The Cajal body (CB) is a nuclear structure closely associated with import and biogenesis of small nuclear ribonucleoprotein particles (snRNPs). Here, we tested whether CBs also contain mature snRNPs and whether CB integrity depends on the ongoing snRNP splicing cycle. Sm proteins tagged with photoactivatable and color-maturing variants of fluorescent proteins were used to monitor snRNP behavior in living cells over time; mature snRNPs accumulated in CBs, traveled from one CB to another, and they were not preferentially replaced by newly imported snRNPs. To test whether CB integrity depends on the snRNP splicing cycle, two human orthologues of yeast proteins involved in distinct steps in spliceosome disassembly after splicing, hPrp22 and hNtr1, were depleted by small interfering RNA treatment. Surprisingly, depletion of either protein led to the accumulation of U4/U6 snRNPs in CBs, suggesting that reassembly of the U4/U6.U5 tri-snRNP was delayed. Accordingly, a relative decrease in U5 snRNPs compared with U4/U6 snRNPs was observed in CBs, as well as in nuclear extracts of treated cells. Together, the data show that particular phases of the spliceosome cycle are compartmentalized in living cells, with reassembly of the tri-snRNP occurring in CBs.

Institute of Molecular Genetics Academy of Sciences of the Czech Republic 142 20 Prague 4 Czech Republic

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Achsel T., Brahms H., Kastner B., Bachi A., Wilm M., Luhrmann R. A doughnut-shaped heteromer of human Sm-like proteins binds to the 3′-end of U6 snRNA, thereby facilitating U4/U6 duplex formation in vitro. EMBO J. 1999;18:5789–5802. PubMed PMC

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

Arenas J. E., Abelson J. N. Prp 43: an RNA helicase-like factor involved in spliceosome disassembly. Proc. Natl. Acad. Sci. USA. 1997;94:11798–11802. PubMed PMC

Bell M., Schreiner S., Damianov A., Reddy R., Bindereif A. p110, a novel human U6 snRNP protein and U4/U6 snRNP recycling factor. EMBO J. 2002;21:2724–2735. PubMed PMC

Black D. L., Pinto A. L. U5 small nuclear ribonucleoprotein: RNA structure analysis and ATP-dependent interaction with U4/U6. Mol. Cell. Biol. 1989;9:3350–3359. PubMed PMC

Blencowe B. J., Carmo-Fonseca M., Behrens S. E., Luhrmann R., Lamond A. I. Interaction of the human autoantigen p150 with splicing snRNPs. J. Cell Sci. 1993;105:685–697. PubMed

Boon K. L., Auchynnikava T., Edwalds-Gilbert G., Barrass J. D., Droop A. P., Dez C., Beggs J. D. Yeast ntr1/spp382 mediates prp43 function in postspliceosomes. Mol. Cell. Biol. 2006;26:6016–6023. PubMed PMC

Carmo-Fonseca M., Pepperkok R., Carvalho M. T., Lamond A. I. Transcription-dependent colocalization of the U1, U2, U4/U6, and U5 snRNPs in coiled bodies. J. Cell Biol. 1992;117:1–14. PubMed PMC

Carvalho T., Almeida F., Calapez A., Lafarga M., Berciano M. T., Carmo-Fonseca M. The spinal muscular atrophy disease gene product, SMN: a link between snRNP biogenesis and the Cajal (coiled) body. J. Cell Biol. 1999;147:715–728. PubMed PMC

Chan S. P., Kao D. I., Tsai W. Y., Cheng S. C. The Prp19p-associated complex in spliceosome activation. Science. 2003;302:279–282. PubMed

Chen C. H., Kao D. I., Chan S. P., Kao T. C., Lin J. Y., Cheng S. C. Functional links between the Prp19-associated complex, U4/U6 biogenesis, and spliceosome recycling. RNA. 2006;12:765–774. PubMed PMC

Company M., Arenas J., Abelson J. Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomes. Nature. 1991;349:487–493. PubMed

Darzacq X., Jady B. E., Verheggen C., Kiss A. M., Bertrand E., Kiss T. Cajal body-specific small nuclear RNAs: a novel class of 2′-O-methylation and pseudouridylation guide RNAs. EMBO J. 2002;21:2746–2756. PubMed PMC

Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983;11:1475–1489. PubMed 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., Misteli T. In vivo kinetics of Cajal body components. J. Cell Biol. 2004;164:831–842. PubMed PMC

Fabrizio P., Laggerbauer B., Lauber J., Lane W. S., Luhrmann R. An evolutionarily conserved U5 snRNP-specific protein is a GTP-binding factor closely related to the ribosomal translocase EF-2. EMBO J. 1997;16:4092–4106. PubMed PMC

Gerlich D., Beaudouin J., Kalbfuss B., Daigle N., Eils R., Ellenberg J. Global chromosome positions are transmitted through mitosis in mammalian cells. Cell. 2003;112:751–764. PubMed

Ghetti A., Company M., Abelson J. Specificity of Prp24 binding to RNA: a role for Prp24 in the dynamic interaction of U4 and U6 snRNAs. RNA. 1995;1:132–145. PubMed PMC

Girard C., Neel H., Bertrand E., Bordonne R. Depletion of SMN by RNA interference in HeLa cells induces defects in Cajal body formation. Nucleic Acids Res. 2006;34:2925–2932. PubMed PMC

Jady B. E., Darzacq X., Tucker K. E., Matera A. G., Bertrand E., Kiss T. Modification of Sm small nuclear RNAs occurs in the nucleoplasmic Cajal body following import from the cytoplasm. EMBO J. 2003;22:1878–1888. PubMed PMC

Jurica M. S., Moore M. J. Pre-mRNA splicing: awash in a sea of proteins. Mol Cell. 2003;12:5–14. PubMed

Kambach C., Walke S., Young R., Avis J. M., de la Fortelle E., Raker V. A., Luhrmann R., Li J., Nagai K. Crystal structures of two Sm protein complexes and their implications for the assembly of the spliceosomal snRNPs. Cell. 1999;96:375–387. PubMed

Kiss T. Small nucleolar RNAs: an abundant group of noncoding RNAs with diverse cellular functions. Cell. 2002;109:145–148. PubMed

Kiss T. Biogenesis of small nuclear RNPs. J. Cell Sci. 2004;117:5949–5951. PubMed

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

Lauber J., Plessel G., Prehn S., Will C. L., Fabrizio P., Groning K., Lane W. S., Luhrmann R. The human U4/U6 snRNP contains 60 and 90kD proteins that are structurally homologous to the yeast splicing factors Prp4p and Prp3p. RNA. 1997;3:926–941. PubMed PMC

Lemm I., Girard C., Kuhn A. N., Watkins N. J., Schneider M., Bordonne R., Luhrmann R. Ongoing U snRNP Biogenesis Is Required for the Integrity of Cajal Bodies. Mol. Biol. Cell. 2006;17:3221–3231. PubMed PMC

Listerman I., Bledau A. S., Grishina I., Neugebauer K. M. Extragenic accumulation of RNA polymerase II enhances transcription by RNA polymerase III. PLoS Genet. 2007;3:e212. PubMed PMC

Listerman I., Sapra A. K., Neugebauer K. M. Cotranscriptional coupling of splicing factor recruitment and precursor messenger RNA splicing in mammalian cells. Nat. Struct. Mol. Biol. 2006;13:815–822. PubMed

Liu J. L., Gall J. G. U bodies are cytoplasmic structures that contain uridine-rich small nuclear ribonucleoproteins and associate with P bodies. Proc. Natl. Acad. Sci. USA. 2007;104:11655–11659. PubMed PMC

Makarov E. M., Makarova O. V., Urlaub H., Gentzel M., Will C. L., Wilm M., Luhrmann R. Small nuclear ribonucleoprotein remodeling during catalytic activation of the spliceosome. Science. 2002;298:2205–2208. PubMed

Makarova O. V., Makarov E. M., Liu S., Vornlocher H. P., Luhrmann R. Protein 61K, encoded by a gene (PRPF31) linked to autosomal dominant retinitis pigmentosa, is required for U4/U6center dotU5 tri-snRNP formation and pre-mRNA splicing. EMBO J. 2002;21:1148–1157. PubMed PMC

Martin A., Schneider S., Schwer B. Prp43 is an essential RNA-dependent ATPase required for release of lariat-intron from the spliceosome. J. Biol. Chem. 2002;277:17743–17750. PubMed

Matera A. G., Shpargel K. B. Pumping RNA: nuclear bodybuilding along the RNP pipeline. Curr. Opin. Cell Biol. 2006;18:317–324. PubMed

Mayes A. E., Verdone L., Legrain P., Beggs J. D. Characterization of Sm-like proteins in yeast and their association with U6 snRNA. EMBO J. 1999;18:4321–4331. PubMed PMC

Meister G., Eggert C., Fischer U. SMN-mediated assembly of RNPs: a complex story. Trends Cell Biol. 2002;12:472–478. PubMed

Narayanan U., Achsel T., Luhrmann R., Matera A. G. Coupled in vitro import of U snRNPs and SMN, the spinal muscular atrophy protein. Mol. Cell. 2004;16:223–234. PubMed

Narayanan U., Ospina J. K., Frey M. R., Hebert M. D., Matera A. G. SMN, the spinal muscular atrophy protein, forms a pre-import snRNP complex with snurportin1 and importin beta. Hum. Mol. Genet. 2002;11:1785–1795. PubMed PMC

Nesic D., Tanackovic G., Kramer A. A role for Cajal bodies in the final steps of U2 snRNP biogenesis. J. Cell Sci. 2004;117:4423–4433. PubMed

Neugebauer K. M. On the importance of being co-transcriptional. J. Cell Sci. 2002;115:3865–3871. PubMed

Ohno M., Shimura Y. A human RNA helicase-like protein, HRH1, facilitates nuclear export of spliced mRNA by releasing the RNA from the spliceosome. Genes Dev. 1996;10:997–1007. PubMed

Patterson G. H., Lippincott-Schwartz J. Selective photolabeling of proteins using photoactivatable GFP. Methods. 2004;32:445–450. PubMed

Paushkin S., Gubitz A. K., Massenet S., Dreyfuss G. The SMN complex, an assemblyosome of ribonucleoproteins. Curr. Opin. Cell Biol. 2002;14:305–312. PubMed

Raghunathan P. L., Guthrie C. A spliceosomal recycling factor that reanneals U4 and U6 small nuclear ribonucleoprotein particles. Science. 1998;279:857–860. PubMed

Raker V. A., Plessel G., Luhrmann R. The snRNP core assembly pathway: identification of stable core protein heteromeric complexes and an snRNP subcore particle in vitro. EMBO J. 1996;15:2256–2269. PubMed PMC

Schaffert N., Hossbach M., Heintzmann R., Achsel T., Luhrmann R. RNAi knockdown of hPrp31 leads to an accumulation of U4/U6 di-snRNPs in Cajal bodies. EMBO J. 2004;23:3000–3009. PubMed PMC

Shpargel K. B., Matera A. G. Gemin proteins are required for efficient assembly of Sm-class ribonucleoproteins. Proc. Natl. Acad. Sci. USA. 2005;102:17372–17377. PubMed PMC

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

Sleeman J. A regulatory role for CRM1 in the multi-directional trafficking of splicing snRNPs in the mammalian nucleus. J. Cell Sci. 2007;120:1540–1550. PubMed

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

Sleeman J. E., Lamond A. I. Newly assembled snRNPs associate with coiled bodies before speckles, suggesting a nuclear snRNP maturation pathway. Curr. Biol. 1999;9:1065–1074. PubMed

Staley J. P., Guthrie C. Mechanical devices of the spliceosome: motors, clocks, springs, and things. Cell. 1998;92:315–326. PubMed

Stanek D., Neugebauer K. M. Detection of snRNP assembly intermediates in Cajal bodies by fluorescence resonance energy transfer. J. Cell Biol. 2004;166:1015–1025. PubMed PMC

Stanek D., Neugebauer K. M. The Cajal body: a meeting place for spliceosomal snRNPs in the nuclear maze. Chromosoma. 2006;115:343–354. PubMed

Stanek D., Rader S. D., Klingauf M., Neugebauer K. M. Targeting of U4/U6 small nuclear RNP assembly factor SART3/p110 to Cajal bodies. J. Cell Biol. 2003;160:505–516. PubMed PMC

Tanaka N., Aronova A., Schwer B. Ntr1 activates the Prp43 helicase to trigger release of lariat-intron from the spliceosome. Genes Dev. 2007;21:2312–2325. PubMed PMC

Terns M. P., Terns R. M. Macromolecular complexes: SMN–the master assembler. Curr. Biol. 2001;11:R862–R864. PubMed

Terskikh A., et al. “Fluorescent timer”: protein that changes color with time. Science. 2000;290:1585–1588. PubMed

Tsai R. T., Fu R. H., Yeh F. L., Tseng C. K., Lin Y. C., Huang Y. H., Cheng S. C. Spliceosome disassembly catalyzed by Prp43 and its associated components Ntr1 and Ntr2. Genes Dev. 2005;19:2991–3003. PubMed PMC

Tsai R. T., Tseng C. K., Lee P. J., Chen H. C., Fu R. H., Chang K. J., Yeh F. L., Cheng S. C. Dynamic interactions of Ntr1-Ntr2 with Prp43 and with U5 govern the recruitment of Prp43 to mediate spliceosome disassembly. Mol. Cell. Biol. 2007;27:8027–8037. PubMed PMC

Tycowski K. T., Kolev N. G., Conrad N. K., Fok V., Steitz J. A. The ever-growing world of small nuclear ribonucleoproteins. In: Gesteland R. F., Cech T. R., Atkins J. F., editors. The RNA world. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2006. pp. 327–368.

Verdone L., Galardi S., Page D., Beggs J. D. Lsm proteins promote regeneration of pre-mRNA splicing activity. Curr. Biol. 2004;14:1487–1491. PubMed

Wang C., Meier U. T. Architecture and assembly of mammalian H/ACA small nucleolar and telomerase ribonucleoproteins. EMBO J. 2004;23:1857–1867. PubMed PMC

Wen X., Lei Y. P., Zhou Y. L., Okamoto C. T., Snead M. L., Paine M. L. Structural organization and cellular localization of tuftelin-interacting protein 11 (TFIP11) Cell Mol. Life Sci. 2005;62:1038–1046. PubMed PMC

Will C. L., Luhrmann R. Spliceosomal UsnRNP biogenesis, structure and function. Curr. Opin. Cell Biol. 2001;13:290–301. PubMed

Will C. L., Luhrmann R. Spliceosome structure and function. In: Gesteland R. F., Cech T. R., Atkins J. F., editors. The RNA world. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2006. pp. 369–400.

Yu Y. T., Sharl E. C., Smith C. M., Steitz J. A. The growing world of small nuclear ribonucleoproteins. In: Gesteland C., Atkins, editors. The RNA world. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1999. pp. 487–524.

Zhang Y., Muyrers J. P., Testa G., Stewart A. F. DNA cloning by homologous recombination in Escherichia coli. Nat. Biotechnol. 2000;18:1314–1317. PubMed

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