Cajal bodies (CBs) are nuclear non-membrane bound organelles where small nuclear ribonucleoprotein particles (snRNPs) undergo their final maturation and quality control before they are released to the nucleoplasm. However, the molecular mechanism how immature snRNPs are targeted and retained in CBs has yet to be described. Here, we microinjected and expressed various snRNA deletion mutants as well as chimeric 7SK, Alu or bacterial SRP non-coding RNAs and provide evidence that Sm and SMN binding sites are necessary and sufficient for CB localization of snRNAs. We further show that Sm proteins, and specifically their GR-rich domains, are important for accumulating snRNPs in CBs. Accordingly, core snRNPs containing the Sm proteins, but not naked snRNAs, restore the formation of CBs after their depletion. Finally, we show that immature but not fully assembled snRNPs are able to induce CB formation and that microinjection of an excess of U2 snRNP-specific proteins, which promotes U2 snRNP maturation, chases U2 snRNA from CBs. We propose that the accessibility of the Sm ring represents the molecular basis for the quality control of the final maturation of snRNPs and the sequestration of immature particles in CBs.

Institute of Microbiology Czech Academy of Sciences Prague Czech Republic

Institute of Molecular Genetics Czech Academy of Sciences Prague Czech Republic

Max Planck Institute for Biophysical Chemistry Göttingen Germany

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Pellizzoni L., Charroux B., Dreyfuss G.. SMN mutants of spinal muscular atrophy patients are defective in binding to snRNP proteins. Proc. Natl. Acad. Sci. U.S.A. 1999; 96:11167–11172. PubMed PMC

Eggert C., Chari A., Laggerbauer B., Fischer U.. Spinal muscular atrophy: the RNP connection. Trends Mol. Med. 2006; 12:113–121. PubMed

Ruzickova S., Stanek D.. Mutations in spliceosomal proteins and retina degeneration. RNA Biol. 2017; 14:544–552. PubMed PMC

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

Gruss O.J., Meduri R., Schilling M., Fischer U.. UsnRNP biogenesis: mechanisms and regulation. Chromosoma. 2017; 126:577–593. PubMed

Massenet S., Pellizzoni L., Paushkin S., Mattaj I.W., Dreyfuss G.. The SMN complex is associated with snRNPs throughout their cytoplasmic assembly pathway. Mol. Cell. Biol. 2002; 22:6533–6541. PubMed PMC

Jin W., Wang Y., Liu C.P., Yang N., Jin M., Cong Y., Wang M., Xu R.M.. Structural basis for snRNA recognition by the double-WD40 repeat domain of Gemin5. Genes Dev. 2016; 30:2391–2403. PubMed PMC

Xu C., Ishikawa H., Izumikawa K., Li L., He H., Nobe Y., Yamauchi Y., Shahjee H.M., Wu X.H., Yu Y.T. et al. . Structural insights into Gemin5-guided selection of pre-snRNAs for snRNP assembly. Genes Dev. 2016; 30:2376–2390. PubMed PMC

Golembe T.J., Yong J., Dreyfuss G.. Specific sequence features, recognized by the SMN complex, identify snRNAs and determine their fate as snRNPs. Mol. Cell. Biol. 2005; 25:10989–11004. PubMed PMC

Yong J., Kasim M., Bachorik J.L., Wan L., Dreyfuss G.. Gemin5 delivers snRNA precursors to the SMN complex for snRNP biogenesis. Mol. Cell. 2010; 38:551–562. PubMed PMC

Chari A., Golas M.M., Klingenhager M., Neuenkirchen N., Sander B., Englbrecht C., Sickmann A., Stark H., Fischer U.. An assembly chaperone collaborates with the SMN complex to generate spliceosomal SnRNPs. Cell. 2008; 135:497–509. PubMed

Grimm C., Chari A., Pelz J.P., Kuper J., Kisker C., Diederichs K., Stark H., Schindelin H., Fischer U.. Structural basis of assembly chaperone-mediated snRNP formation. Mol. Cell. 2013; 49:692–703. PubMed

Fischer U., Englbrecht C., Chari A.. Biogenesis of spliceosomal small nuclear ribonucleoproteins. Wiley Interdiscipl. Rev. RNA. 2011; 2:718–731. PubMed

Mattaj I.W. Cap trimethylation of U snRNA is cytoplasmic and dependent on U snRNP protein binding. Cell. 1986; 46:905–911. PubMed

Matera A.G., Wang Z.. A day in the life of the spliceosome. Nat. Rev. Mol. Cell Biol. 2014; 15:108–121. PubMed PMC

Hamm J., Darzynkiewicz E., Tahara S.M., Mattaj I.W.. The trimethylguanosine cap structure of U1 snRNA is a component of a bipartite nuclear targeting signal. Cell. 1990; 62:569–577. PubMed

Narayanan U., Achsel T., Lührmann 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

Raimer A.C., Gray K.M., Matera A.G.. SMN - A chaperone for nuclear RNP social occasions?. RNA Biol. 2016; 14:1–11. PubMed PMC

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

Ospina J.K., Gonsalvez G.B., Bednenko J., Darzynkiewicz E., Gerace L., Matera A.G.. Cross-talk between snurportin1 subdomains. Mol. Biol. Cell. 2005; 16:4660–4671. 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

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., 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

Nesic D., Kramer A.. Domains in human splicing factors SF3a60 and SF3a66 required for binding to SF3a120, assembly of the 17S U2 snRNP, and prespliceosome formation. Mol. Cell. Biol. 2001; 21:6406–6417. PubMed PMC

Novotny I., Blazikova M., Stanek D., Herman P., Malinsky J.. In vivo kinetics of U4/U6.U5 tri-snRNP formation in Cajal bodies. Mol. Biol. Cell. 2011; 22:513–523. PubMed PMC

Will C.L., Urlaub H., Achsel T., Gentzel M., Wilm M., Luhrmann R.. Characterization of novel SF3b and 17S U2 snRNP proteins, including a human Prp5p homologue and an SF3b DEAD-box protein. EMBO J. 2002; 21:4978–4988. PubMed PMC

Darzacq X., Jady B.E., Verheggen C., Kiss A.M., Bertrand E., Kiss T.. Cajal body-speci c small nuclear RNAs: a novel class of 2 0 -O-methylation and pseudouridylation guide RNAs. EMBO J. 2002; 21:2746–2756. 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

Stanek D. Cajal bodies and snRNPs - friends with benefits. RNA Biol. 2017; 14:671–679. PubMed PMC

Malinova A., Cvackova Z., Mateju D., Horejsi Z., Abeza C., Vandermoere F., Bertrand E., Stanek D., Verheggen C.. Assembly of the U5 snRNP component PRPF8 is controlled by the HSP90/R2TP chaperones. J. Cell Biol. 2017; 216:1579–1596. PubMed PMC

Bizarro J., Dodre M., Huttin A., Charpentier B., Schlotter F., Branlant C., Verheggen C., Massenet S., Bertrand E.. NUFIP and the HSP90/R2TP chaperone bind the SMN complex and facilitate assembly of U4-specific proteins. Nucleic Acids Res. 2015; 43:8973–8989. PubMed PMC

Cloutier P., Poitras C., Durand M., Hekmat O., Fiola-Masson E., Bouchard A., Faubert D., Chabot B., Coulombe B.. R2TP/Prefoldin-like component RUVBL1/RUVBL2 directly interacts with ZNHIT2 to regulate assembly of U5 small nuclear ribonucleoprotein. Nat. Commun. 2017; 8:15615. PubMed PMC

Novotny I., Malinova A., Stejskalova E., Mateju D., Klimesova K., Roithova A., Sveda M., Knejzlik Z., Stanek D.. SART3-dependent accumulation of incomplete spliceosomal snRNPs in Cajal bodies. Cell Rep. 2015; 10:429–440. PubMed

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

Hnilicova J., Jirat Matejckova J., Sikova M., Pospisil J., Halada P., Panek J., Krasny L.. Ms1, a novel sRNA interacting with the RNA polymerase core in mycobacteria. Nucleic Acids Res. 2014; 42:11763–11776. PubMed PMC

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

Lorenz R., Bernhart S.H., Honer Zu Siederdissen C., Tafer H., Flamm C., Stadler P.F., Hofacker I.L.. ViennaRNA Package 2.0. Algorithms Mol. Biol. 2011; 6:26. PubMed PMC

Huranova M., Hnilicova J., Fleischer B., Cvackova Z., Stanek D.. A mutation linked to retinitis pigmentosa in HPRP31 causes protein instability and impairs its interactions with spliceosomal snRNPs. Hum. Mol. Genet. 2009; 18:2014–2023. PubMed

Sumpter V., Kahrs A., Fischer U., Kornstadt U., Luhrmann R.. Invitro reconstitution of U1 and U2 Snrnps from isolated proteins and Snrna. Mol. Biol. Rep. 1992; 16:229–240. PubMed

Segault V., Will C.L., Sproat B.S., Luhrmann R.. In vitro reconstitution of mammalian U2 and U5 snRNPs active in splicing: Sm proteins are functionally interchangeable and are essential for the formation of functional U2 and U5 snRNPs. EMBO J. 1995; 14:4010–4021. PubMed PMC

Malatesta M., Fakan S., Fischer U.. The Sm core domain mediates targeting of U1 snRNP to subnuclear compartments involved in transcription and splicing. Exp. Cell Res. 1999; 249:189–198. PubMed

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

Brosi R., Groning K., Behrens S.E., Luhrmann R., Kramer A.. Interaction of mammalian splicing factor SF3a with U2 snRNP and relation of its 60-kD subunit to yeast PRP9. Science. 1993; 262:102–105. PubMed

Boelens W., Scherly D., Beijer R.P., Jansen E.J., Dathan N.A., Mattaj I.W., van Venrooij W.J.. A weak interaction between the U2A' protein and U2 snRNA helps to stabilize their complex with the U2B" protein. Nucleic Acids Res. 1991; 19:455–460. PubMed PMC

Williams S.G., Hall K.B.. Human U2B″ protein binding to snRNA stemloops. Biophys. Chem. 2011; 159:82–89. PubMed PMC

Yong J., Golembe T.J., Battle D.J., Pellizzoni L., Dreyfuss G.. snRNAs contain specific SMN-binding domains that are essential for snRNP assembly. Mol. Cell. Biol. 2004; 24:2747–2756. PubMed PMC

Battle D.J., Kasim M., Yong J., Lotti F., Lau C.K., Mouaikel J., Zhang Z., Han K., Wan L., Dreyfuss G.. The SMN complex: an assembly machine for RNPs. Cold Spring Harb. Symp. Quant. Biol. 2006; 71:313–320. PubMed

Carmo-Fonseca M., Tollervey D., Pepperkok R., Barabino S.M., Merdes A., Brunner C., Zamore P.D., Green M.R., Hurt E., Lamond A.I.. Mammalian nuclei contain foci which are highly enriched in components of the pre-mRNA splicing machinery. EMBO J. 1991; 10:195–206. PubMed PMC

Matera A.G., Ward D.C.. Nucleoplasmic organization of small nuclear ribonucleoproteins in cultured human cells. J. Cell Biol. 1993; 121:715–727. PubMed PMC

Stejskalova E., Stanek D.. The splicing factor U1-70K interacts with the SMN complex and is required for nuclear gem integrity. J. Cell Sci. 2014; 127:3909–3915. PubMed

Tanackovic G., Kramer A.. Human Splicing Factor SF3a, but Not SF1, Is Essential for Pre-mRNA Splicing In Vivo. Mol. Biol. Cell. 2005; 16:1366–1377. PubMed PMC

So B.R., Wan L., Zhang Z., Li P., Babiash E., Duan J., Younis I., Dreyfuss G.. A U1 snRNP-specific assembly pathway reveals the SMN complex as a versatile hub for RNP exchange. Nat. Struct. Mol. Biol. 2016; 23:225–230. PubMed PMC

Peterlin B.M., Brogie J.E., Price D.H.. 7SK snRNA: a noncoding RNA that plays a major role in regulating eukaryotic transcription. Wiley Interdiscipl. Rev. RNA. 2012; 3:92–103. PubMed PMC

Golembe T.J., Yong J., Battle D.J., Feng W., Wan L., Dreyfuss G.. Lymphotropic Herpesvirus saimiri uses the SMN complex to assemble Sm cores on its small RNAs. Mol. Cell. Biol. 2005; 25:602–611. PubMed PMC

Maraia R.J., Driscoll C.T., Bilyeu T., Hsu K., Darlington G.J.. Multiple dispersed loci produce small cytoplasmic Alu RNA. Mol. Cell. Biol. 1993; 13:4233–4241. PubMed PMC

Ivanova E., Berger A., Scherrer A., Alkalaeva E., Strub K.. Alu RNA regulates the cellular pool of active ribosomes by targeted delivery of SRP9/14 to 40S subunits. Nucleic Acids Res. 2015; 43:2874–2887. PubMed PMC

Hallais M., Pontvianne F., Andersen P.R., Clerici M., Lener D., Benbahouche Nel H., Gostan T., Vandermoere F., Robert M.C., Cusack S. et al. . CBC-ARS2 stimulates 3′-end maturation of multiple RNA families and favors cap-proximal processing. Nat. Struct. Mol. Biol. 2013; 20:1358–1366. PubMed

Yong J., Wan L., Dreyfuss G.. Why do cells need an assembly machine for RNA-protein complexes?. Trends Cell Biol. 2004; 14:226–232. PubMed

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

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

Friesen W.J., Paushkin S., Wyce A., Massenet S., Pesiridis G.S., Van Duyne G., Rappsilber J., Mann M., Dreyfuss G.. The methylosome, a 20S complex containing JBP1 and pICln, produces dimethylarginine-modified Sm proteins. Mol. Cell. Biol. 2001; 21:8289–8300. PubMed PMC

Mouaikel J., Verheggen C., Bertrand E., Tazi J., Bordonne R.. Hypermethylation of the cap structure of both yeast snRNAs and snoRNAs requires a conserved methyltransferase that is localized to the nucleolus. Mol. Cell. 2002; 9:891–901. PubMed

Fischer U., Sumpter V., Sekine M., Satoh T., Luhrmann R.. Nucleo-cytoplasmic transport of U snRNPs: definition of a nuclear location signal in the Sm core domain that binds a transport receptor independently of the m3G cap. EMBO J. 1993; 12:573–583. PubMed PMC

Girard C., Mouaikel J., Neel H., Bertrand E., Bordonne R.. Nuclear localization properties of a conserved protuberance in the Sm core complex. Exp. Cell Res. 2004; 299:199–208. PubMed

Heyn P., Salmonowicz H., Rodenfels J., Neugebauer K.M.. Activation of transcription enforces the formation of distinct nuclear bodies in zebrafish embryos. RNA Biol. 2017; 14:752–760. PubMed PMC

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

Hebert M.D., Szymczyk P.W., Shpargel K.B., Matera A.G.. Coilin forms the bridge between Cajal bodies and SMN, the spinal muscular atrophy protein. Genes Dev. 2001; 15:2720–2729. PubMed PMC

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

Toyota C.G., Davis M.D., Cosman A.M., Hebert M.D.. Coilin phosphorylation mediates interaction with SMN and SmB′. Chromosoma. 2010; 119:205–215. PubMed PMC

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

Pek J.W., Anand A., Kai T.. Tudor domain proteins in development. Development. 2012; 139:2255–2266. PubMed

Machyna M., Kehr S., Straube K., Kappei D., Buchholz F., Butter F., Ule J., Hertel J., Stadler P., Neugebauer K.M.. Global identification of coilin binding partners reveals hundreds of small non-coding RNAs that traffic through Cajal bodies. Mol. Cell. 2014; 56:389–399. PubMed

Broome H.J., Hebert M.D.. Coilin displays differential affinity for specific RNAs in vivo and is linked to telomerase RNA biogenesis. J. Mol. Biol. 2013; 425:713–724. PubMed PMC

Kramer A., Gruter P., Groning K., Kastner B.. Combined biochemical and electron microscopic analyses reveal the architecture of the mammalian U2 snRNP. J. Cell Biol. 1999; 145:1355–1368. PubMed PMC

Nguyen T.H.D., Galej W.P., Bai X.-C., Oubridge C., Newman A.J., Scheres S.H.W., Nagai K.. Cryo-EM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 A resolution. Nature. 2016; 530:298–302. PubMed PMC

Agafonov D.E., Kastner B., Dybkov O., Hofele R.V., Liu W.T., Urlaub H., Luhrmann R., Stark H.. Molecular architecture of the human U4/U6.U5 tri-snRNP. Science. 2016; 351:1416–1420. PubMed

Pomeranz Krummel D.A., Oubridge C., Leung A.K., Li J., Nagai K.. Crystal structure of human spliceosomal U1 snRNP at 5.5 A resolution. Nature. 2009; 458:475–480. PubMed PMC

Weber G., Trowitzsch S., Kastner B., Luhrmann R., Wahl M.C.. Functional organization of the Sm core in the crystal structure of human U1 snRNP. EMBO J. 2010; 29:4172–4184. PubMed PMC

De novo and inherited dominant variants in U4 and U6 snRNAs cause retinitis pigmentosa

Coilin and Cajal bodies

The SMN complex drives structural changes in human snRNAs to enable snRNP assembly

DIS3L2 and LSm proteins are involved in the surveillance of Sm ring-deficient snRNAs