Open Access

Solution structure of the Drosha double-stranded RNA-binding domain

  • Geoffrey A Mueller1Email author,
  • Matthew T Miller1,
  • Eugene F DeRose1,
  • Mahua Ghosh1,
  • Robert E London1 and
  • Traci M Tanaka Hall1Email author
Contributed equally
Silence20101:2

DOI: 10.1186/1758-907X-1-2

Received: 11 May 2009

Accepted: 12 January 2010

Published: 12 January 2010

Abstract

Background

Drosha is a nuclear RNase III enzyme that initiates processing of regulatory microRNA. Together with partner protein DiGeorge syndrome critical region 8 (DGCR8), it forms the Microprocessor complex, which cleaves precursor transcripts called primary microRNA to produce hairpin precursor microRNA. In addition to two RNase III catalytic domains, Drosha contains a C-terminal double-stranded RNA-binding domain (dsRBD). To gain insight into the function of this domain, we determined the nuclear magnetic resonance (NMR) solution structure.

Results

We report here the solution structure of the dsRBD from Drosha (Drosha-dsRBD). The αβββα fold is similar to other dsRBD structures. A unique extended loop distinguishes this domain from other dsRBDs of known structure.

Conclusions

Despite uncertainties about RNA-binding properties of the Drosha-dsRBD, its structure suggests it retains RNA-binding features. We propose that this domain may contribute to substrate recognition in the Drosha-DGCR8 Microprocessor complex.

Background

MicroRNA (miRNA) are small regulatory RNAs derived from longer RNA transcripts called primary miRNA (pri-miRNA) ([1], reviewed recently in [2]). Pri-miRNA are cleaved by an RNase III family enzyme called Drosha to produce hairpin precursor miRNA (pre-miRNA) [3]. Pre-miRNA are transported to the cytoplasm [47] and further processed by Dicer enzymes to produce mature miRNA [813]. Drosha contains two RNase III domains that form the enzyme's catalytic center. At the C-terminus is a double-stranded RNA-binding domain (dsRBD), which is essential for pri-miRNA processing [14].

To process pri-miRNA, Drosha forms an enzyme complex with a partner protein DiGeorge syndrome critical region 8 (DGCR8; also known as Pasha in Drosophila and Caenorhabditis elegans) [1417], which contains two dsRBDs. DGCR8 has been proposed to be a crucial factor for recognition of pri-miRNA substrate via its dsRBDs [18]. A crystal structure of the tandem dsRBDs of DGCR8 revealed closely interacting domains whose conformation would not be expected to change upon RNA binding [19]. A model for RNA recognition suggests that the two domains bind to portions of the pri-miRNA that are distant from each other. It is not known whether the dsRBD of Drosha is also important for substrate RNA binding or serves another function, since little to no RNA-binding activity has been observed for Drosha and the dsRBD is not necessary for interaction with DGCR8 [14, 18, 20, 21]. To gain insight into the function of Drosha-dsRBD, we determined the solution structure of this domain. The structure suggests it retains RNA-binding features. We suggest this domain may participate in RNA interaction with DGCR8 in the context of the microprocessor complex.

Results and Discussion

The solution structure of Drosha-dsRBD comprises an α helix (Ser1263 to Thr1271), followed by three β strands forming an antiparallel β sheet (Leu1283 to Gly1314), and terminating with a second α helix (Ile1317 to Lys1331) (Figure 1a-c). This αβββα fold is consistent with the core structures of other members of the dsRBD family [22]. Residues highly conserved among dsRBDs and important for the fold are found in the Drosha-dsRBD (boxed in Figure 1c) [22]. A unique feature of the Drosha-dsRBD is an extended α1-β1 loop. This loop is compact in all other known dsRBD structures. The α1-β1 loop shows some of the lowest {1H}-15N-nuclear Overhauser effects (NOEs) (Figure 2), indicating it is dynamic on a fast time scale (picoseconds to nanoseconds).
https://static-content.springer.com/image/art%3A10.1186%2F1758-907X-1-2/MediaObjects/13101_2009_Article_2_Fig1_HTML.jpg
Figure 1

Nuclear magnetic resonance (NMR) solution structure of Drosha-double-stranded RNA-binding domain (dsRBD). (a) Ribbon diagram of the lowest energy-minimized structure of Drosha-dsRBD. Regions of dsRBDs that typically interact with RNA are highlighted with light blue and labeled as in the text. (b) Superposition of the Cα traces of the 10 lowest energy-minimized structures of Drosha-dsRBD. (c) Structure-based sequence alignment of Drosha-dsRBD and selected dsRBDs. Amino acid residues that do not structurally align with Drosha-dsRBD are shown in lower case letters. Secondary structural elements and amino acid numbers for Drosha-dsRBD are indicated. Boxed residues are well conserved among dsRBDs. Typical RNA-interacting regions are indicated with brackets, and RNA-interacting residues are in bold. Sequences of Aquifex aeolicus RNase III (AaRnIII) [24], Saccharomyces cerevisiae Rnt1p [23, 35], Xenopus laevis Xlrbpa-2 [26], Drosophila melanogaster Staufen dsRBD-3 [25], and DiGeorge syndrome critical region 8 (DGCR8) protein dsRBD 1 (DGCR8-1) and 2 (DGCR8-2)[19] are shown. (d, e) Electrostatic surface representation calculated using the APBS package [36] of the lowest energy-minimized structure of Drosha-dsRBD. Red (negative) is set at - 3 kT/e and blue (positive) is set at 3 kT/e. RNA is from A. aeolicus RNase III in complex with dsRNA substrate [PDB:2EZ6]. Panel e is rotated 180° relative to the other panels. This figure was prepared with the PyMol package [37].

https://static-content.springer.com/image/art%3A10.1186%2F1758-907X-1-2/MediaObjects/13101_2009_Article_2_Fig2_HTML.jpg
Figure 2

Heteronuclear nuclear Overhauser effect (NOE) { 1 H}- 15 N measured at 14.1 T. The ratio of measured intensity with and without presaturation is plotted versus residue, for those residues with well isolated (non-degenerate) chemical shifts.

Sequence features important for RNA recognition are also conserved in Drosha-dsRBD. In structures of dsRBDs in complex with RNA, the domain binds to one face of a dsRNA helix, and three regions are important for RNA recognition: β1 (region 1), the β1-β2 loop (region 2), and the β3-α2 loop (region 3) [22]. Helix α1 and the β1-β2 loop interact with successive minor grooves of the dsRNA, and the β3-α2 loop interacts with the intervening major groove. RNA interacting residues in region 1 are conserved in Drosha-dsRBD. For example, Lys1262 is equivalent to Lys271 in Saccharomyces cerevisiae Rnt1p, which contacts the RNA substrate, and mutation of Rnt1p-Lys271 to alanine severely suppresses in vivo RNA processing [23]. This lysine residue is conserved in dsRBDs associated with RNase III enzymes. Similarly, Gln1267 is equivalent to Aquifex aeolicus RNase III Glu158, Rnt1p Ser376, Xenopus laevis Xlrbpa-2 Glu119, and Drosophila melanogaster Staufen Glu7, which contact the RNA backbone [2326]. In region 2, His1294 and Arg1296 are equivalent to His141 and Arg143 in Xlrbpa-2, which contact the RNA in the subsequent minor groove [26]. A cluster of basic and polar side chains in region 3 typically contacts the major groove. Drosha-dsRBD lacks a high density of basic residues in this region (Figure 1c); thus it is possible that interactions with the major groove are minimal or comprise mainly polar interactions.

The distribution of charged side chains on the surface of the protein is also consistent with RNA binding (Figure 1d, e). A positively charged region could facilitate the binding of the negatively charged phosphate backbone of an RNA molecule. This region extends to the opposite side of the dsRBD, which is not the typical RNA-binding surface. We superimposed Drosha-dsRBD and the dsRBD of A. aeolicus RNase III [24] to illustrate how Drosha-dsRBD could bind to a dsRNA (Figure 1d, e). Given the electrostatic surface and the presence of specific RNA-interacting residues, Drosha-dsRBD appears capable of binding RNA, despite the inability to demonstrate interaction of Drosha or its dsRBD with pri-mRNA [18, 20, 21]. From the model in Figure 1d, the extended α1-β1 loop in Drosha-dsRBD could interact with the RNA, adding a new substrate recognition feature. However, the loop is negatively charged, and although this does not exclude nucleic acid interaction [27, 28], alternatively it could facilitate intermolecular or intramolecular protein-protein interaction. Both this loop and the β1-β2 loop are not positioned to allow direct interactions with the straight, regular RNA duplex in the model. The substrates of Drosha are hairpin pri-miRNA with mismatched and bulged bases that would form irregular structures. Thus the substrate RNA could be bent and the protein loops could alter conformation to allow interaction.

DGCR8 contains two dsRBDs, which recognize pri-miRNA [1820]. In the crystal structure of the tandem dsRBDs of DGCR8, the dsRBDs likely bind to separate dsRNA regions on the pri-miRNA [19]. Pri-miRNA contain long hairpin loops with several distinguishing characteristics: The 5' and 3' ends are unstructured basal segments, an approximate 11-bp lower stem proceeds from the basal segments to the cleavage site, and on the other side of the cleavage site is an approximate 22-bp upper stem that ends with a terminal loop [18]. These features are important for substrate recognition and/or cleavage site location [21, 29]. The reported affinity of DGCR8 for pri-miRNA is relatively weak (Kd = 2 mM) [19], and full-length Drosha or Drosha-dsRBD exhibit poor, if any, binding to RNA on their own [18, 20, 21]. Perhaps the Drosha-DGCR8 complex has greater affinity and specificity with each dsRBD fine tuning substrate recognition by binding to a specific feature of the pri-miRNA. For example, the dsRBDs of DGCR8 may recognize the upper and lower stem regions of the pri-miRNA near the basal segments and terminal loop, respectively, while Drosha-dsRBD may bind to the central region near the cleavage site, as is observed with A. aeolicus RNase III [24]. Additional biochemical and structural studies are needed to understand fully how each dsRBD participates in substrate recognition. Such studies would benefit from abundant pure Drosha/DGCR8 complex.

Conclusions

We have determined the solution structure of Drosha-dsRBD. The structure is similar to other RNA-binding dsRBDs, and features important for RNA recognition are conserved. A long loop between α1 and β1 is unique to Drosha-dsRBD. We propose Drosha-dsRBD may participate in RNA recognition in the Drosha-DGCR8 complex, despite little to no RNA binding on its own.

Methods

Protein expression and purification

Human Drosha-dsRBD [EMBL:AF189011] (amino acids 1,259 to 1,337, Addgene plasmid no. 108,208; Addgene, Cambridge, MA, USA) was expressed with a C-terminal His6 tag using pET21c(+). Protein was expressed in BL21(DE3) cells induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 3 to 5 h at 37°C. Single-labeled and double-labeled proteins were generated by growth in M9 minimal media including combinations of 13C-labeled glucose and/or15N-labeled ammonium chloride. Protein was purified using Ni2+-NTA resin followed by separation on a Resource Q anion exchange column (GE Healthcare, Uppsala, Sweden). For nuclear magnetic resonance (NMR) analysis, the purified protein was pooled and exchanged into a buffer comprising 25 mM tris(hydroxymethyl)aminomethane (Tris)(D11) pH 7.0, 100 mM KCl, 1 mM dithiothreitol (DTT)(D10), 1 mM ethylenediaminetetraacetic acid (EDTA), 10% D2O, and 10 mm dimethylsilapentanesulfonate (DSS) by concentration and dilution.

NMR structure determination and refinement

Standard triple resonance NMR experiments were utilized to assign the backbone and side-chain resonances of the proteins [30]. Proton chemical shift assignment was 86% complete as assessed by the CYANA package [31]. Of note, the only NOE experiment acquired was the 4D simultaneous 13C/15N nuclear Overhauser enhancement spectroscopy (NOESY) [32]. Customized scripts were written to unalias peaks and write formatted files for structure calculation and automated assignment with CYANA. The structures were subsequently refined with the XPLOR-NIH package [33] utilizing the hydrogen bond distance angle (HBDA) module [34] for hydrogen bond restraints. Structural statistics are given in Table 1.
Table 1

Structural statistics for the 10 lowest energy-minimized conformers of Drosha-double-stranded RNA-binding domain (dsRBD)

Statistic

Value

NOE distance restraints:

 

Intraresidue

98

Sequential

111

Medium range (i, i + 2 to 4)

74

Long range (i, i>4)

206

Total

489

Dihedral restraints:

189

Hydrogen bond (HBDA):

27

Ensemble RMSD:

 

Backbone secondary structure

0.44

Heavy atoms secondary structure

0.86

Violations:

 

NOE

0

Dihedral

0

HBDA

1.4

RMS experimental:

 

NOE

0.02 ± 0.005

Dihedral

0.415 ± 0.077

HBDA

0.006 ± 0.006

RMS covalent geometry:

 

Bonds

0.003 ± 0.000

Angles

0.514 ± 0.009

Impropers

0.362 ± 0.013

Ramachandran space:

 

Most favored region

81.6%

Additionally allowed

17.0%

Generously allowed

1.4%

Disallowed

0

HBDA = hydrogen bond distance angle; NOE = nuclear Overhauser effect; RMSD = root mean square deviation.

Accession numbers

The PDB coordinates [PDB:2KHX] and NMR assignments (16,256) have been deposited to the Research Collaboratory for Structural Bioinformatics (RCSB) and BioMagResBank (BMRB) databases, respectively.

Notes

Declarations

Acknowledgements

We are grateful to Alison Mead for assistance with protein purification. This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences.

Authors’ Affiliations

(1)
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health

References

  1. Lee Y, Jeon K, Lee JT, Kim S, Kim VN: MicroRNA maturation: stepwise processing and subcellular localization. EMBO J. 2002, 21: 4663-4670. 10.1093/emboj/cdf476.PubMed CentralView ArticlePubMedGoogle Scholar
  2. Kim VN, Han J, Siomi MC: Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol. 2009, 10: 126-139. 10.1038/nrm2632.View ArticlePubMedGoogle Scholar
  3. Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, Lee J, Provost P, Radmark O, Kim S, Kim VN: The nuclear RNase III Drosha initiates microRNA processing. Nature. 2003, 425: 415-419. 10.1038/nature01957.View ArticlePubMedGoogle Scholar
  4. Bohnsack MT, Czaplinski K, Gorlich D: Exportin 5 is a RanGTP-dependent dsRNA-binding protein that mediates nuclear export of pre-miRNAs. RNA. 2004, 10: 185-191. 10.1261/rna.5167604.PubMed CentralView ArticlePubMedGoogle Scholar
  5. Lund E, Guttinger S, Calado A, Dahlberg JE, Kutay U: Nuclear export of microRNA precursors. Science. 2004, 303: 95-98. 10.1126/science.1090599.View ArticlePubMedGoogle Scholar
  6. Park MY, Wu G, Gonzalez-Sulser A, Vaucheret H, Poethig RS: Nuclear processing and export of microRNAs in Arabidopsis. Proc Natl Acad Sci USA. 2005, 102: 3691-3696. 10.1073/pnas.0405570102.PubMed CentralView ArticlePubMedGoogle Scholar
  7. Yi R, Qin Y, Macara IG, Cullen BR: Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev. 2003, 17: 3011-3016. 10.1101/gad.1158803.PubMed CentralView ArticlePubMedGoogle Scholar
  8. Bernstein E, Caudy AA, Hammond SM, Hannon GJ: Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature. 2001, 409: 363-366. 10.1038/35053110.View ArticlePubMedGoogle Scholar
  9. Grishok A, Pasquinelli AE, Conte D, Li N, Parrish S, Ha I, Baillie DL, Fire A, Ruvkun G, Mello CC: Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell. 2001, 106: 23-34. 10.1016/S0092-8674(01)00431-7.View ArticlePubMedGoogle Scholar
  10. Hutvagner G, McLachlan J, Pasquinelli AE, Balint E, Tuschl T, Zamore PD: A cellular function for the RNA-interference enzyme dicer in the maturation of the let-7 small temporal RNA. Science. 2001, 293: 834-838. 10.1126/science.1062961.View ArticlePubMedGoogle Scholar
  11. Ketting RF, Fischer SE, Bernstein E, Sijen T, Hannon GJ, Plasterk RH: Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev. 2001, 15: 2654-2659. 10.1101/gad.927801.PubMed CentralView ArticlePubMedGoogle Scholar
  12. Knight SW, Bass BL: A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans. Science. 2001, 293: 2269-2271. 10.1126/science.1062039.PubMed CentralView ArticlePubMedGoogle Scholar
  13. Park W, Li J, Song R, Messing J, Chen X: CARPEL FACTORY, a dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Curr Biol. 2002, 12: 1484-1495. 10.1016/S0960-9822(02)01017-5.View ArticlePubMedGoogle Scholar
  14. Han J, Lee Y, Yeom KH, Kim YK, Jin H, Kim VN: The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev. 2004, 18: 3016-3027. 10.1101/gad.1262504.PubMed CentralView ArticlePubMedGoogle Scholar
  15. Denli AM, Tops BB, Plasterk RH, Ketting RF, Hannon GJ: Processing of primary microRNAs by the microprocessor complex. Nature. 2004, 432: 231-235. 10.1038/nature03049.View ArticlePubMedGoogle Scholar
  16. Gregory RI, Yan KP, Amuthan G, Chendrimada T, Doratotaj B, Cooch N, Shiekhattar R: The microprocessor complex mediates the genesis of microRNAs. Nature. 2004, 432: 235-240. 10.1038/nature03120.View ArticlePubMedGoogle Scholar
  17. Landthaler M, Yalcin A, Tuschl T: The human DiGeorge syndrome critical region gene 8 and Its D. melanogaster homolog are required for miRNA biogenesis. Curr Biol. 2004, 14: 2162-2167. 10.1016/j.cub.2004.11.001.View ArticlePubMedGoogle Scholar
  18. Han J, Lee Y, Yeom KH, Nam JW, Heo I, Rhee JK, Sohn SY, Cho Y, Zhang BT, Kim VN: Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell. 2006, 125: 887-901. 10.1016/j.cell.2006.03.043.View ArticlePubMedGoogle Scholar
  19. Sohn SY, Bae WJ, Kim JJ, Yeom KH, Kim VN, Cho Y: Crystal structure of human DGCR8 core. Nat Struct Mol Biol. 2007, 14: 847-853. 10.1038/nsmb1294.View ArticlePubMedGoogle Scholar
  20. Yeom KH, Lee Y, Han J, Suh MR, Kim VN: Characterization of DGCR8/Pasha, the essential cofactor for Drosha in primary miRNA processing. Nucleic Acids Res. 2006, 34: 4622-4629. 10.1093/nar/gkl458.PubMed CentralView ArticlePubMedGoogle Scholar
  21. Zeng Y, Cullen BR: Efficient processing of primary microRNA hairpins by Drosha requires flanking nonstructured RNA sequences. J Biol Chem. 2005, 280: 27595-27603. 10.1074/jbc.M504714200.View ArticlePubMedGoogle Scholar
  22. Tian B, Bevilacqua PC, Diegelman-Parente A, Mathews MB: The double-stranded-RNA-binding motif: interference and much more. Nat Rev Mol Cell Biol. 2004, 5: 1013-1023. 10.1038/nrm1528.View ArticlePubMedGoogle Scholar
  23. Wu H, Henras A, Chanfreau G, Feigon J: Structural basis for recognition of the AGNN tetraloop RNA fold by the double-stranded RNA-binding domain of Rnt1p RNase III. Proc Natl Acad Sci USA. 2004, 101: 8307-8312. 10.1073/pnas.0402627101.PubMed CentralView ArticlePubMedGoogle Scholar
  24. Gan J, Tropea JE, Austin BP, Court DL, Waugh DS, Ji X: Structural insight into the mechanism of double-stranded RNA processing by ribonuclease III. Cell. 2006, 124: 355-366. 10.1016/j.cell.2005.11.034.View ArticlePubMedGoogle Scholar
  25. Ramos A, Bayer P, Varani G: Determination of the structure of the RNA complex of a double-stranded RNA-binding domain from Drosophila Staufen protein. Biopolymers. 1999, 52: 181-196. 10.1002/1097-0282(1999)52:4<181::AID-BIP1003>3.0.CO;2-5.View ArticlePubMedGoogle Scholar
  26. Ryter JM, Schultz SC: Molecular basis of double-stranded RNA-protein interactions: structure of a dsRNA-binding domain complexed with dsRNA. EMBO J. 1998, 17: 7505-7513. 10.1093/emboj/17.24.7505.PubMed CentralView ArticlePubMedGoogle Scholar
  27. Lahm A, Suck D: DNase I-induced DNA conformation. 2 A structure of a DNase I-octamer complex. J Mol Biol. 1991, 222: 645-667. 10.1016/0022-2836(91)90502-W.View ArticlePubMedGoogle Scholar
  28. Ledvina PS, Yao N, Choudhary A, Quiocho FA: Negative electrostatic surface potential of protein sites specific for anionic ligands. Proc Natl Acad Sci USA. 1996, 93: 6786-6791. 10.1073/pnas.93.13.6786.PubMed CentralView ArticlePubMedGoogle Scholar
  29. Zeng Y, Yi R, Cullen BR: Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha. EMBO J. 2005, 24: 138-148. 10.1038/sj.emboj.7600491.PubMed CentralView ArticlePubMedGoogle Scholar
  30. Mueller GA, Moon AF, Derose EF, Havener JM, Ramsden DA, Pedersen LC, London RE: A comparison of BRCT domains involved in nonhomologous end-joining: introducing the solution structure of the BRCT domain of polymerase lambda. DNA Repair (Amst). 2008, 7: 1340-1351. 10.1016/j.dnarep.2008.04.018.View ArticleGoogle Scholar
  31. Herrmann T, Guntert P, Wuthrich K: Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA. J Mol Biol. 2002, 319: 209-227. 10.1016/S0022-2836(02)00241-3.View ArticlePubMedGoogle Scholar
  32. Xu Y, Long D, Yang D: Rapid data collection for protein structure determination by NMR spectroscopy. J Am Chem Soc. 2007, 129: 7722-7723. 10.1021/ja071442e.View ArticlePubMedGoogle Scholar
  33. Schwieters CD, Kuszewski JJ, Tjandra N, Clore GM: The Xplor-NIH NMR molecular structure determination package. J Magn Reson. 2003, 160: 65-73. 10.1016/S1090-7807(02)00014-9.View ArticlePubMedGoogle Scholar
  34. Grishaev A, Bax A: An empirical backbone-backbone hydrogen-bonding potential in proteins and its applications to NMR structure refinement and validation. J Am Chem Soc. 2004, 126: 7281-7292. 10.1021/ja0319994.View ArticlePubMedGoogle Scholar
  35. Leulliot N, Quevillon-Cheruel S, Graille M, van Tilbeurgh H, Leeper TC, Godin KS, Edwards TE, Sigurdsson ST, Rozenkrants N, Nagel RJ, Ares M, Varani G: A new alpha-helical extension promotes RNA binding by the dsRBD of Rnt1p RNAse III. EMBO J. 2004, 23: 2468-2477. 10.1038/sj.emboj.7600260.PubMed CentralView ArticlePubMedGoogle Scholar
  36. Baker NA, Sept D, Joseph S, Holst MJ, McCammon JA: Electrostatics of nanosystems: application to microtubules and the ribosome. Proc Natl Acad Sci USA. 2001, 98: 10037-10041. 10.1073/pnas.181342398.PubMed CentralView ArticlePubMedGoogle Scholar
  37. DeLano WL: The PyMOL molecular graphics system. 2002, San Carlos, CA, USA: DeLano ScientificGoogle Scholar

Copyright

© Mueller et al; licensee BioMed Central Ltd. 2010

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Advertisement