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
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.
Available from: Min-Ju Kang
- "Nucleolin is another multifunctional protein capable of interacting with DNA and RNA. With an apparent molecular weight of 100 kDa and a length of 710 amino acids, nucleolin has several different domains: an N-terminal segment with multiple phosphorylation sites, a central domain with four RNA-recognition motifs (RRMs) and a C-terminal arginine–glycine-rich (RGG) domain (21–24). Among its functions associated with binding DNA, nucleolin can induce chromatin decondensation by the remodeling complex SWI/SNF (switch/sucrose non-fermentable in yeast), facilitates transcription and modulates DNA replication (23,25,26). "
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ABSTRACT: RNA-binding proteins (RBPs) regulate gene expression at many post-transcriptional levels, including mRNA stability and translation. The RBP nucleolin, with four RNA-recognition motifs, has been implicated in cell proliferation, carcinogenesis and viral infection. However, the subset of nucleolin target mRNAs and the influence of nucleolin on their expression had not been studied at a transcriptome-wide level. Here, we globally identified nucleolin target transcripts, many of which encoded cell growth- and cancer-related proteins, and used them to find a signature motif on nucleolin target mRNAs. Surprisingly, this motif was very rich in G residues and was not only found in the 3'-untranslated region (UTR), but also in the coding region (CR) and 5'-UTR. Nucleolin enhanced the translation of mRNAs bearing the G-rich motif, since silencing nucleolin did not change target mRNA stability, but decreased the size of polysomes forming on target transcripts and lowered the abundance of the encoded proteins. In summary, nucleolin binds G-rich sequences in the CR and UTRs of target mRNAs, many of which encode cancer proteins, and enhances their translation.
Nucleic Acids Research 07/2011; 39(19):8513-30. DOI:10.1093/nar/gkr488 · 9.11 Impact Factor
Available from: ncbi.nlm.nih.gov
- "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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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 · 6.84 Impact Factor
Available from: Michael Toft Overgaard
- ") 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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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 · 4.94 Impact Factor
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