Solution structure of the complex formed by the two N-terminal RNA-binding domains of nucleolin and a pre-rRNA target

Department of Chemistry and Biochemistry, Molecular Biology Institute, University of California, Los Angeles, CA 90095-1569, USA.
Journal of Molecular Biology (Impact Factor: 4.33). 05/2004; 337(4):799-816. DOI: 10.1016/j.jmb.2004.01.056
Source: PubMed

ABSTRACT Nucleolin is a 70 kDa multidomain protein involved in several steps of eukaryotic ribosome biogenesis. In vitro selection in combination with mutagenesis and structural analysis identified binding sites in pre-rRNA with the consensus (U/G)CCCG(A/G) in the context of a hairpin structure, the nucleolin recognition element (NRE). The central region of the protein contains four tandem RNA-binding domains (RBDs), of which the first two are responsible for the RNA-binding specificity and affinity for NREs. Here, we present the solution structure of the 28 kDa complex formed by the two N-terminal RNA-binding domains of nucleolin (RBD12) and a natural pre-rRNA target, b2NRE. The structure demonstrates that the sequence-specific recognition of the pre-rRNA NRE is achieved by intermolecular hydrogen bonds and stacking interactions involving mainly the beta-sheet surfaces of the two RBDs and the linker residues. A comparison with our previously determined NMR structure of RBD12 in complex with an in vitro selected RNA target, sNRE, shows that although the sequence-specific recognition of the loop consensus nucleotides is the same in the two complexes, they differ in several aspects. While the protein makes numerous specific contacts to the non-consensus nucleotides in the loop E motif (S-turn) in the upper part of the sNRE stem, nucleolin RBD12 contacts only consensus nucleotides in b2NRE. The absence of these upper stem contacts from the RBD12/b2NRE complex results in a much less stable complex, as demonstrated by kinetic analyses. The role of the loop E motif in high-affinity binding is supported by gel-shift analyses with a series of sNRE mutants. The less stable interaction of RBD12 with the natural RNA target is consistent with the proposed role of nucleolin as a chaperone that interacts transiently with pre-rRNA to prevent misfolding.

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    • "The acidic/Ser-rich region induces nucleolar chromatin decondensation through interaction with histone H1 (Jordon, 1987; Erard et al., 1988) and binds nontranscribed spacer regions in DNA that separate rRNA gene repeats to organize rDNA chromatin for transcription by RNA polymerase I (Olson and Thompson, 1983; Bouche et al., 1984; Egyhazi et al., 1988; Ghisolfi-Nieto et al., 1996). The RRM (also called RBD) domain consists of approximately 80 amino acid residues containing two highly conserved regions, called RNP motifs, that interact specifically with RNA, particularly with the external transcribed spacer region of primary rRNA transcripts (Ghisolfi et al., 1992; Burd and Dreyfuss, 1994; Johansson et al., 2004). A phylogenetic tree of the plant and yeast proteins was constructed using the neighbor-joining method and then analyzed using 1,000 replicates of bootstrap analysis. "
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    • ") and a naturally occurring sequence (b2NRE) (Johansson et al. 2004) with high affinity and sequence specificity. A substantial fraction of the binding interactions of the RNA are with amino acid residues of the polypeptide linker between the RRM FIGURE 3. YxiN RBD with conserved residues highlighted. "
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    • "As nucleolar tau, nucleolin localizes at the dense fibrillar component of nucleoli. It interacts with nascent pre-rRNA transcripts as well as with several ribosomal proteins and has been implicated in chromatin structure, rDNA transcription, rRNA maturation, ribosome assembly and nucleo-cytoplasmic transport (Bouvet et al., 1998; Ginisty et al., 1999; Roger et al., 2003; Johansson et al., 2004). Colocalization of tau with nucleolin suggests that these two proteins could interact with one another, reinforcing the hypothesis of a possible role of tau during nucleolar organization. "
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