Supplementary material
Letter to the editor: Resonance assignments of the two N-terminal RNA recognition motifs (RRM) of the human heterogeneous nuclear ribonucleoprotein F (HnRNP F)
Cyril Dominguez & Frédéric H.-T. Allain*
Institute of Molecular Biology and Biophysics, Swiss Federal Institute of Technology Zürich, ETH-Hönggerberg, CH-8093 Zürich
*To whom correspondence should be addressed:
Phone: 041 44 633 3940
Fax: 041 44 633 1294
Email:
Keywords: NMR resonance assignment, RNA recognition motif, alternative splicing, G-tract recognition
Character count with space: 6831
Biological context:
HnRNP F is an RNA binding protein belonging to the HnRNP H protein family that contains four highly homologous members, namely HnRNP H, HnRNP H’, HnRNP F, and HnRNP 2H9. These proteins contain two (2H9) or three (H, H’, and F) RNA recognition motifs (RRM) and two glycine rich auxiliary domains (Honore et al., 1995). Members of this family specifically recognize poly-G RNA sequences (G-tracts) (Swanson and Dreyfuss, 1988) that are frequent splicing recognition elements found both in introns and exons and are crucial for splicing regulation (McCullough and Berget, 1997, Grabowski, 2004). Mutations in regulatory sequences containing G-tracts have been correlated with many diseases (reviewed in (Faustino and Cooper, 2003)) and in some cases, these mutations directly affect (disrupt or enhance) the binding of HnRNP H and F to the pre-mRNA (Pagani et al., 2003, Buratti et al., 2004).
HnRNP F is a 45 kDa protein composed of two N-terminal RRMs (residues 1 to 90 and 103 to 194) followed by a glycine-rich motif (residues 195 to 276), a third RRM (residues 277 to 366) and a second glycine-rich motif (residues 367 to 415). RRMs are among the most abundant protein domains in eukaryotes, consisting of approximately 80-90 amino acids that adopt a typical babbab fold (for review, see (Maris et al., 2005). The RNA is usually bound on the b-sheet surface of the RRM. The RRMs of members of the hnRNP H family were originally termed quasi-RRM (qRRM) because their RNP sequences deviate significantly from the consensus (Honore et al., 1995). Although, several structures of RRM in complex with RNA have been determined (for review, see (Maris et al., 2005)), there is no structural information on G-tract recognition by RRMs.
To gain insight into G-tract recognition by RRMs, we initiated the structural study of a complex between HnRNP F and an RNA containing G-tracts using NMR spectroscopy. In many cases, it was demonstrated that two RRMs separated by short linkers bind RNA cooperatively with high affinity (Maris et al., 2005). We therefore decided to focus our study on the first two RRMs of HnRNP F that are separated by a 12 amino acids linker. Here we report, as a first step in this investigation, the 1H, 15N, and 13C resonance assignments of the two N-terminal RRMs of human HnRNP F (Figure 1).
Material and Methods:
The DNA sequence encoding the first two RRM domains of HnRNP F (residues 1-194) was subcloned into a Pet15b vector at the Xho1 and BamH1 restriction sites. The protein fragment is therefore fused to an N-terminal hexa-histidine tag. This fusion protein was overexpressed in BL21 (DE3) in M9 minimum medium, containing 15NH4Cl and 13C-glucose as the sole nitrogen and carbon source. Cells were grown at 37 ºC until an OD600 ~ 0.7 and then the protein expression was induced by adding IPTG to a final concentration of 1mM. The cells were harvested after two hours of expression. The protein was purified using a Ni-NTA affinity column, dialyzed against the NMR Buffer (25mM Na2HPO4, 50mM NaCl, pH 6.2) and concentrated to ~0.5 mM.
We also subcloned and expressed the individual RRMs of HnRNP F (residues 1-102 and 103-194) using the same protocol in order to confirm the assignment of the longest construct because RRM1 and RRM2 share 45% identity and contain repeated sequences in the predicted b-strands.
All NMR experiments were carried out at 30 ºC using Bruker DRX-500 MHz equipped with a cryoprobe, DRX-600 MHz and Avance-900 MHz spectrometers. Sequence-specific backbone assignments were achieved using 2D 15N-1H HSQC, 2D 13C-1H HSQC, 3D HNCA, 3D HNCACB, 3D HN(CO)CA and 3D CBCA(CO)NH experiments (for review, see (Sattler et al., 1999)). 1H and 13C side-chain assignments were performed using 3D H(C)CH-TOCSY, 3D (H)CCH-TOCSY, 3D 15N-1H NOESY-HSQC, and 3D 13C-1H NOESY-HSQC. Amino side-chain resonances of asparagine and glutamine were assigned using 3D 15N-1H NOESY-HSQC. Aromatic proton assignments were performed using 2D 1H-1H TOCSY and 2D 1H-1H NOESY in 100% D2O. All NOESY spectra were recorded with a mixing time of 150 ms, the 3D TOCSY spectra with a mixing time of 23 ms, and the 2D TOCSY with a mixing time of 50 ms.
Extent of assignment:
The 15N-1H HSQC spectrum of the 15N/13C-labeled RRM12 of HnRNP F is shown in Figure 1. All 15N, 13C and 1H backbone and side chain atoms were assigned except for Pro5, Glu6, Gly7, His80, and Lys173. Additionally, the side-chain resonances assignment of Met1, Met2, Leu3, Gly79, Arg90, Met93, Pro102, Glu166 and of the aromatics of His89, His172 and His178 could not be unambiguously assigned due to spectral overlap. In total, 94% of all observable atoms were assigned. The chemical shifts have been deposited in the BioMagResBank database (accession number 6745 ).
Acknowledgements:
We are grateful to L. Skrisovska (ETH Zurich) for helpful discussions and to Dr. D. Black (HHMI-UCLA) for sending us the clone containing the DNA sequence coding for the human hnRNP F. This investigation was supported by grants from the Structural Biology National Center of Competence in Research and by the Roche Research Fund for Biology at the ETH Zurich to FHTA. FHTA is an EMBO Young Investigator.
References:
Buratti, E., Baralle, M., De Conti, L., Baralle, D., Romano, M., Ayala, Y. M. and Baralle, F. E. (2004) Nucleic Acids Res, 32, 4224-36.
Faustino, N. A. and Cooper, T. A. (2003) Genes Dev, 17, 419-37.
Grabowski, P. J. (2004) Biochem Soc Trans, 32, 924-7.
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Maris, C., Dominguez, C. and Allain, F. H. (2005) Febs J, 272, 2118-31.
McCullough, A. J. and Berget, S. M. (1997) Mol Cell Biol, 17, 4562-71.
Pagani, F., Buratti, E., Stuani, C. and Baralle, F. E. (2003) J Biol Chem, 278, 26580-8.
Sattler, M., Schleucher, J. and Griesinger, C. (1999) Prog. NMR Spec., 34, 93-158.
Swanson, M. S. and Dreyfuss, G. (1988) Mol Cell Biol, 8, 2237-41.
Figure 1: 2D 15N-1H HSQC of uniformly 15N-13C labeled HnRNP F RRM 1 and 2 in 25mM sodium phosphate buffer (pH 6.2), 50mM NaCl, acquired at 313K on a Bruker Avance 900 MHz spectrometer. Assignments are labeled by their one letter residue name followed by the residue number.
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