR8 has

been shown to play a central role in pri-miRNA recognition

and processing (Han et al. 2004; Landthaler et al. 2004).

Drosha and DGCR8 copurify from cell extracts and together

constitute the ‘‘minimal’’ Microprocessor, responsible for

the initiation step of pri-miRNA biogenesis (Gregory et al.

2004). Through a largely unknown process, the Micropro-

cessor recognizes and binds the miRNA-containing hairpin

at its single-stranded(ss)-double stranded (ds) RNA junction

B. M. Roth � M. Hennig (&)

Department of Biochemistry and Molecular Biology, Medical

University of South Carolina, Charleston, 70 President St,

DD213, PO Box 250509, South Carolina 29425, USA

e-mail: hennig@musc.edu

123

Biomol NMR Assign

DOI 10.1007/s12104-012-9406-x

positioning Drosha’s catalytic RNase III domains to cleave

the pri-miRNA approximately one helical turn from the ss-

dsRNA junction (Han et al. 2006).

The *175 amino acid tandem dsRBD domain (511–685)

is the signature motif of RNase III-associated accessory

proteins. Crystal structures of dsRBD in complex with two

collinearly stacking, GC-rich dsRNA substrates helped to

define canonical interaction interfaces involving the a-heli-

cal and b1-b2 loop regions of a typical abbba dsRBD.

Besides its tandem C-terminal RBDs, DGCR8 features a

WW motif overlapping with the C352-mediated heme-

binding/dimerization domain (Faller et al. 2007), and an

N-terminal nuclear localization signal. Undoubtedly, the two

C-terminal dsRBDs of DGCR8 play a key role in pri-miRNA

substrate recognition, but answering the question whether

Drosha and DGCR8 function in mutually exclusive or

cooperative fashion awaits detailed structural information on

DGCR8-pri-miRNA complexes. Sohn et al. (2007) reported

the crystal structure of the DGCR8 ‘‘core’’, (residues

493–720) comprising both dsRBDs and the flexible linker

(residues 493–720). Their analysis revealed that the dsRBDs

are arranged in a pseudo two-fold symmetry stabilized by

hydrophobic interactions and hydrogen bonding between

both dsRBDs and a C-terminal loop/helix (residues

684–698) (Fig. 2, a7). Absent from the crystal structure,

however, are residues that correspond to flexible regions,

most notably the dsRBD1-dsRBD2 linker and a canonical

RNA-binding region located in the b5–b6 loop (Fig. 2).

Although this work has confirmed the canonical abbbaarchitecture of each dsRBD, how pri-miRNA are recognized

and distinguished from the myriad of ss-dsRNA junction in

other RNA transcripts by DGCR8 and whether the dsRBDs

coordinate the binding of a single RNA substrates remains

unknown. If a single, rod-like pri-miRNA substrate would be

recognized then the tandem dsRBD1-dsRBD2 would have to

undergo a significant conformational change upon binding

suggesting that NMR dynamics investigations could help to

decipher the pri-miRNA binding mechanism.

Here we report the 1HN, 13C, and 15N backbone reso-

nance assignments and secondary structure of the 24 kDa

human DGCR8core in solution. These will serve as a

foundation for further investigation of DGCR8 in terms of

protein dynamics and DGCR8/pri-miRNA complex

interactions.

Methods and experiments

Expression and purification of deuterated 2H,15N,(13C)-

DGCR8core

We have generated a 214 amino acid (24 kDa) truncated

version of the DGCR8core consisting of residues 493–706.

DGCR8core was amplified from pFLAG/HA-DGCR8

plasmid (Addgene #10921; Landthaler et al. 2004) with the

TEV protease recognition sequence ENLYFQS incorpo-

rated in the 50 primer: (50- GAAAACCTGTACTTT-

CAGtcagtgcaagatgcacccacaaagaaag-30 and 50- tcagaccatct

tgctgctctcacggccatacatgcgc-30; uppercase represents TEV

recognition sequence). The PCR product was used to

generate a pENTR-TEV/core Gateway entry vector by

TOPO-TA cloning with Invitrogen’s pCR8/GW/TOPO

vector. pENTR-TEV/core was recombined with a cus-

tomized Gatweway p(H)GB1-GTW destination vector that

incorporated the TEV/core sequence in frame with an

N-terminal His6-GB1 protein. Positive clones were trans-

formed into E. coli BL21-CodonPlus (DE3)-RIPL compe-

tent cells (Stratagene). Deuterated cultures of E. coli

BL21-CodonPlus (DE3)-RIPL cells expressing DGCR8core

were grown from a single colony in 2 ml LB/Amp100 at

37 �C overnight. These cultures were then adapted to

growth in 500 ml of [99.9 % D2O]-15N, (13C)-enriched M9

minimal media with a succession of small-volume cultures

grown at 37 �C. Protonated glucose carbon sources were

used and expression was induced with 1 mM IPTG for 4 h

at 37 �C, then purified with the HisTrapFF affinity column

(GE Healthcare). Partially purified fusion proteins were

cleaved with TEV protease overnight at room temperature

and reapplied to the HisTrap column. The flow-through

contained highly purified protein that was subsequently

exchanged into NMR buffer.

NMR spectroscopy

Even in the absence of the previously identified dimeriza-

tion domain of DGCR8 and at modest concentration,

DGCR8core exhibits a strong tendency to dimerize in

solution. In agreement with previously published work,

size-exclusion chromatography (SEC) analysis yielded an

estimated molecular weight (MW) of 31.0 kDa for the

DGCR8core, significantly larger than the theoretical MW of

24.2 kDa (data not shown). To address the resulting line

broadening, we have expressed deuterated 15N, 13C-

DGCR8core and utilized TROSY (transverse relaxation

optimized spectroscopy)-based pulse sequences to enhance

sensitivity and resolution. NMR spectra were acquired in

500 ll of 90 % H2O/10 % D2O at 25 �C on back-

exchanged 2H,15N- as well as 2H,15N,13C-labeled

DGCR8core samples (ca. 300 lM in 20 mM Na-phosphate

buffer, pH 7.0, 150 mM KCl, and 5 mM TCEP).

Three-dimensional, clean TROSY-based (Pervushin

et al. 1997; Schulte-Herbruggen and Sorensen 2000),

constant-time (CT)-HNCA, sequential CT-HNCA, CT-

HN(CA)CB, CT-HN(COCA)CB, HNCO and HN(CA)CO

were recorded on a TCI-cryoprobe-equipped Bruker

Avance800 spectrometer to establish sequential

B. M. Roth, M. Hennig

123

connectivities based on a pairwise comparison of intra- and

inter-residue 13Ca, 13Cb and 13C’ chemical shifts. Subse-

quently, assignments were confirmed using through-space1HN and 15N correlations derived from modified 3D

HSQC-NOESY-TROSY and NOESY-TROSY experi-

ments. The NMR data were processed using nmrPipe, and

analyzed with CcpNMR Analysis software.

Assignments and data deposition

The well-dispersed 2D 1H-15N TROSY spectrum of

DGCR8core is shown in Fig. 1. In total, 800 of 845 (95 %)

backbone resonances were assigned, including 96 % of1HN protons and 15N amides, 95 % of Ca, 90 % of Cb, and

97 % of C’ resonances. Residues in flexible regions (the

dsRBD1-dsRBD2 linker and the b5-b6 loop) that were

elusive in the crystal structure were readily observed. 15N

assignments for the N-terminal residue (S493), nine pro-

lines and eight other residues (H140, N156, Q157, K175,

N176, K177, S185, and N196) have not been obtained,

presumably due to line broadening as a result of solvent

exchange or intermediate conformational exchange. 1H

chemical shifts were externally referenced to DSS, with

heteronuclear 13C and 15N chemical shifts referenced

indirectly according to the X/1H ratio in DSS. Assignments

Fig. 1 Quality of the NMR spectra obtained for DGCR8core showing an 800 MHz 1H,15N TROSY spectrum recorded at 25 �C. An enlarged

view of the most crowded region of the spectrum is show in the top-left corner. Assignments of the signals from backbone amide groups are

indicated by residue type and number; tryptophan sidechain indole resonances are marked with e

Fig. 2 Plot of the secondary chemical shifts used to derive the

secondary structure of DGCR8core. Differences of chemical shift

deviations of Ca and Cb carbons (Dd(Ca)–(Dd(Cb)) with respect to

corresponding random coil values are plotted against amino acid

residue number. Locations of the a-helices and b-strands identified by

TALOS? are indicated with boxes and labeled a1-7 and b1-7

Backbone 1HN, 13C, and 15N resonance assignments

123

have been deposited in the BioMagResBank database

(http://www.bmrb.wisc.edu) under accession number

17773.

Backbone Ca Cb and C’ assignments were used in

chemical shift index (Wishart and Sykes 1994) and TA-

LOS? (Shen et al. 2009) analyses as shown in Fig. 2; the

resulting secondary structure elements closely resemble

those of the crystal structure (Sohn et al. 2007) with minor

differences in secondary structure boundaries. The pre-

dicted secondary structure of human DGCR8core in solution

includes seven a-helices (a1: residues 512–524, a2: resi-

dues 562–576, a3: residues 593–598, a4: residues 606–614,

a5: residues 620–629, a6: residues 670–684, and a7: resi-

dues 691–697) and seven b-strands (b1: residues 503–505,

b2: residues 529–535, b3: residues 544–548, b4: residues

551–558, b5: residues 639–644, b6: residues 651–657, and

b7: residues 661–666).

Acknowledgments This work was supported by the National Sci-

ence Foundation [MCB 0845512 to M.H.]. We thank T. Tuschl for the

pFLAG/HA-DGCR8 construct and members of the Hennig laboratory

for stimulating discussions and comments on the manuscript and

acknowledge the support of the Hollings Marine Laboratory NMR

Facility for this work.

References

Faller M, Matsunaga M, Yin S, Loo JA, Guo F (2007) Heme is

involved in microRNA processing. Nat Struct Mol Biol 14(1):

23–29

Gregory RI, Yan KP, Amuthan G, Chendrimada T, Doratotaj B,

Cooch N, Shiekhattar R (2004) The Microprocessor complex

mediates the genesis of microRNAs. Nature 432(7014):235–240

Han J, Lee Y, Yeom KH, Kim YK, Jin H, Kim VN (2004) The

Drosha-DGCR8 complex in primary microRNA processing.

Genes Dev 18(24):3016–3027

Han J, Lee Y, Yeom KH, Nam JW, Heo I, Rhee JK, Sohn SY, Cho Y,

Zhang BT, Kim VN (2006) Molecular basis for the recognition

of primary microRNAs by the Drosha-DGCR8 complex. Cell

125(5):887–901

Landthaler M, Yalcin A, Tuschl T (2004) The human DiGeorge

syndrome critical region gene 8 and Its D. melanogaster

homolog are required for miRNA biogenesis. Curr Biol 14(23):

2162–2167

Pervushin K, Riek R, Wider G, Wuthrich K (1997) Attenuated T2

relaxation by mutual cancellation of dipole–dipole coupling and

chemical shift anisotropy indicates an avenue to NMR structures

of very large biological macromolecules in solution. Proc Natl

Acad Sci USA 94(23):12366–12371

Schulte-Herbruggen T, Sorensen OW (2000) Clean TROSY: com-

pensation for relaxation-induced artifacts. J Magn Reson 144(1):

123–128

Shen Y, Delaglio F, Cornilescu G, Bax A (2009) TALOS ? : a hybrid

method for predicting protein backbone torsion angles from

NMR chemical shifts. J Biomol NMR 44(4):213–223

Sohn SY, Bae WJ, Kim JJ, Yeom KH, Kim VN, Cho Y (2007) Crystal

structure of human DGCR8 core. Nat Struct Mol Biol 14(9):

847–853

Wishart DS, Sykes BD (1994) The 13C chemical-shift index: a simple

method for the identification of protein secondary structure using

13C chemical-shift data. J Biomol NMR 4(2):171–180

B. M. Roth, M. Hennig

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