Coilin is a conserved protein essential for integrity of nuclear membrane-less inclusions called Cajal bodies. Here, we report an amino acid substitution (p.K496E) found in a widely-used human EGFP-coilin construct that has a dominant-negative effect on Cajal body formation. We show that this coilin-K496E variant fails to rescue Cajal bodies in cells lacking endogenous coilin, whereas the wild-type construct restores Cajal bodies in mouse and human coilin-knockout cells. In cells containing endogenous coilin, both the wild-type and K496E variant proteins accumulate in Cajal bodies. However, high-level overexpression of coilin-K496E causes Cajal body disintegration. Thus, a mutation in the C-terminal region of human coilin can disrupt Cajal body assembly. Caution should be used when interpreting data from coilin plasmids that are derived from this variant (currently deposited at Addgene).

Faculty of Science Charles University Prague 14220 Czech Republic

Institute of Molecular Genetics Czech Academy of Science Prague Czech Republic

Integrative Program for Biological and Genome Sciences and Department of Biology University of North Carolina Chapel Hill NC 27599 3280 USA

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Akimov, V., Barrio-Hernandez, I., Hansen, S. V. F., Hallenborg, P., Pedersen, A. K., Bekker-Jensen, D. B., Puglia, M., Christensen, S. D. K., Vanselow, J. T., Nielsen, M. M.et al. (2018). UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites. Nat. Struct. Mol. Biol. 25, 631-640. 10.1038/s41594-018-0084-y PubMed DOI

Andrade, L. E., Chan, E. K., Raska, I., Peebles, C. L., Roos, G. and Tan, E. M. (1991). Human autoantibody to a novel protein of the nuclear coiled body: immunological characterization and cDNA cloning of p80-coilin. J. Exp. Med. 173, 1407-1419. 10.1084/jem.173.6.1407 PubMed DOI PMC

Bohmann, K., Ferreira, J. A. and Lamond, A. I. (1995). Mutational analysis of p80 coilin indicates a functional interaction between coiled bodies and the nucleolus. J. Cell Biol. 131, 817-831. 10.1083/jcb.131.4.817 PubMed DOI PMC

Cajal, S. R. (1903). Un sencillo metodo de coloracion seletiva del reticulo protoplasmatico y sus efectos en los diversos organos nerviosos de vertebrados e invertebrados. Trab. Lab. Invest. Biol. (Madrid) 2, 129-221.

Cantarero, L., Sanz-García, M., Vinograd-Byk, H., Renbaum, P., Levy-Lahad, E. and Lazo, P. A. (2015). VRK1 regulates Cajal body dynamics and protects coilin from proteasomal degradation in cell cycle. Sci. Rep. 5, 10543. 10.1038/srep10543 PubMed DOI PMC

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

Carrero, Z. I., Velma, V., Douglas, H. E. and Hebert, M. D. (2011). Coilin phosphomutants disrupt Cajal body formation, reduce cell proliferation and produce a distinct coilin degradation product. PLoS ONE 6, e25743. 10.1371/journal.pone.0025743 PubMed DOI PMC

Chan, E. K. L., Takano, S., Andrade, L. E. C., Hamel, J. C. and Matera, A. G. (1994). Structure, expression and chromosomal localization of human p80-coilin gene. Nucleic Acids Res. 22, 4462-4469. 10.1093/nar/22.21.4462 PubMed DOI PMC

Chen, Y., Deng, Z., Jiang, S., Hu, Q., Liu, H., Songyang, Z., Ma, W., Chen, S. and Zhao, Y. (2015). Human cells lacking coilin and Cajal bodies are proficient in telomerase assembly, trafficking and telomere maintenance. Nucleic Acids Res. 43, 385-395. 10.1093/nar/gku1277 PubMed DOI PMC

Collier, S., Pendle, A., Boudonck, K., van Rij, T., Dolan, L. and Shaw, P. (2006). A distant coilin homologue is required for the formation of cajal bodies in Arabidopsis. Mol. Biol. Cell. 17, 2942-2951. 10.1091/mbc.e05-12-1157 PubMed DOI PMC

Hebert, M. D. (2013). Signals controlling Cajal body assembly and function. Int. J. Biochem. Cell Biol. 45, 1314-1317. 10.1016/j.biocel.2013.03.019 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., Szymczyk, P. W., Shpargel, K. B. and Matera, A. G. (2001). Coilin forms the bridge between Cajal bodies and SMN, the spinal muscular atrophy protein. Genes Dev. 15, 2720-2729. 10.1101/gad.908401 PubMed DOI PMC

Kasparek, P., Krausova, M., Haneckova, R., Kriz, V., Zbodakova, O., Korinek, V. and Sedlacek, R. (2014). Efficient gene targeting of the Rosa26 locus in mouse zygotes using TALE nucleases. FEBS Lett. 588, 3982-3988. 10.1016/j.febslet.2014.09.014 PubMed DOI

Kim, W., Bennett, E. J., Huttlin, E. L., Guo, A., Li, J., Possemato, A., Sowa, M. E., Rad, R., Rush, J., Comb, M. J.et al. (2011). Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol. Cell. 44, 325-340. 10.1016/j.molcel.2011.08.025 PubMed DOI PMC

Liu, J., Hebert, M. D., Ye, Y., Templeton, D. J., Kung, H. and Matera, A. G. (2000). Cell cycle-dependent localization of the CDK2-cyclin E complex in Cajal (coiled) bodies. J. Cell Sci. 113, 1543-1552. 10.1242/jcs.113.9.1543 PubMed DOI

Liu, J. L., Wu, Z., Nizami, Z., Deryusheva, S., Rajendra, T. K., Beumer, K. J., Gao, H., Matera, A. G., Carroll, D. and Gall, J. G. (2009). Coilin is essential for Cajal body organization in Drosophila melanogaster. Mol. Biol. Cell 20, 1661-1670. 10.1091/mbc.e08-05-0525 PubMed DOI PMC

Lumpkin, R. J., Gu, H., Zhu, Y., Leonard, M., Ahmad, A. S., Clauser, K. R., Meyer, J. G., Bennett, E. J. and Komives, E. A. (2017). Site-specific identification and quantitation of endogenous SUMO modifications under native conditions. Nat. Commun. 8, 1171. 10.1038/s41467-017-01271-3 PubMed DOI PMC

Lyon, C. E., Bohmann, K., Sleeman, J. and Lamond, A. I. (1997). Inhibition of protein dephosphorylation results in the accumulation of splicing snRNPs and coiled bodies within the nucleolus. Exp. Cell Res. 230, 84-93. 10.1006/excr.1996.3380 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

Navascues, J., Bengoechea, R., Tapia, O., Casafont, I., Berciano, M. T. and Lafarga, M. (2008). SUMO-1 transiently localizes to Cajal bodies in mammalian neurons. J. Struct. Biol. 163, 137-146. 10.1016/j.jsb.2008.04.013 PubMed DOI

Raška, 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

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., Lyon, C. E., Platani, M., Kreivi, J. P. and Lamond, A. I. (1998). Dynamic interactions between splicing snRNPs, coiled bodies and nucleoli revealed using snRNP protein fusions to the green fluorescent protein. Exp. Cell Res. 243, 290-304. 10.1006/excr.1998.4135 PubMed DOI

Stanek, D. (2017). Cajal bodies and snRNPs - friends with benefits. RNA Biol. 14, 671-679. 10.1080/15476286.2016.1231359 PubMed DOI PMC

Stanek, D. and Neugebauer, K. M. (2004). Detection of snRNP assembly intermediates in Cajal bodies by fluorescence resonance energy transfer. J. Cell Biol. 166, 1015-1025. 10.1083/jcb.200405160 PubMed DOI PMC

Stern, J. L., Zyner, K. G., Pickett, H. A., Cohen, S. B. and Bryan, T. M. (2012). Telomerase recruitment requires both TCAB1 and Cajal bodies independently. Mol. Cell Biol. 32, 2384-2395. 10.1128/MCB.00379-12 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

Sveda, M., Častoralova, M., Lipov, J., Ruml, T. and Knejzlík, Z. (2013). Human UBL5 protein interacts with coilin and meets the Cajal bodies. Biochem. Biophys. Res. Commun. 436, 240-245. 10.1016/j.bbrc.2013.05.083 PubMed DOI

Tammsalu, T., Matic, I., Jaffray, E. G., Ibrahim, A. F. M., Tatham, M. H. and Hay, R. T. (2014). Proteome-wide identification of SUMO2 modification sites. Sci. Signal. 7, rs2. 10.1126/scisignal.2005146 PubMed DOI PMC

Tapia, O., Bengoechea, R., Berciano, M. T. and Lafarga, M. (2010). Nucleolar targeting of coilin is regulated by its hypomethylation state. Chromosoma 119, 527-540. 10.1007/s00412-010-0276-7 PubMed DOI

Toyota, C. G., Davis, M. D., Cosman, A. M. and Hebert, M. D. (2010). Coilin phosphorylation mediates interaction with SMN and SmB'. Chromosoma 119, 205-215. 10.1007/s00412-009-0249-x PubMed DOI PMC

Tucker, K. E., Massello, L. K., Gao, L., Barber, T. J., Hebert, M. D., Chan, E. K. and Matera, A. G. (2000). Structure and characterization of the murine p80 coilin gene, Coil. J. Struct. Biol. 129, 269-277. 10.1006/jsbi.2000.4234 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. and Conlon, R. A. (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, 293-308. 10.1083/jcb.200104083 PubMed DOI PMC

Tuma, R. S., Stolk, J. A. and Roth, M. B. (1993). Identification and characterization of a sphere organelle protein. J. Cell Biol. 122, 767-773. 10.1083/jcb.122.4.767 PubMed DOI PMC

Wu, Z., Murphy, C. and Gall, J. G. (1994). Human p80-coilin is targeted to sphere organelles in the amphibian germinal vesicle. Mol. Biol. Cell 5, 1119-1127. 10.1091/mbc.5.10.1119 PubMed DOI PMC

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

Coilin and Cajal bodies