High Affinity Interactions of Nucleolin with G-G-paired rDNA*

Nucleolin is a very abundant eukaryotic protein that localizes to the nucleolus, where the rDNA undergoes transcription, replication, and recombination and where rRNA processing occurs. The top (non-template) strand of the rDNA is very guanine-rich and has considerable potential to form structures stabilized by G-G pairing. We have assayed binding of endogenous and recombinant nucleolin to synthetic oligonucleotides in which G-rich regions have formed intermolecular G-G pairs to produce either two-stranded G2 or four-stranded G4 DNA. We report that nucleolin binds G-G-paired DNA with very high affinity; the dissociation constant for interaction with G4 DNA is K D = 1 nm. Two separate domains of nucleolin can interact with G-G-paired DNA, the four RNA binding domains and the C-terminal Arg-Gly-Gly repeats. Both domains bind G4 DNA with high specificity and recognize G4 DNA structure independent of sequence context. The high affinity of the nucleolin/G4 DNA interaction identifies G-G-paired structures as natural binding targets of nucleolin in the nucleolus. The ability of two independent domains of nucleolin to bind G-G-paired structures suggests that nucleolin can function as an architectural factor in rDNA transcription, replication, or recombination.

[1]  F. Amalric,et al.  Phosphorylation of nucleolin by a nucleolar type NII protein kinase. , 1987, Biochemistry.

[2]  Dipankar Sen,et al.  A sodium-potassium switch in the formation of four-stranded G4-DNA , 1990, Nature.

[3]  J. Labbé,et al.  Mitosis-specific phosphorylation of nucleolin by p34cdc2 protein kinase , 1990, Molecular and cellular biology.

[4]  H. Bourbon,et al.  Nucleolin, the major nucleolar protein of growing eukaryotic cells: an unusual protein structure revealed by the nucleotide sequence. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[5]  L. Créancier,et al.  Determination of the functional domains involved in nucleolar targeting of nucleolin. , 1993, Molecular biology of the cell.

[6]  W. Gilbert,et al.  Formation of parallel four-stranded complexes by guanine-rich motifs in DNA and its implications for meiosis , 1988, Nature.

[7]  D A Sinclair,et al.  Accelerated aging and nucleolar fragmentation in yeast sgs1 mutants. , 1997, Science.

[8]  S. Gerbi.  Small nucleolar RNA. , 1995, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[9]  H. Bourbon,et al.  Structure of the mouse nucleolin gene. The complete sequence reveals that each RNA binding domain is encoded by two independent exons. , 1988, Journal of molecular biology.

[10]  A. Rich,et al.  Crystal structure of four-stranded Oxytricha telomeric DNA , 1992, Nature.

[11]  D. Sen,et al.  Novel DNA superstructures formed by telomere-like oligomers. , 1992, Biochemistry.

[12]  S. H. Ghaffari,et al.  Protein and cDNA sequence of a glycine-rich, dimethylarginine-containing region located near the carboxyl-terminal end of nucleolin (C23 and 100 kDa). , 1986, The Journal of biological chemistry.

[13]  E. Nigg,et al.  Protein localization to the nucleolus: a search for targeting domains in nucleolin. , 1993, Journal of cell science.

[14]  J. Belasco,et al.  RNA recognition by the joint action of two nucleolin RNA‐binding domains: genetic analysis and structural modeling , 1997, The EMBO journal.

[15]  E. Bradbury,et al.  GAA instability in Friedreich's Ataxia shares a common, DNA-directed and intraallelic mechanism with other trinucleotide diseases. , 1998, Molecular cell.

[16]  R. Cook,et al.  Clustering of glycine and NG,NG-dimethylarginine in nucleolar protein C23. , 1985, Biochemistry.

[17]  B Sollner-Webb,et al.  Metazoan rDNA enhancer acts by making more genes transcriptionally active , 1996, The Journal of cell biology.

[18]  D. Davies,et al.  Helix formation by guanylic acid. , 1962, Proceedings of the National Academy of Sciences of the United States of America.

[19]  N. Maizels,et al.  A Specific Isoform of hnRNP D Interacts with DNA in the LR1 Heterodimer: Canonical RNA Binding Motifs in a Sequence-specific Duplex DNA Binding Protein* , 1998, The Journal of Biological Chemistry.

[20]  N. Maizels,et al.  G4 DNA Binding by LR1 and Its Subunits, Nucleolin and hnRNP D, A Role for G-G pairing in Immunoglobulin Switch Recombination* , 1999, The Journal of Biological Chemistry.

[21]  L. Guarente,et al.  Nucleolar localization of the Werner syndrome protein in human cells. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[22]  L. Guarente,et al.  Extrachromosomal rDNA Circles— A Cause of Aging in Yeast , 1997, Cell.

[23]  H. Bourbon,et al.  Nucleolin gene organization in rodents: highly conserved sequences within three of the 13 introns. , 1990, Gene.

[24]  W. Gilbert,et al.  Sequencing end-labeled DNA with base-specific chemical cleavages. , 1980, Methods in enzymology.

[25]  E. Birney,et al.  Analysis of the RNA-recognition motif and RS and RGG domains: conservation in metazoan pre-mRNA splicing factors. , 1993, Nucleic acids research.

[26]  P C Moody,et al.  The high-resolution crystal structure of a parallel-stranded guanine tetraplex. , 1994, Science.

[27]  E. Nigg,et al.  cDNA sequences of chicken nucleolin/C23 and NO38/B23, two major nucleolar proteins. , 1990, Nucleic acids research.

[28]  P. Bouvet,et al.  Nucleolin is a sequence-specific RNA-binding protein: characterization of targets on pre-ribosomal RNA. , 1996, Journal of molecular biology.

[29]  N. Maizels,et al.  Nucleolin is one component of the B cell-specific transcription factor and switch region binding protein, LR1. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[30]  O. Mcbride,et al.  Genomic organization and chromosomal localization of the human nucleolin gene. , 1990, The Journal of biological chemistry.

[31]  N. Maizels,et al.  The Saccharomyces cerevisiae Sgs1 helicase efficiently unwinds G-G paired DNAs. , 1999, Nucleic acids research.

[32]  J. Keene,et al.  RNA recognition: towards identifying determinants of specificity. , 1991, Trends in biochemical sciences.

[33]  P. Bouvet,et al.  Nucleolin functions in the first step of ribosomal RNA processing , 1998, The EMBO journal.

[34]  M. Ptashne.  How eukaryotic transcriptional activators work , 1988, Nature.

[35]  H. Prats,et al.  Protein kinase NII and the regulation of rDNA transcription in mammalian cells. , 1989, Nucleic acids research.

[36]  P. Bouvet,et al.  Nucleolin Interacts with Several Ribosomal Proteins through Its RGG Domain* , 1998, The Journal of Biological Chemistry.

[37]  J. R. Williamson,et al.  G-quartet structures in telomeric DNA. , 1994, Annual review of biophysics and biomolecular structure.

[38]  Gwyn Jordan.  At the heart of the nucleolus , 1987, Nature.

[39]  H. Pollard,et al.  Cloning and sequencing of the human nucleolin cDNA , 1989, FEBS letters.

[40]  T. Cech,et al.  Monovalent cation-induced structure of telomeric DNA: The G-quartet model , 1989, Cell.

[41]  N. Maizels,et al.  The Bloom’s Syndrome Helicase Unwinds G4 DNA* , 1998, The Journal of Biological Chemistry.

[42]  Michele L. Rankin,et al.  A complete nucleolin cDNA sequence from Xenopus laevis , 1993, Nucleic Acids Res..

[43]  M. Gray,et al.  Werner helicase is localized to transcriptionally active nucleoli of cycling cells. , 1998, Experimental cell research.

[44]  G. Serin,et al.  Two RNA-binding Domains Determine the RNA-binding Specificity of Nucleolin* , 1997, The Journal of Biological Chemistry.

[45]  P. Moore,et al.  Tetramerization of an RNA oligonucleotide containing a GGGG sequence , 1991, Nature.

[46]  P. DiMario,et al.  The Gly/Arg-rich (GAR) domain of Xenopus nucleolin facilitates in vitro nucleic acid binding and in vivo nucleolar localization. , 1993, Molecular biology of the cell.