title: Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA.
abstract: The nucleolus is a membrane-less organelle formed through liquid-liquid phase separation of its components from the surrounding nucleoplasm. Here, we show that nucleophosmin (NPM1) integrates within the nucleolus via a multi-modal mechanism involving multivalent interactions with proteins containing arginine-rich linear motifs (R-motifs) and ribosomal RNA (rRNA). Importantly, these R-motifs are found in canonical nucleolar localization signals. Based on a novel combination of biophysical approaches, we propose a model for the molecular organization within liquid-like droplets formed by the N-terminal domain of NPM1 and R-motif peptides, thus providing insights into the structural organization of the nucleolus. We identify multivalency of acidic tracts and folded nucleic acid binding domains, mediated by N-terminal domain oligomerization, as structural features required for phase separation of NPM1 with other nucleolar components in vitro and for localization within mammalian nucleoli. We propose that one mechanism of nucleolar localization involves phase separation of proteins within the nucleolus.
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, United States.
authors.forename: Diana M
authors.initials: DM
authors.lastname: Mitrea
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, United States.|Integrative Biomedical Sciences Program, University of Tennessee Health Sciences Center, Memphis, United States.
authors.forename: Jaclyn A
authors.initials: JA
authors.lastname: Cika
authors.affiliation: Department of Immunology, St. Jude Children's Research Hospital, Memphis, United States.
authors.forename: Clifford S
authors.initials: CS
authors.lastname: Guy
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, United States.
authors.forename: David
authors.initials: D
authors.lastname: Ban
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, United States.
authors.forename: Priya R
authors.initials: PR
authors.lastname: Banerjee
authors.affiliation: Biology and Biomedical Sciences Group, Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, United States.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Stanley
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, United States.|Molecular Interactions Analysis Shared Resource, St. Jude Children's Research Hospital, Memphis, United States.
authors.forename: Amanda
authors.initials: A
authors.lastname: Nourse
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, United States.
authors.forename: Ashok A
authors.initials: AA
authors.lastname: Deniz
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, United States.|Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Sciences Center, Memphis, United States.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Kriwacki
mesh_terms: D002466:Cell Nucleolus / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D000090243:Nucleophosmin; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D012335:RNA, Ribosomal / Q000378:metabolism*
chemical_list: C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D012335:RNA, Ribosomal; D000090243:Nucleophosmin
keywords: NPM1/B23/nucleophosmin; biophysics; human; intrinsically disordered proteins; nucleolar localization; nucleolus; phase separation; single molecule fluorescence; structural biology
references.citation: Biochem J. 2004 Mar 15;378(Pt 3):929-37
references.pmid: 14678012
references.citation: Angew Chem Int Ed Engl. 2013 Apr 2;52(14):3911-5
references.pmid: 23450638
references.citation: Mol Cell Biol. 2004 Feb;24(3):985-96
references.pmid: 14729947
references.citation: Nat Protoc. 2013 Mar;8(3):602-26
references.pmid: 23449254
references.citation: J Magn Reson. 2006 Jan;178(1):72-6
references.pmid: 16188473
references.citation: Oncogene. 2010 Mar 25;29(12):1821-34
references.pmid: 20101230
references.citation: J Biol Chem. 2000 Apr 21;275(16):12061-8
references.pmid: 10766838
references.citation: Proc Natl Acad Sci U S A. 2009 Apr 7;106(14):5645-50
references.pmid: 19293380
references.citation: J Biomol NMR. 1995 Nov;6(3):277-93
references.pmid: 8520220
references.citation: Mol Cell Biol. 2005 Oct;25(20):8874-86
references.pmid: 16199867
references.citation: Curr Biol. 2015 Mar 2;25(5):641-6
references.pmid: 25702583
references.citation: Nature. 2005 Sep 1;437(7055):147-53
references.pmid: 16007073
references.citation: Biochem Biophys Res Commun. 1996 Dec 4;229(1):295-8
references.pmid: 8954121
references.citation: J Biol Chem. 1994 Dec 9;269(49):30994-8
references.pmid: 7527039
references.citation: FEBS Lett. 2013 Jul 11;587(14):2254-9
references.pmid: 23742937
references.citation: Blood. 2006 Jun 1;107(11):4514-23
references.pmid: 16455950
references.citation: J Biomol NMR. 1999 Feb;13(2):101-12
references.pmid: 10070752
references.citation: J Biomol NMR. 1997 Apr;9(3):287-98
references.pmid: 9204557
references.citation: Nucleic Acids Res. 2009 Jan;37(1):1-13
references.pmid: 19033363
references.citation: PLoS One. 2012;7(12):e52476
references.pmid: 23285058
references.citation: Virology. 2014 Mar;452-453:212-22
references.pmid: 24606698
references.citation: Nat Biotechnol. 2004 Dec;22(12):1567-72
references.pmid: 15558047
references.citation: Biochemistry. 1989 Nov 14;28(23):8972-9
references.pmid: 2690953
references.citation: Nature. 2013 Jun 20;498(7454):390-4
references.pmid: 23783631
references.citation: J Am Chem Soc. 2010 Mar 31;132(12):4295-309
references.pmid: 20199098
references.citation: Chem Rev. 2004 Aug;104(8):3623-40
references.pmid: 15303831
references.citation: J Biol Chem. 2006 Jul 7;281(27):18463-72
references.pmid: 16679321
references.citation: Angew Chem Int Ed Engl. 2014 Jul 7;53(28):7354-9
references.pmid: 24862735
references.citation: J Am Chem Soc. 2008 Dec 10;130(49):16518-20
references.pmid: 19049453
references.citation: Nucleic Acids Res. 2013 Mar 1;41(5):3228-39
references.pmid: 23328624
references.citation: Oncogene. 2009 Nov 26;28(47):4201-11
references.pmid: 19734942
references.citation: Nature. 2012 Mar 15;483(7389):336-40
references.pmid: 22398450
references.citation: J Biol Chem. 2008 Aug 22;283(34):23326-32
references.pmid: 18511415
references.citation: Oncogene. 2011 Jun 9;30(23):2595-609
references.pmid: 21278791
references.citation: Curr Protoc Protein Sci. 2013 Feb;Chapter 20:Unit20.12
references.pmid: 23377850
references.citation: Nat Rev Mol Cell Biol. 2007 Jul;8(7):574-85
references.pmid: 17519961
references.citation: PLoS One. 2015;10(5):e0126420
references.pmid: 25997164
references.citation: Biophys J. 2000 Mar;78(3):1606-19
references.pmid: 10692345
references.citation: Biochem Res Int. 2011;2011:195209
references.pmid: 21152184
references.citation: Proc Natl Acad Sci U S A. 2011 Mar 15;108(11):4334-9
references.pmid: 21368180
references.citation: J Biol Chem. 2000 Aug 11;275(32):24451-7
references.pmid: 10829026
references.citation: Nucleic Acids Res. 2010 Nov;38(21):7388-99
references.pmid: 20663773
references.citation: J Biol Chem. 2014 Dec 12;289(50):34601-19
references.pmid: 25349213
references.citation: Nucleic Acids Res. 2009 Jan;37(Database issue):D181-4
references.pmid: 18984612
references.citation: J Cell Sci. 2006 Sep 1;119(Pt 17):3676-85
references.pmid: 16912078
references.citation: Proc Natl Acad Sci U S A. 2014 Mar 25;111(12):4466-71
references.pmid: 24616519
references.citation: J Biomol NMR. 2012 Dec;54(4):355-76
references.pmid: 23086713
references.citation: J Am Chem Soc. 2011 Sep 14;133(36):14184-7
references.pmid: 21848255
references.citation: J Am Chem Soc. 2005 Sep 21;127(37):12764-5
references.pmid: 16159246
references.citation: Nat Protoc. 2009;4(1):44-57
references.pmid: 19131956
references.citation: Nucleic Acids Res. 2014 Jan;42(2):1180-95
references.pmid: 24106084
references.citation: Mol Cell Biol. 1991 May;11(5):2567-75
references.pmid: 2017166
references.citation: Cancer Res. 2005 Nov 1;65(21):9834-42
references.pmid: 16267006
references.citation: J Biomol NMR. 2012 Jul;53(3):209-21
references.pmid: 22689066
references.citation: J Cell Biol. 2001 Apr 2;153(1):169-76
references.pmid: 11285283
references.citation: J Mol Biol. 2016 May 22;428(10 Pt A):2060-90
references.pmid: 26523678
references.citation: Nucleic Acids Res. 2014 Jan;42(Database issue):D191-8
references.pmid: 24253303
references.citation: Exp Cell Res. 1992 Nov;203(1):174-81
references.pmid: 1426041
references.citation: J Am Chem Soc. 2012 Feb 29;134(8):3792-803
references.pmid: 22280219