The Cajal body (CB) is a conserved non-membrane nuclear structure where several steps of small nuclear RNP particle (snRNP) biogenesis take place. It has been proposed that CB formation follows a liquid-liquid phase separation model, but this hypothesis has never been rigorously tested. Here, we applied live-cell imaging to show that the key CB assembly factor coilin is mobile within the CB, and we revealed a diffusion barrier that limits the coilin exchange between CBs and the nucleoplasm. We generated single aa mutations and demonstrated that RNA-dependent coilin oligomerization and coilin interaction with snRNP are essential for CB formation and maintenance. We applied these data to formulate a mathematical model that links the movement of coilin within the nucleoplasm, CB, and across the boundary with its oligomerization and snRNP binding. Our results illustrate CB as a structure dynamically responding to snRNP assembly and recycling.

Cell Biology Yale University New Haven CT USA

Faculty of Science Charles University Prague Prague Czech Republic

Institute of Molecular Genetics Czech Academy of Sciences Prague Czech Republic

Molecular Biophysics and Biochemistry Yale University New Haven CT USA

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Almeida, F., Saffrich R., Ansorge W., and Carmo-Fonseca M.. 1998. Microinjection of anti-coilin antibodies affects the structure of coiled bodies. J. Cell Biol. 142:899–912. 10.1083/jcb.142.4.899 PubMed DOI PMC

Arias Escayola, D., Zhang C., Nischwitz E., Schärfen L., Dörner K., Straube K., Kutay U., Butter F., and Neugebauer K.M.. 2025. Identification of coilin interactors reveals coordinated control of Cajal body number and structure. J. Cell Biol. 224:e202305081. 10.1083/jcb.202305081 PubMed DOI PMC

Banani, S.F., Rice A.M., Peeples W.B., Lin Y., Jain S., Parker R., and Rosen M.K.. 2016. Compositional control of phase-separated cellular bodies. Cell. 166:651–663. 10.1016/j.cell.2016.06.010 PubMed DOI PMC

Basello, D.A., Matera A.G., and Staněk D.. 2022. A point mutation in human coilin prevents Cajal body formation. J. Cell Sci. 135:jcs259587. 10.1242/jcs.259587 PubMed DOI PMC

Beechem, J.M., Knutson J.R., Ross J.B.A., Turner B.W., and Brand L.. 1983. Global resolution of heterogeneous decay by phase modulation fluorometry– mixtures and proteins. Biochemistry. 22:6054–6058. 10.1021/bi00295a002 DOI

Carmo-Fonseca, M., Ferreira J., and Lamond A.I.. 1993. Assembly of snRNP-containing coiled bodies is regulated in interphase and mitosis--evidence that the coiled body is a kinetic nuclear structure. J. Cell Biol. 120:841–852. 10.1083/jcb.120.4.841 PubMed DOI PMC

Cihlarova, Z., Kubovciak J., Sobol M., Krejcikova K., Sachova J., Kolar M., Stanek D., Barinka C., Yoon G., Caldecott K.W., and Hanzlikova H.. 2022. BRAT1 links Integrator and defective RNA processing with neurodegeneration. Nat. Commun. 13:5026. 10.1038/s41467-022-32763-6 PubMed DOI PMC

Courchaine, E., Gelles-Watnick S., Machyna M., Straube K., Sauyet S., Enright J., and Neugebauer K.M.. 2022. The coilin N-terminus mediates multivalent interactions between coilin and Nopp140 to form and maintain Cajal bodies. Nat. Commun. 13:6005. 10.1038/s41467-022-33434-2 PubMed DOI PMC

Courchaine, E.M., Lu A., and Neugebauer K.M.. 2016. Droplet organelles? EMBO J. 35:1603–1612. 10.15252/embj.201593517 PubMed DOI PMC

Cvacková, Z., Albring K.F., Koberna K., Ligasová A., Huber O., Raska I., and Stanek D.. 2008. Pontin is localized in nucleolar fibrillar centers. Chromosoma. 117:487–497. 10.1007/s00412-008-0170-8 PubMed DOI PMC

Decker, C.J., Burke J.M., Mulvaney P.K., and Parker R.. 2022. RNA is required for the integrity of multiple nuclear and cytoplasmic membrane-less RNP granules. EMBO J. 41:e110137. 10.15252/embj.2021110137 PubMed DOI PMC

Di Rienzo, C., Cardarelli F., Di Luca M., Beltram F., and Gratton E.. 2016. Diffusion tensor analysis by two-dimensional pair correlation of fluorescence fluctuations in cells. Biophys. J. 111:841–851. 10.1016/j.bpj.2016.07.005 PubMed DOI PMC

Dundr, M., Hebert M.D., Karpova T.S., Stanek D., Xu H., Shpargel K.B., Meier U.T., Neugebauer K.M., Matera A.G., and Misteli T.. 2004. In vivo kinetics of Cajal body components. J. Cell Biol. 164:831–842. 10.1083/jcb.200311121 PubMed DOI PMC

Eisenfeld, J., and Ford C.C.. 1979. A systems-theory approach to the analysis of multiexponential fluorescence decay. Biophys. J. 26:73–83. 10.1016/S0006-3495(79)85236-4 PubMed DOI PMC

Enwerem, I.I., Velma V., Broome H.J., Kuna M., Begum R.A., and Hebert M.D.. 2014. Coilin association with Box C/D scaRNA suggests a direct role for the Cajal body marker protein in scaRNP biogenesis. Biol. Open. 3:240–249. 10.1242/bio.20147443 PubMed DOI PMC

Ferreira, J.A., Carmo-Fonseca M., and Lamond A.I.. 1994. Differential interaction of splicing snRNPs with coiled bodies and interchromatin granules during mitosis and assembly of daughter cell nuclei. J. Cell Biol. 126:11–23. 10.1083/jcb.126.1.11 PubMed DOI PMC

Frey, M.R., and Matera A.G.. 1995. Coiled bodies contain U7 small nuclear RNA and associate with specific DNA sequences in interphase human cells. Proc. Natl. Acad. Sci. USA. 92:5915–5919. 10.1073/pnas.92.13.5915 PubMed DOI PMC

Frey, M.R., and Matera A.G.. 2001. RNA-mediated interaction of Cajal bodies and U2 snRNA genes. J. Cell Biol. 154:499–509. 10.1083/jcb.200105084 PubMed DOI PMC

Girard, C., Neel H., Bertrand E., and Bordonné R.. 2006. Depletion of SMN by RNA interference in HeLa cells induces defects in Cajal body formation. Nucleic Acids Res. 34:2925–2932. 10.1093/nar/gkl374 PubMed DOI PMC

Guillén-Boixet, J., Kopach A., Holehouse A.S., Wittmann S., Jahnel M., Schlüßler R., Kim K., Trussina I.R.E.A., Wang J., Mateju D., et al. 2020. RNA-induced conformational switching and clustering of G3BP drive stress granule assembly by condensation. Cell. 181:346–361.e17. 10.1016/j.cell.2020.03.049 PubMed DOI PMC

Haaf, T., and Ward D.C.. 1996. Inhibition of RNA polymerase II transcription causes chromatin decondensation, loss of nucleolar structure, and dispersion of chromosomal domains. Exp. Cell Res. 224:163–173. 10.1006/excr.1996.0124 PubMed DOI

Handwerger, K.E., Cordero J.A., and Gall J.G.. 2005. Cajal bodies, nucleoli, and speckles in the Xenopus oocyte nucleus have a low-density, sponge-like structure. Mol. Biol. Cell. 16:202–211. 10.1091/mbc.e04-08-0742 PubMed DOI PMC

Handwerger, K.E., Murphy C., and Gall J.G.. 2003. Steady-state dynamics of Cajal body components in the Xenopus germinal vesicle. J. Cell Biol. 160:495–504. 10.1083/jcb.200212024 PubMed DOI PMC

Hebert, M.D., and Matera A.G.. 2000. Self-association of coilin reveals a common theme in nuclear body localization. Mol. Biol. Cell. 11:4159–4171. 10.1091/mbc.11.12.4159 PubMed DOI PMC

Hebert, M.D., Shpargel K.B., Ospina J.K., Tucker K.E., and Matera A.G.. 2002. Coilin methylation regulates nuclear body formation. Dev. Cell. 3:329–337. 10.1016/S1534-5807(02)00222-8 PubMed DOI

Hedde, P.N., Staaf E., Singh S.B., Johansson S., and Gratton E.. 2019. Pair correlation analysis maps the dynamic two-dimensional organization of natural killer cell receptors at the synapse. ACS Nano. 13:14274–14282. 10.1021/acsnano.9b07486 PubMed DOI PMC

Irizarry, R. 2004. LARES: An artificial chemical process approach for optimization. Evol. Comput. 12:435–459. 10.1162/1063656043138897 PubMed DOI

Jacobs, E.Y., Frey M.R., Wu W., Ingledue T.C., Gebuhr T.C., Gao L., Marzluff W.F., and Matera A.G.. 1999. Coiled bodies preferentially associate with U4, U11, and U12 small nuclear RNA genes in interphase HeLa cells but not with U6 and U7 genes. Mol. Biol. Cell. 10:1653–1663. 10.1091/mbc.10.5.1653 PubMed DOI PMC

Kaiser, T.E., Intine R.V., and Dundr M.. 2008. De novo formation of a subnuclear body. Science. 322:1713–1717. 10.1126/science.1165216 PubMed DOI

Kato, M., Han T.W., Xie S., Shi K., Du X., Wu L.C., Mirzaei H., Goldsmith E.J., Longgood J., Pei J., et al. 2012. Cell-free formation of RNA granules: Low complexity sequence domains form dynamic fibers within hydrogels. Cell. 149:753–767. 10.1016/j.cell.2012.04.017 PubMed DOI PMC

Klimešová, K., Vojáčková J., Radivojević N., Vandermoere F., Bertrand E., Verheggen C., and Staněk D.. 2021. TSSC4 is a component of U5 snRNP that promotes tri-snRNP formation. Nat. Commun. 12:3646. 10.1038/s41467-021-23934-y PubMed DOI PMC

Klingauf, M., Stanek D., and Neugebauer K.M.. 2006. Enhancement of U4/U6 small nuclear ribonucleoprotein particle association in Cajal bodies predicted by mathematical modeling. Mol. Biol. Cell. 17:4972–4981. 10.1091/mbc.e06-06-0513 PubMed DOI PMC

Knutson, J.R., Beechem J.M., and Brand L.. 1983. Simultaneous analysis of multiple fluorescence decay curves—a global approach. Chem. Phys. Lett. 102:501–507. 10.1016/0009-2614(83)87454-5 DOI

Koo, D.H., Zhao H., and Jiang J.. 2016. Chromatin-associated transcripts of tandemly repetitive DNA sequences revealed by RNA-FISH. Chromosome Res. 24:467–480. 10.1007/s10577-016-9537-5 PubMed DOI

Krchnáková, Z., Thakur P.K., Krausová M., Bieberstein N., Haberman N., Müller-McNicoll M., and Stanek D.. 2019. Splicing of long non-coding RNAs primarily depends on polypyrimidine tract and 5′ splice-site sequences due to weak interactions with SR proteins. Nucleic Acids Res. 47:911–928. 10.1093/nar/gky1147 PubMed DOI PMC

Lemm, I., Girard C., Kuhn A.N., Watkins N.J., Schneider M., Bordonné R., and Lührmann R.. 2006. Ongoing U snRNP biogenesis is required for the integrity of Cajal bodies. Mol. Biol. Cell. 17:3221–3231. 10.1091/mbc.e06-03-0247 PubMed DOI PMC

Lerner, E.A., Lerner M.R., Janeway C.A. Jr., and Steitz J.A.. 1981. Monoclonal antibodies to nucleic acid-containing cellular constituents: Probes for molecular biology and autoimmune disease. Proc. Natl. Acad. Sci. USA. 78:2737–2741. 10.1073/pnas.78.5.2737 PubMed DOI PMC

Lin, Y., Protter D.S., Rosen M.K., and Parker R.. 2015. Formation and maturation of phase-separated liquid droplets by RNA-binding proteins. Mol. Cell. 60:208–219. 10.1016/j.molcel.2015.08.018 PubMed DOI PMC

Machyna, M., Heyn P., and Neugebauer K.M.. 2013. Cajal bodies: Where form meets function. Wiley Interdiscip. Rev. RNA. 4:17–34. 10.1002/wrna.1139 PubMed DOI

Machyna, M., Kehr S., Straube K., Kappei D., Buchholz F., Butter F., Ule J., Hertel J., Stadler P.F., and Neugebauer K.M.. 2014. The coilin interactome identifies hundreds of small noncoding RNAs that traffic through Cajal bodies. Mol. Cell. 56:389–399. 10.1016/j.molcel.2014.10.004 PubMed DOI

Machyna, M., Neugebauer K.M., and Staněk D.. 2015. Coilin: The first 25 years. RNA Biol. 12:590–596. 10.1080/15476286.2015.1034923 PubMed DOI PMC

Makarov, V., Rakitina D., Protopopova A., Yaminsky I., Arutiunian A., Love A.J., Taliansky M., and Kalinina N.. 2013. Plant coilin: Structural characteristics and RNA-binding properties. PLoS One. 8:e53571. 10.1371/journal.pone.0053571 PubMed DOI PMC

Malacrida, L., Hedde P.N., Torrado B., and Gratton E.. 2020. Barriers to diffusion in cells: Visualization of membraneless particles in the nucleus. Biophysicist (Rockv.). 1:9. 10.35459/tbp.2019.000111 PubMed DOI PMC

McSwiggen, D.T., Hansen A.S., Teves S.S., Marie-Nelly H., Hao Y., Heckert A.B., Umemoto K.K., Dugast-Darzacq C., Tjian R., and Darzacq X.. 2019a. Evidence for DNA-mediated nuclear compartmentalization distinct from phase separation. Elife. 8:e47098. 10.7554/eLife.47098 PubMed DOI PMC

McSwiggen, D.T., Mir M., Darzacq X., and Tjian R.. 2019b. Evaluating phase separation in live cells: Diagnosis, caveats, and functional consequences. Genes Dev. 33:1619–1634. 10.1101/gad.331520.119 PubMed DOI PMC

Mitrea, D.M., and Kriwacki R.W.. 2016. Phase separation in biology; functional organization of a higher order. Cell Commun. Signal. 14:1. 10.1186/s12964-015-0125-7 PubMed DOI PMC

Molliex, A., Temirov J., Lee J., Coughlin M., Kanagaraj A.P., Kim H.J., Mittag T., and Taylor J.P.. 2015. Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell. 163:123–133. 10.1016/j.cell.2015.09.015 PubMed DOI PMC

Novotný, I., Blažíková M., Staněk D., Herman P., and Malinsky J.. 2011. In vivo kinetics of U4/U6·U5 tri-snRNP formation in Cajal bodies. Mol. Biol. Cell. 22:513–523. 10.1091/mbc.e10-07-0560 PubMed DOI PMC

Novotný, I., Malinová A., Stejskalová E., Matějů D., Klimešová K., Roithová A., Švéda M., Knejzlík Z., and Staněk D.. 2015. SART3-Dependent accumulation of incomplete spliceosomal snRNPs in Cajal bodies. Cell Rep. 10:429–440. 10.1016/j.celrep.2014.12.030 PubMed DOI

Ochs, R.L., Lischwe M.A., Spohn W.H., and Busch H.. 1985. Fibrillarin: A new protein of the nucleolus identified by autoimmune sera. Biol. Cell. 54:123–133. 10.1111/j.1768-322X.1985.tb00387.x PubMed DOI

Patel, A., Lee H.O., Jawerth L., Maharana S., Jahnel M., Hein M.Y., Stoynov S., Mahamid J., Saha S., Franzmann T.M., et al. 2015. A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell. 162:1066–1077. 10.1016/j.cell.2015.07.047 PubMed DOI

Pena, E., Berciano M.T., Fernandez R., Ojeda J.L., and Lafarga M.. 2001. Neuronal body size correlates with the number of nucleoli and Cajal bodies, and with the organization of the splicing machinery in rat trigeminal ganglion neurons. J. Comp. Neurol. 430:250–263. 10.1002/1096-9861(20010205)430:2<250::AID-CNE1029>3.0.CO;2-L PubMed DOI

Platani, M., Goldberg I., Swedlow J.R., and Lamond A.I.. 2000. In vivo analysis of Cajal body movement, separation, and joining in live human cells. J. Cell Biol. 151:1561–1574. 10.1083/jcb.151.7.1561 PubMed DOI PMC

Poser, I., Sarov M., Hutchins J.R., Hériché J.K., Toyoda Y., Pozniakovsky A., Weigl D., Nitzsche A., Hegemann B., Bird A.W., et al. 2008. BAC TransgeneOmics: A high-throughput method for exploration of protein function in mammals. Nat. Methods. 5:409–415. 10.1038/nmeth.1199 PubMed DOI PMC

Raska, I., Andrade L.E., Ochs R.L., Chan E.K., Chang C.M., Roos G., and Tan E.M.. 1991. Immunological and ultrastructural studies of the nuclear coiled body with autoimmune antibodies. Exp. Cell Res. 195:27–37. 10.1016/0014-4827(91)90496-H PubMed DOI

Riback, J.A., Zhu L., Ferrolino M.C., Tolbert M., Mitrea D.M., Sanders D.W., Wei M.T., Kriwacki R.W., and Brangwynne C.P.. 2020. Composition-dependent thermodynamics of intracellular phase separation. Nature. 581:209–214. 10.1038/s41586-020-2256-2 PubMed DOI PMC

Roithová, A., Klimešová K., Pánek J., Will C.L., Lührmann R., Stanek D., and Girard C.. 2018. The Sm-core mediates the retention of partially-assembled spliceosomal snRNPs in Cajal bodies until their full maturation. Nucleic Acids Res. 46:3774–3790. 10.1093/nar/gky070 PubMed DOI PMC

Roithová, A., and Staněk D.. 2019. Analysis of spliceosomal snRNA localization in human hela cells using microinjection. J. Vis. Exp. 10.3791/59797 PubMed DOI

Schwille, P., Haupts U., Maiti S., and Webb W.W.. 1999. Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation. Biophys. J. 77:2251–2265. 10.1016/S0006-3495(99)77065-7 PubMed DOI PMC

Shanbhag, R., Kurabi A., Kwan J.J., and Donaldson L.W.. 2010. Solution structure of the carboxy-terminal Tudor domain from human Coilin. FEBS Lett. 584:4351–4356. 10.1016/j.febslet.2010.09.034 PubMed DOI

Shpargel, K.B., Ospina J.K., Tucker K.E., Matera A.G., and Hebert M.D.. 2003. Control of Cajal body number is mediated by the coilin C-terminus. J. Cell Sci. 116:303–312. 10.1242/jcs.00211 PubMed DOI

Sleeman, J.E., Ajuh P., and Lamond A.I.. 2001. snRNP protein expression enhances the formation of Cajal bodies containing p80-coilin and SMN. J. Cell Sci. 114:4407–4419. 10.1242/jcs.114.24.4407 PubMed DOI

Sleeman, J.E., Trinkle-Mulcahy L., Prescott A.R., Ogg S.C., and Lamond A.I.. 2003. Cajal body proteins SMN and Coilin show differential dynamic behaviour in vivo. J. Cell Sci. 116:2039–2050. 10.1242/jcs.00400 PubMed DOI

Smith, K.P., Carter K.C., Johnson C.V., and Lawrence J.B.. 1995. U2 and U1 snRNA gene loci associate with coiled bodies. J. Cell. Biochem. 59:473–485. 10.1002/jcb.240590408 PubMed DOI

Smith, K.P., and Lawrence J.B.. 2000. Interactions of U2 gene loci and their nuclear transcripts with Cajal (coiled) bodies: Evidence for PreU2 within Cajal bodies. Mol. Biol. Cell. 11:2987–2998. 10.1091/mbc.11.9.2987 PubMed DOI PMC

Staněk, D. 2017. Cajal bodies and snRNPs - friends with benefits. RNA Biol. 14:671–679. 10.1080/15476286.2016.1231359 PubMed DOI PMC

Staněk, D., and Fox A.H.. 2017. Nuclear bodies: News insights into structure and function. Curr. Opin. Cell Biol. 46:94–101. 10.1016/j.ceb.2017.05.001 PubMed DOI

Stanek, D., Pridalová-Hnilicová J., Novotný I., Huranová M., Blazíková M., Wen X., Sapra A.K., and Neugebauer K.M.. 2008. Spliceosomal small nuclear ribonucleoprotein particles repeatedly cycle through Cajal bodies. Mol. Biol. Cell. 19:2534–2543. 10.1091/mbc.e07-12-1259 PubMed DOI PMC

Strzelecka, M., Trowitzsch S., Weber G., Lührmann R., Oates A.C., and Neugebauer K.M.. 2010. Coilin-dependent snRNP assembly is essential for zebrafish embryogenesis. Nat. Struct. Mol. Biol. 17:403–409. 10.1038/nsmb.1783 PubMed DOI

Suzuki, T., Izumi H., and Ohno M.. 2010. Cajal body surveillance of U snRNA export complex assembly. J. Cell Biol. 190:603–612. 10.1083/jcb.201004109 PubMed DOI PMC

Takata, H., Nishijima H., Maeshima K., and Shibahara K.. 2012. The integrator complex is required for integrity of Cajal bodies. J. Cell Sci. 125:166–175. 10.1242/jcs.090837 PubMed DOI

Tucker, K.E., Berciano M.T., Jacobs E.Y., LePage D.F., Shpargel K.B., Rossire J.J., Chan E.K.L., Lafarga M., Conlon R.A., and Matera A.G.. 2001. Residual Cajal bodies in coilin knockout mice fail to recruit Sm snRNPs and SMN, the spinal muscular atrophy gene product. J. Cell Biol. 154:293–307. 10.1083/jcb.200104083 PubMed DOI PMC

Velma, V., Broome H.J., and Hebert M.D.. 2012. Regulated specific proteolysis of the Cajal body marker protein coilin. Chromosoma. 121:629–642. 10.1007/s00412-012-0387-4 PubMed DOI PMC

Wang, Q., Sawyer I.A., Sung M.H., Sturgill D., Shevtsov S.P., Pegoraro G., Hakim O., Baek S., Hager G.L., and Dundr M.. 2016. Cajal bodies are linked to genome conformation. Nat. Commun. 7:10966. 10.1038/ncomms10966 PubMed DOI PMC

Widengren, J., Mets U., and Rigler R.. 1995. Fluorescence correlation spectroscopy of triplet-states in solution - a theoretical and experimental-study. J. Phys. Chem. 99:13368–13379. 10.1021/j100036a009 DOI

Xu, H., Pillai R.S., Azzouz T.N., Shpargel K.B., Kambach C., Hebert M.D., Schümperli D., and Matera A.G.. 2005. The C-terminal domain of coilin interacts with Sm proteins and U snRNPs. Chromosoma. 114:155–166. 10.1007/s00412-005-0003-y PubMed DOI PMC

Zhu, L., and Brangwynne C.P.. 2015. Nuclear bodies: The emerging biophysics of nucleoplasmic phases. Curr. Opin. Cell Biol. 34:23–30. 10.1016/j.ceb.2015.04.003 PubMed DOI PMC