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

  • L.A. Hanakahi
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  • Hui Sun
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  • Nancy Maizels
    Correspondence
    To whom correspondence should be addressed: Depts. of Molecular Biophysics and Biochemistry and Genetics, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510-8024. Tel.: 203-432-5641; Fax: 203-432-3047;
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  • Author Footnotes
    * This research was supported by National Institutes of Health Grants R01 GM39799 and P01 CA16038 (to N. M.) and a Ford Foundation postdoctoral fellowship (to L. A. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    § Present address: Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms Herts. EN63LD, United Kingdom.
Open AccessPublished:May 28, 1999DOI:https://doi.org/10.1074/jbc.274.22.15908
      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.
      Transcription and processing of rRNA occur within a specialized subnuclear compartment, the nucleolus. In cells that are actively transcribing the rDNA, nucleoli appear to be composed of three compartments: the fibrillar center, which contains DNA that is not being transcribed; the dense fibrillar component, where rDNA transcription occurs; and the peripheral granular component, where pre-rRNA processing and pre-ribosome assembly take place (
      • Puvion-Dutilleul F.
      • Bachellerie J.-P.
      • Puvion E.
      ,
      • Puvion-Dutilleul F.
      • Puvion E.
      • Bachellerie J.-P.
      ). In proliferating cells, RNA polymerase I (pol I)
      The abbreviations used are: pol I, polymerase I; RBD, RNA binding domain; MBP, maltose-binding protein; ETS, external transcribed spacer
      1The abbreviations used are: pol I, polymerase I; RBD, RNA binding domain; MBP, maltose-binding protein; ETS, external transcribed spacer
      and other components of the transcription complex localize to the dense fibrillar component, whereas molecules essential for rRNA processing, like fibrillarin and the small nucleolar RNAs, localize to the peripheral granular component (for review, see Refs.
      • Fakan S.
      • Puvion E.
      ,
      • Jordan G.
      ,
      • Gerbi S.A.
      ). The rate at which the rDNA is transcribed in actively dividing cells is remarkable. Electron microscopic analysis shows that during active rDNA transcription in metazoan cells, the spacing between pol I complexes is only 100 base pairs (
      • Osheim Y.
      • Mougey E.B.
      • Windle J.
      • Anderson M.
      • O'Reilly M.
      • Miller O.L.
      • Beyer A.
      • Sollner-Webb B.
      ).
      One of the most abundant proteins in the nucleoli of vertebrate cells is the highly conserved protein, nucleolin. Mammalian nucleolin is 709 amino acids in length and consists of an unusual grouping of sequence and structural motifs (
      • Lapeyre B.
      • Bourbon H.
      • Amalric F.
      ,
      • Bourbon H.-M.
      • Lapeyre B.
      • Amalric F.
      ,
      • Bourbon H.-M.
      • Amalric F.
      ,
      • Srivastava M.
      • McBride O.W.
      • Fleming P.J.
      • Pollard H.B.
      • Burns A.L.
      ,
      • Srivastava M.
      • Fleming P.J.
      • Pollard H.
      • Burns A.L.
      ,
      • Maridor G.
      • Nigg E.A.
      ,
      • Rankin M.L.
      • Heine M.A.
      • Xiao S.
      • LeBlanc M.D.
      • Nelson J.W.
      • DiMario P.J.
      ,
      • Hanakahi L.A.
      • Dempsey L.A.
      • Li M.-J.
      • Maizels N.
      ). The N-terminal region of nucleolin houses several long stretches of acidic residues with the potential to function as “acid blobs” in activation of transcription (
      • Ptashne M.
      ). The central region of nucleolin contains four RNA binding domains (RBDs; also called RNA recognition motifs or RRMs). RBDs are common among proteins that interact with single-stranded nucleic acids (
      • Kenan D.J.
      • Query C.C.
      • Keene J.D.
      ,
      • Birney E.
      • Kumar S.
      • Krainer A.R.
      ), and the RBDs of nucleolin are believed to mediate interactions of nucleolin with RNA (
      • Ghisolfi-Nieto L.
      • Joseph G.
      • Puvion-Dutilleul F.
      • Amalric F.
      • Bouvet P.
      ,
      • Ginisty H.
      • Amalric F.
      • Bouvet P.
      ,
      • Serin G.
      • Joseph G.
      • Ghisolfi L.
      • Bauzan M.
      • Erard M.
      • Amalric F.
      • Bouvet P.
      ,
      • Bouvet P.
      • Jain C.
      • Belasco J.B.
      • Amalric F.
      • Erard M.
      ,
      • Bouvet P.
      • Diaz J.-J.
      • Kindbeiter K.
      • Madjar J.-J.
      • Amalric F.
      ). The C terminus of nucleolin contains nine repeats of the tripeptide motif arginine-glycine-glycine (RGG), in which the arginine residues are dimethylated (
      • Lischwe M.A.
      • Cook R.G.
      • Ahn Y.S.
      • Yeoman L.C.
      • Busch H.
      ,
      • Lapeyre B.
      • Amalric F.
      • Ghaffari S.H.
      • Venkatarama Rao S.V.
      • Dumbar T.S.
      • Olson M.O.J.
      ).
      The distribution of nucleolin within the nucleolus is unusual. Whereas proteins like pol I and fibrillarin appear to be restricted to a single compartment of the nucleolus, nucleolin is abundant within both the dense fibrillar component and the granular component (for review, see Ref.
      • Jordan G.
      ). The presence of nucleolin in the peripheral granular component is consistent with the participation of nucleolin in rRNA processing and ribosome assembly (
      • Ginisty H.
      • Amalric F.
      • Bouvet P.
      ,
      • Serin G.
      • Joseph G.
      • Ghisolfi L.
      • Bauzan M.
      • Erard M.
      • Amalric F.
      • Bouvet P.
      ,
      • Bouvet P.
      • Jain C.
      • Belasco J.B.
      • Amalric F.
      • Erard M.
      ,
      • Bouvet P.
      • Diaz J.-J.
      • Kindbeiter K.
      • Madjar J.-J.
      • Amalric F.
      ). The fact that nucleolin is abundant within the dense fibrillar component suggests that nucleolin also functions in other processes, including transcription, replication, or recombination of the rDNA. Nonetheless, conserved and specific interactions of nucleolin with the duplex rDNA have not been reported.
      The rDNA transcription unit includes the regions that template mature 18, 5.8, and 28 S RNAs and external and internal transcribed spacer regions (Fig. 1 A). In all eukaryotes, the entire transcribed region of the rDNA is very rich in the base guanine (34.2% in humans) within the spacers, as well as within the regions that template the mature rRNAs. The G-richness is restricted to a single strand, the non-template strand, and most guanines are within runs that contain three or more consecutive Gs (Fig. 1 B).
      Figure thumbnail gr1
      Figure 1The human rDNA is G-rich. A,the 13.4-kilobase human rRNA transcription unit. Regions that template 18, 5.8, and 28 S rRNAs are shaded; the external transcribed spacers (ETS) and internal transcribed spacers (ITS) are unfilled. B, graphical representation of the occurrence of G-runs within the transcribed region of the human rDNA. The 13.4-kilobase region shown inA was searched for runs of three or more G residues using the program FINDPATTERNS of the GCG suite of programs. The number of G-runs per 500-base pair (bp) interval is shown. Both the top (non-template) strand of the rDNA and the pre-rRNA transcript will contain many runs of Gs, as shown.
      Single-stranded DNAs that contain runs of three or more consecutive guanine residues readily self-associate in vitro to form structures stabilized by G-G pairing (
      • Sen D.
      • Gilbert W.
      ,
      • Sen D.
      • Gilbert W.
      ,
      • Williamson J.R.
      • Raghuraman M.K.
      • Cech T.R.
      ,
      • Kang C.-H.
      • Zhang X.
      • Ratliff R.
      • Moyzis R.
      • Rich A.
      ,
      • Kim J.
      • Cheong C.
      • Moore P.B.
      ,
      • Wang Y.
      • Patel D.J.
      ,
      • Laughlan G.
      • Murchie A.I.
      • Norman D.G.
      • Moore M.H.
      • Moody P.C.
      • Lilley D.M.
      • Luisi B.
      ). In these structures, guanines interact via Hoogsteen bonding to form planar rings called G quartets (Fig. 2 A), and the G quartets stack upon each other to stabilize higher order structures (Fig. 2 B). That guanine-guanine interactions could occur readily in solution was first established nearly 40 years ago (
      • Gellert M.
      • Lipsett M.N.
      • Davies D.R.
      ). Although G-G-paired DNA has not been directly observed in vivo, G-G-paired structures form rapidly and spontaneouslyin vitro and are very stable once formed. Because of its sequence, the G-rich strand of the rDNA has considerable potential to form G-G-paired structures (Fig. 2 A). Formation of such structures may be stimulated by the unwinding and localized denaturation that accompanies rDNA transcription.
      Figure thumbnail gr2
      Figure 2Formation of G-G-paired DNA. A, top view of G quartet shows four guanine residues forming a planar array stabilized by Hoogsteen bonding. B, G4 DNA and G2 DNA. Stacking of the planar G quartets in four-stranded G4 DNA (left) and the two-stranded hairpin conformation of G2 DNA (right) are shown. C, dimethyl sulfate (DMS) protection verified formation of G4 structures by the ETS-1 oligonucleotide. Brackets denote the G-runs, which are accessible in the single-stranded (ss) ETS-1 oligonucleotide but protected in G4 DNA (G4).
      The observations presented above have led us to investigate the interaction of nucleolin with G-G-paired DNA. Here we report that mammalian nucleolin binds tightly and specifically to both four-stranded G4 DNA and two-stranded G2 DNA. The dissociation constant for binding is KD = 1 nm, which represents a remarkably high affinity for interaction of a eukaryotic protein with nucleic acid. Mutational analysis shows that two separable domains of nucleolin can bind G4 DNA, one comprised of the four RBDs (RBD-1,2,3,4) and the other comprised of the C-terminal Arg-Gly-Gly repeats (RGG9). These results suggest that G-G-paired DNA is a natural binding target of nucleolin within the nucleolus. Nucleolin may, therefore, be an architectural factor that functions to organize the G-rich non-template strand of the rDNA during transcription, replication, or recombination.

      DISCUSSION

      We have shown that the abundant nucleolar protein, nucleolin, binds G-G-paired DNA with very high affinity (KD= 1 nm). Nucleolin can bind to both four-stranded G4 DNA and two-stranded G2 DNA, and nucleolin recognizes G-G-paired structures independent of sequence context. The remarkably high binding affinities suggest that G-G-paired structures are binding targets of nucleolin in vivo. The observation that nucleolin binds G-G-paired structures independent of sequence context shows that this protein will be able to bind G-G-paired structures wherever they might form within the G-rich rDNA.

      Dynamic Formation of G-G-paired DNA in the Nucleolus

      Most nuclear DNA is double-stranded, and complementary base pairing will normally protect duplex DNA from forming G-G-paired structures. However, duplex DNA becomes transiently single-stranded during three critical and dynamic processes: transcription, replication, and recombination. Cells have developed sophisticated mechanisms to prevent DNA from adopting alternative structures, including a variety of proteins that bind to transiently exposed single-stranded regions. Nonetheless, these mechanisms are not foolproof. For example, there is considerable evidence that triplet repeat expansion results from formation of non-Watson-Crick structures during replication (see Ref.
      • Gacy A.M.
      • Goellner G.M.
      • Spiro C.
      • Chen X.
      • Gupta G.
      • Bradbury E.M.
      • Dyer R.B.
      • Mikesell M.J.
      • Yao J.Z.
      • Johnson A.J.
      • Richter A.
      • Melancon S.B.
      • McMurray C.T.
      and references therein).
      The sequence composition and the strand asymmetry of the rDNA provide it with considerable potential to form G-G-paired structures. The rDNA is G-rich on the top (non-template) strand, not only within the region transcribed into pre-rRNA but also within the spacers (Fig. 1). During active transcription, pol I molecules pack at extremely high density on the rDNA repeats; electron micrographic analysis shows that the spacing between pol I complexes is only 100 base pairs (
      • Osheim Y.
      • Mougey E.B.
      • Windle J.
      • Anderson M.
      • O'Reilly M.
      • Miller O.L.
      • Beyer A.
      • Sollner-Webb B.
      ). Transcription at this level requires that a considerable fraction of the rDNA duplex be denatured. We hypothesize that G-G-paired structures form within the G-rich top strand of the rDNA during transcription or when the duplex is transiently denatured during replication or recombination. G-G-paired structures are very stable once formed (
      • Sen D.
      • Gilbert W.
      ) and would not be predicted to dissociate spontaneously in vivo.
      Other experiments provide further support for the notion of a dynamic process of formation and unwinding of G-G-paired structures within the active rDNA. We have recently shown that G-G-paired DNA is the preferred substrate of two eukaryotic helicases, the human BLM helicase, which is deficient in Bloom's syndrome (
      • Sun H.
      • Karow J.K.
      • Hickson I.D.
      • Maizels N.
      ), and theSaccharomyces cerevisiae Sgs1p helicase (
      • Sun H.
      • Bennett R.J.
      • Maizels N.
      ). Both these helicases are members of the highly conserved RecQ helicase family. Moreover, S. cerevisiae Sgs1p localizes predominantly to the nucleolus (
      • Sinclair D.A.
      • Guarente L.
      ,
      • Sinclair D.A.
      • Mills K.
      • Guarente L.
      ), where it could function to maintain the structure of the G-rich rDNA. The human functional homolog of Sgs1p in S. cerevisiae appears to be the WRN helicase (deficient in Werner's syndrome). Like Sgs1p, WRN is a RecQ family helicase that is predominantly nucleolar in localization (
      • Marciniak R.A.
      • Lombard D.B.
      • Johnson F.B.
      • Guarente L.
      ,
      • Gray M.D.
      • Wang L.
      • Youssoufian H.
      • Martin G.M.
      • Oshima J.
      ). Unwinding activity mapped to the conserved helicase core domain of Sgs1p (
      • Sun H.
      • Bennett R.J.
      • Maizels N.
      ), strongly suggesting that preferential activity on G-G-paired substrates may be a general property of helicases in this family. It is therefore very likely that WRN will also prove to be active on G-G-paired rDNA substrates.

      Nucleolin as an Architectural Factor in rDNA Transcription, Replication, or Recombination

      Two separable domains within nucleolin can bind G-G-paired structures, one comprised of the RBDs 1, 2, 3, and 4 and the other comprised of the C-terminal RGG9domain. The presence of two independent G-G DNA binding domains would contribute to the ability of nucleolin to organize G-G-paired regions. Nucleolin may thus be an architectural factor, in effect forming a scaffolding for the structured G-rich strand. The presence of long acidic runs in the N terminus of nucleolin is consistent with its function in transcription, but nucleolin is a complex molecule with multiple distinct domains, and it may have multiple functions. We have identified nucleolin as one component of a heterodimeric protein, LR1, induced specifically in B cells activated for immunoglobulin heavy chain switch recombination (
      • Hanakahi L.A.
      • Dempsey L.A.
      • Li M.-J.
      • Maizels N.
      ,
      • Dempsey L.A.
      • Hanakahi L.A.
      • Maizels N.
      ,
      • Dempsey L.A.
      • Sun H.
      • Hanakahi L.A.
      • Maizels N.
      ). The rDNA repeats must undergo active recombination to maintain homogeneity of this gene family, and one function of nucleolin may be to stimulate or regulate recombination of the rDNA.

      Nucleolin in the Nucleolus

      Nucleolin is abundant in the peripheral granular component of the nucleolus, where rRNA processing occurs, and also in the central dense fibrillar component of the nucleolus, where rDNA transcription occurs (for review, see Ref.
      • Jordan G.
      ). Reported functions of nucleolin in rRNA processing (
      • Bouvet P.
      • Jain C.
      • Belasco J.B.
      • Amalric F.
      • Erard M.
      ) and ribosome assembly (
      • Lapeyre B.
      • Amalric F.
      • Ghaffari S.H.
      • Venkatarama Rao S.V.
      • Dumbar T.S.
      • Olson M.O.J.
      ) are consistent with its presence in the nucleolar peripheral granular component. Function in rDNA transcription, replication, and/or recombination is consistent with the observed localization of nucleolin within the nucleolar central dense fibrillar component. The N terminus of nucleolin contains long acidic regions of as many as 38 aspartate and glutamate residues in an uninterrupted stretch, which could function as acid blobs (
      • Ptashne M.
      ) to activate transcription by pol I. The N terminus of nucleolin also contains sites for the mitosis-specific cdc2 kinase (
      • Belenguer P.
      • Caizergues-Ferrer M.
      • Labbe J.
      • Doree M.
      • Amalric F.
      ) and casein kinase II (
      • Caizergues-Ferrer M.
      • Belenguer P.
      • Lapeyre B.
      • Amalric F.
      • Wallace M.O.
      • Olson M.O.J.
      ,
      • Belenguer P.
      • Baldin V.
      • Mathieu C.
      • Prats H.
      • Bensaid M.
      • Bouche G.
      • Amalric F.
      ). Both of these kinases phosphorylate histone H1, and they could analogously regulate nucleolin in response to cell cycle-dependent controls.
      Many proteins have been identified which contain RBDs and RGG motifs, but the mutational analysis of nucleolin makes it unlikely that high affinity binding to G-G-paired DNA is a common property of all RBD/RGG proteins. Most RBD-containing proteins contain only two or three RBDs, and deletion of two of the RBDs of nucleolin to produce Nuc-1,2, Nuc-2,3, or Nuc-3,4 greatly diminished binding affinity (Fig. 7). Similarly, whereas many proteins contain RGG motifs, nucleolin is unusual in that it contains nine repeats of the RGG motif, and deletion analysis showed that Nuc-RGG4 does not bind G4 DNA.
      The broad nucleolar distribution of nucleolin has led to considerable interest regarding its mode of localization within the nucleolus. The two domains of nucleolin that bind G-G-paired DNA (RBD-1,2,3,4 and RGG9) are also essential for nucleolar localization (
      • Heine M.A.
      • Rankin M.L.
      • DiMario P.J.
      ,
      • Schmidt-Zachermann M.S.
      • Nigg E.A.
      ,
      • Créancier L.
      • Prats H.
      • Zanibellato C.
      • Amalric F.
      • Bugler B.
      ), whereas the N-terminal acidic region is dispensable. The ability to interact with G-G-paired nucleic acids may, therefore, be essential to localization or retention of nucleolin within the nucleolus.

      Acknowledgments

      We are grateful to Dr. W. P. Russ for invaluable discussions.

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