The EMBO Journal vol. 1 1 no.9 pp.3289 - 3295, 1992
Interaction of the RNA-binding domain of the hnRNP C proteins with RNA
Matthias Gorlach, Michael Wittekind1, Robert A.Beckman', Luciano Muellerl and Gideon Dreyfuss2 Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6148 and 'Bristol-Myers Squibb, Macromolecular NMR Department, Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ 08543-4000, USA 2Corresponding author
Communicated by I.W.Mattaj
The hnRNP C proteins are among the most abundant and avid pre-mRNA-binding proteins and they contain a consensus sequence RNA-binding domain (RBD) that is found in a large number of RNA-binding proteins. The interaction of the RBD of the hnRNP C proteins with an RNA oligonucleotide [r(U)8] was monitored by nuclear magnetic resonance (NMR). '5N and 13C/15Nlabelled hnRNP C protein RBD was mixed with r(U)8 and one- and two-dimensional (iD and 2D) NMR spectra were recorded in a titration experiment. NMR studies of the uncomplexed 93 amino acid hnRNP C RBD (Wittekind et al., 1992) have shown that it has a compact folded structure (3c3alo0af), which is typical for the RBD of this family of proteins and which is comprised of a four-stranded antiparallel fl-sheet, two c-helices and relatively unstructured amino- and carboxy-terminal regions. Sequential assignments of the polypeptide mainchain atoms of the hnRNP C RBD-r(U)8 complex revealed that these typical structural features are maintained in the complex, but significant perturbations of the chemical shifts of aniide group atoms occur in a large number of residues. Most of these residues are in the ,Bsheet region and especially in the terminal regions of the RBD. In contrast, chemical shifts of the residues of the well conserved a-helices, with the exception of Lys3O, are not significantly perturbed. These observations localize the candidate residues of the RBD that are involved in the interaction with the RNA. They suggest that most of the amino acids directly involved in binding to RNA are located on the f-sheet and the contiguous amino- and carboxy-terminal regions of the RBD. These regions therefore appear to provide an exposed 'platform' for extensive interactions with the RNA. Key words: multi-dimensional NMR/pre-mRNA binding
protein/ribonucleoprotein/RNA-binding/three-dimensional structure
The RNA-binding domain (RBD) is composed of -90 amino acids and it is found in divergent eukaryotes in nuclear, cytoplasmic and organellar RNA-binding proteins. The hallmarks of the RBD are two well conserved sequences, RNP1 and RNP2, spaced 30 amino acids from each other (Dreyfuss et al., 1988; Bandziulis et al., 1989; Mattaj, 1989; Query et al., 1989; Kenan et al., 1991). This domain has been shown to confer the specific RNA-binding activity of this family of RNA-binding proteins (Lutz-Freyermuth et al., 1990; Query et al., 1989; Scherly et al., 1989; Nietfeld et al., 1990; Scherly et al., 1990; Burd et al., 1991). The hnRNP Cl and C2 proteins are among the most avid pre-mRNA-binding proteins (Piniol-Roma et al., 1988; Swanson and Dreyfuss, 1988a,b) and are very likely to be involved in pre-mRNA processing (Choi et al., 1986; Swanson and Dreyfuss, 1988a; Wilusz et al., 1988; Wilusz and Shenk, 1990). Their single RBD, which is also one of the smallest RBDs because it lacks the loop region preceeding RNP1 (Bandziulis et al., 1989; Kenan et al., 1991), as well as their status as major hnRNP proteins, makes the C proteins a prototype of the family of the consensus sequence RBD proteins. One of the key questions in understanding the function of RNP proteins centers around the activity of the RNAbinding domain. Specifically, in order to understand in detail the interaction of the RBD with RNA, it is essential to identify the residues within the RNA-binding domain that are involved in the interaction with RNA. Recently, we determined the structure of the RNA-binding domain of the hnRNP C proteins in solution using multi-dimensional heteronuclear NMR techniques (Wittekind et al., 1992; see also Figure 3). Together with the determination of the threedimensional structure of one of the RNA-binding domain of the small nuclear ribonucleoprotein (snRNP) Ul A protein by X-ray crystallography (Nagai et al., 1990) and studies of its global fold by NMR (Hoffman et al., 1991), these structural studies have shown that a CS-RBD has a fl(3(3ac4a domain structure in which the RNP1 and RNP2 sequences are located on adjacent antiparallel strands. Here we have carried out NMR studies on the interaction of the RNAbinding domain of the hnRNP C proteins with a small RNA substrate, r(U)8. Substantial perturbations of chemical shifts in a large number of residues were observed and their positions in the RBD make it possible to localize the candidate amino acids that are involved in the interaction with the RNA. Interestingly, the majority of the affected residues are located on the four-stranded fl-sheet and on the contiguous and relatively unstructured (in the uncomplexed RBD) amino- and carboxy-terminal regions of the domain. -
Introduction
Results
Many of the RNA-binding proteins involved in RNA processing in eukaryotes have a common consensus sequence RNA-binding domain (CS-RBD, Bandziulis et al., 1989).
Earlier studies on the hnRNP C proteins showed that they bind specifically to poly(U) and to stretches of five or more U-residues in the context of pre-mRNA substrates (Swanson
Oxford University Press
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Interaction of the RNA-binding domain of the hnRNP C proteins with RNA
Matthias Gorlach, Michael Wittekind1, Robert A.Beckman', Luciano Muellerl and Gideon Dreyfuss2 Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6148 and 'Bristol-Myers Squibb, Macromolecular NMR Department, Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ 08543-4000, USA 2Corresponding author
Communicated by I.W.Mattaj
The hnRNP C proteins are among the most abundant and avid pre-mRNA-binding proteins and they contain a consensus sequence RNA-binding domain (RBD) that is found in a large number of RNA-binding proteins. The interaction of the RBD of the hnRNP C proteins with an RNA oligonucleotide [r(U)8] was monitored by nuclear magnetic resonance (NMR). '5N and 13C/15Nlabelled hnRNP C protein RBD was mixed with r(U)8 and one- and two-dimensional (iD and 2D) NMR spectra were recorded in a titration experiment. NMR studies of the uncomplexed 93 amino acid hnRNP C RBD (Wittekind et al., 1992) have shown that it has a compact folded structure (3c3alo0af), which is typical for the RBD of this family of proteins and which is comprised of a four-stranded antiparallel fl-sheet, two c-helices and relatively unstructured amino- and carboxy-terminal regions. Sequential assignments of the polypeptide mainchain atoms of the hnRNP C RBD-r(U)8 complex revealed that these typical structural features are maintained in the complex, but significant perturbations of the chemical shifts of aniide group atoms occur in a large number of residues. Most of these residues are in the ,Bsheet region and especially in the terminal regions of the RBD. In contrast, chemical shifts of the residues of the well conserved a-helices, with the exception of Lys3O, are not significantly perturbed. These observations localize the candidate residues of the RBD that are involved in the interaction with the RNA. They suggest that most of the amino acids directly involved in binding to RNA are located on the f-sheet and the contiguous amino- and carboxy-terminal regions of the RBD. These regions therefore appear to provide an exposed 'platform' for extensive interactions with the RNA. Key words: multi-dimensional NMR/pre-mRNA binding
protein/ribonucleoprotein/RNA-binding/three-dimensional structure
The RNA-binding domain (RBD) is composed of -90 amino acids and it is found in divergent eukaryotes in nuclear, cytoplasmic and organellar RNA-binding proteins. The hallmarks of the RBD are two well conserved sequences, RNP1 and RNP2, spaced 30 amino acids from each other (Dreyfuss et al., 1988; Bandziulis et al., 1989; Mattaj, 1989; Query et al., 1989; Kenan et al., 1991). This domain has been shown to confer the specific RNA-binding activity of this family of RNA-binding proteins (Lutz-Freyermuth et al., 1990; Query et al., 1989; Scherly et al., 1989; Nietfeld et al., 1990; Scherly et al., 1990; Burd et al., 1991). The hnRNP Cl and C2 proteins are among the most avid pre-mRNA-binding proteins (Piniol-Roma et al., 1988; Swanson and Dreyfuss, 1988a,b) and are very likely to be involved in pre-mRNA processing (Choi et al., 1986; Swanson and Dreyfuss, 1988a; Wilusz et al., 1988; Wilusz and Shenk, 1990). Their single RBD, which is also one of the smallest RBDs because it lacks the loop region preceeding RNP1 (Bandziulis et al., 1989; Kenan et al., 1991), as well as their status as major hnRNP proteins, makes the C proteins a prototype of the family of the consensus sequence RBD proteins. One of the key questions in understanding the function of RNP proteins centers around the activity of the RNAbinding domain. Specifically, in order to understand in detail the interaction of the RBD with RNA, it is essential to identify the residues within the RNA-binding domain that are involved in the interaction with RNA. Recently, we determined the structure of the RNA-binding domain of the hnRNP C proteins in solution using multi-dimensional heteronuclear NMR techniques (Wittekind et al., 1992; see also Figure 3). Together with the determination of the threedimensional structure of one of the RNA-binding domain of the small nuclear ribonucleoprotein (snRNP) Ul A protein by X-ray crystallography (Nagai et al., 1990) and studies of its global fold by NMR (Hoffman et al., 1991), these structural studies have shown that a CS-RBD has a fl(3(3ac4a domain structure in which the RNP1 and RNP2 sequences are located on adjacent antiparallel strands. Here we have carried out NMR studies on the interaction of the RNAbinding domain of the hnRNP C proteins with a small RNA substrate, r(U)8. Substantial perturbations of chemical shifts in a large number of residues were observed and their positions in the RBD make it possible to localize the candidate amino acids that are involved in the interaction with the RNA. Interestingly, the majority of the affected residues are located on the four-stranded fl-sheet and on the contiguous and relatively unstructured (in the uncomplexed RBD) amino- and carboxy-terminal regions of the domain. -
Introduction
Results
Many of the RNA-binding proteins involved in RNA processing in eukaryotes have a common consensus sequence RNA-binding domain (CS-RBD, Bandziulis et al., 1989).
Earlier studies on the hnRNP C proteins showed that they bind specifically to poly(U) and to stretches of five or more U-residues in the context of pre-mRNA substrates (Swanson
Oxford University Press
3289
;r.
M.Gorlach et a!.
A
(37 /y
G60'
Slfi/ 0
t6
6
O
E
e.
