Article

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.

0 Followers
 · 
163 Views
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Nucleolin is a major nucleolar protein implicated in many aspects of ribosomal biogenesis, including early events such as processing of the large 35S preribosomal RNA. We found that the Arabidopsis (Arabidopsis thaliana) parallel1 (parl1) mutant, originally identified by its aberrant leaf venation, corresponds to the Arabidopsis nucleolin gene. parl1 mutants display parallel leaf venation, aberrant localization of the provascular marker Athb8:beta-glucuronidase, the auxin-sensitive reporter DR5:beta-glucuronidase, and auxin-dependent growth defects. PARL1 is highly similar to the yeast (Saccharomyces cerevisiae) nucleolin NUCLEAR SIGNAL RECOGNITION 1 (NSR1) multifunctional protein; the Arabidopsis PARL1 gene can rescue growth defects of yeast nsr1 null mutants. This suggests that PARL1 protein may have roles similar to those of the yeast nucleolin in nuclear signal recognition, ribosomal processing, and ribosomal subunit accumulation. Based on the range of auxin-related defects in parl1 mutants, we propose that auxin-dependent organ growth and patterning is highly sensitive to the efficiency of nucleolin-dependent ribosomal processing.
    Plant physiology 06/2007; 144(1):173-86. DOI:10.1104/pp.106.093575 · 7.39 Impact Factor
  • Source
    • ") 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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The YxiN protein of Bacillus subtilis is a member of the DbpA subfamily of prokaryotic DEAD-box RNA helicases. Like DbpA, it binds with high affinity and specificity to segments of 23S ribosomal RNA as short as 32 nucleotides (nt) that include hairpin 92. Several experiments have shown that the 76-residue carboxy-terminal domain of YxiN is responsible for the high-affinity RNA binding. The domain has been crystallized and its structure has been solved to 1.7 Angstroms resolution. The structure reveals an RNA recognition motif (RRM) fold that is found in many eukaryotic RNA binding proteins; the RRM fold was not apparent from the amino acid sequence. The domain has two solvent exposed aromatic residues at sites that correspond to the aromatic residues of the ribonucleoprotein (RNP) motifs RNP1 and RNP2 that are essential for RNA binding in many RRMs. However, mutagenesis of these residues (Tyr404 and Tyr447) to alanine has little effect on RNA affinity, suggesting that the YxiN domain binds target RNAs in a manner that differs from the binding mode commonly found in many eukaryotic RRMs.
    RNA 07/2006; 12(6):959-67. DOI:10.1261/rna.5906 · 4.62 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The microtubule-associated tau protein participates in the organization and integrity of the neuronal cytoskeleton. A nuclear form of tau has been described in neuronal and non-neuronal cells, which displays a nucleolar localization during interphase but is associated with nucleolar-organizing regions in mitotic cells. In the present study, based on immunofluorescence, immuno-FISH and confocal microscopy, we show that nuclear tau is mainly present at the internal periphery of nucleoli, partially colocalizing with the nucleolar protein nucleolin and human AT-rich alpha-satellite DNA sequences organized as constitutive heterochromatin. By using gel retardation, we demonstrate that tau not only colocalizes with, but also specifically binds to, AT-rich alpha-satellite DNA sequences apparently through the recognition of AT- rich DNA stretches. Here we propose a functional role for nuclear tau in relation to the nucleolar organization and/or heterochromatinization of a portion of RNA genes. Since nuclear tau has also been found in neurons from patients with Alzheimer's disease (AD), aberrant nuclear tau could affect the nucleolar organization during the course of AD. We discuss nucleolar tau associated with AT-rich alpha-satellite DNA sequences as a potential molecular link between trisomy 21 and AD.
    Journal of Cell Science 06/2006; 119(10). DOI:10.1242/jcs.02907 · 5.33 Impact Factor
Show more