Download supp - Springer Static Content Server

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: [email protected]
Keywords: NMR resonance assignment, RNA recognition motif, alternative splicing, G-tract
recognition
Character count with space: 6831
1
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  fold (for review, see (Maris et al., 2005). The RNA is usually bound
on the -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
2
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 NiNTA 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 -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
15
N-1H NOESY-HSQC, and 3D
13
C-1H NOESY-HSQC. Amino
side-chain resonances of asparagine and glutamine were assigned using 3D 15N-1H NOESY3
HSQC. Aromatic proton assignments were performed using 2D 1H-1H TOCSY and 2D 1H1
H 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.
Honore, B., Rasmussen, H. H., Vorum, H., Dejgaard, K., Liu, X., Gromov, P., Madsen, P.,
Gesser, B., Tommerup, N. and Celis, J. E. (1995) J Biol Chem, 270, 28780-9.
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.
4
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.
5