Abstract
The nuclear import of the spliceosomal snRNPs U1, U2, U4 and U5, is dependent on the presence of a complex nuclear localization signal (NLS). The latter is composed of the 5'-2,2,7-terminal trimethylguanosine (m3G) cap structure of the U snRNA and the Sm core domain. Here, we describe the isolation and cDNA cloning of a 45 kDa protein, termed snurportin1, which interacts specifically with m3G-cap but not m7G-cap structures. Snurportin1 enhances the m3G-capdependent nuclear import of U snRNPs in both Xenopus laevis oocytes and digitonin-permeabilized HeLa cells, demonstrating that it functions as an snRNP-specific nuclear import receptor. Interestingly, solely the m3G-cap and not the Sm core NLS appears to be recognized by snurportin1, indicating that at least two distinct import receptors interact with the complex snRNP NLS. Snurportin1 represents a novel nuclear import receptor which contains an N-terminal importin beta binding (IBB) domain, essential for function, and a C-terminal m3G-cap-binding region with no structural similarity to the arm repeat domain of importin alpha.
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- Adam S. A., Marr R. S., Gerace L. Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J Cell Biol. 1990 Sep;111(3):807–816. doi: 10.1083/jcb.111.3.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Aitchison J. D., Blobel G., Rout M. P. Kap104p: a karyopherin involved in the nuclear transport of messenger RNA binding proteins. Science. 1996 Oct 25;274(5287):624–627. doi: 10.1126/science.274.5287.624. [DOI] [PubMed] [Google Scholar]
- Bach M., Bringmann P., Lührmann R. Purification of small nuclear ribonucleoprotein particles with antibodies against modified nucleosides of small nuclear RNAs. Methods Enzymol. 1990;181:232–257. doi: 10.1016/0076-6879(90)81125-e. [DOI] [PubMed] [Google Scholar]
- Bochnig P., Reuter R., Bringmann P., Lührmann R. A monoclonal antibody against 2,2,7-trimethylguanosine that reacts with intact, class U, small nuclear ribonucleoproteins as well as with 7-methylguanosine-capped RNAs. Eur J Biochem. 1987 Oct 15;168(2):461–467. doi: 10.1111/j.1432-1033.1987.tb13439.x. [DOI] [PubMed] [Google Scholar]
- Chi N. C., Adam E. J., Adam S. A. Sequence and characterization of cytoplasmic nuclear protein import factor p97. J Cell Biol. 1995 Jul;130(2):265–274. doi: 10.1083/jcb.130.2.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cortes P., Ye Z. S., Baltimore D. RAG-1 interacts with the repeated amino acid motif of the human homologue of the yeast protein SRP1. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7633–7637. doi: 10.1073/pnas.91.16.7633. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cuomo C. A., Kirch S. A., Gyuris J., Brent R., Oettinger M. A. Rch1, a protein that specifically interacts with the RAG-1 recombination-activating protein. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6156–6160. doi: 10.1073/pnas.91.13.6156. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dickmanns A., Bischoff F. R., Marshallsay C., Lührmann R., Ponstingl H., Fanning E. The thermolability of nuclear protein import in tsBN2 cells is suppressed by microinjected Ran-GTP or Ran-GDP, but not by RanQ69L or RanT24N. J Cell Sci. 1996 Jun;109(Pt 6):1449–1457. doi: 10.1242/jcs.109.6.1449. [DOI] [PubMed] [Google Scholar]
- 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 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dingwall C., Laskey R. A. Nuclear targeting sequences--a consensus? Trends Biochem Sci. 1991 Dec;16(12):478–481. doi: 10.1016/0968-0004(91)90184-w. [DOI] [PubMed] [Google Scholar]
- Fischer U., Darzynkiewicz E., Tahara S. M., Dathan N. A., Lührmann R., Mattaj I. W. Diversity in the signals required for nuclear accumulation of U snRNPs and variety in the pathways of nuclear transport. J Cell Biol. 1991 May;113(4):705–714. doi: 10.1083/jcb.113.4.705. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fischer U., Heinrich J., van Zee K., Fanning E., Lührmann R. Nuclear transport of U1 snRNP in somatic cells: differences in signal requirement compared with Xenopus laevis oocytes. J Cell Biol. 1994 Jun;125(5):971–980. doi: 10.1083/jcb.125.5.971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fischer U., Lührmann R. An essential signaling role for the m3G cap in the transport of U1 snRNP to the nucleus. Science. 1990 Aug 17;249(4970):786–790. doi: 10.1126/science.2143847. [DOI] [PubMed] [Google Scholar]
- Fischer U., Sumpter V., Sekine M., Satoh T., Lührmann 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 Feb;12(2):573–583. doi: 10.1002/j.1460-2075.1993.tb05689.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fornerod M., van Deursen J., van Baal S., Reynolds A., Davis D., Murti K. G., Fransen J., Grosveld G. The human homologue of yeast CRM1 is in a dynamic subcomplex with CAN/Nup214 and a novel nuclear pore component Nup88. EMBO J. 1997 Feb 17;16(4):807–816. doi: 10.1093/emboj/16.4.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fridell R. A., Truant R., Thorne L., Benson R. E., Cullen B. R. Nuclear import of hnRNP A1 is mediated by a novel cellular cofactor related to karyopherin-beta. J Cell Sci. 1997 Jun;110(Pt 11):1325–1331. doi: 10.1242/jcs.110.11.1325. [DOI] [PubMed] [Google Scholar]
- Görlich D., Dabrowski M., Bischoff F. R., Kutay U., Bork P., Hartmann E., Prehn S., Izaurralde E. A novel class of RanGTP binding proteins. J Cell Biol. 1997 Jul 14;138(1):65–80. doi: 10.1083/jcb.138.1.65. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Görlich D., Henklein P., Laskey R. A., Hartmann E. A 41 amino acid motif in importin-alpha confers binding to importin-beta and hence transit into the nucleus. EMBO J. 1996 Apr 15;15(8):1810–1817. [PMC free article] [PubMed] [Google Scholar]
- Görlich D., Kostka S., Kraft R., Dingwall C., Laskey R. A., Hartmann E., Prehn S. Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope. Curr Biol. 1995 Apr 1;5(4):383–392. doi: 10.1016/s0960-9822(95)00079-0. [DOI] [PubMed] [Google Scholar]
- Görlich D., Mattaj I. W. Nucleocytoplasmic transport. Science. 1996 Mar 15;271(5255):1513–1518. doi: 10.1126/science.271.5255.1513. [DOI] [PubMed] [Google Scholar]
- Görlich D., Prehn S., Laskey R. A., Hartmann E. Isolation of a protein that is essential for the first step of nuclear protein import. Cell. 1994 Dec 2;79(5):767–778. doi: 10.1016/0092-8674(94)90067-1. [DOI] [PubMed] [Google Scholar]
- Görlich D., Vogel F., Mills A. D., Hartmann E., Laskey R. A. Distinct functions for the two importin subunits in nuclear protein import. Nature. 1995 Sep 21;377(6546):246–248. doi: 10.1038/377246a0. [DOI] [PubMed] [Google Scholar]
- 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 Aug 10;62(3):569–577. doi: 10.1016/0092-8674(90)90021-6. [DOI] [PubMed] [Google Scholar]
- Hodel A. E., Gershon P. D., Shi X., Wang S. M., Quiocho F. A. Specific protein recognition of an mRNA cap through its alkylated base. Nat Struct Biol. 1997 May;4(5):350–354. doi: 10.1038/nsb0597-350. [DOI] [PubMed] [Google Scholar]
- Imamoto N., Shimamoto T., Kose S., Takao T., Tachibana T., Matsubae M., Sekimoto T., Shimonishi Y., Yoneda Y. The nuclear pore-targeting complex binds to nuclear pores after association with a karyophile. FEBS Lett. 1995 Jul 24;368(3):415–419. doi: 10.1016/0014-5793(95)00699-a. [DOI] [PubMed] [Google Scholar]
- Iwase R., Sekine M., Tokumoto Y., Ohshima Y., Hata T. Synthesis of N2, N2, 7-trimethylguanosine cap derivatives. Nucleic Acids Res. 1989 Nov 25;17(22):8979–8989. doi: 10.1093/nar/17.22.8979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Izaurralde E., Adam S. Transport of macromolecules between the nucleus and the cytoplasm. RNA. 1998 Apr;4(4):351–364. [PMC free article] [PubMed] [Google Scholar]
- Izaurralde E., Jarmolowski A., Beisel C., Mattaj I. W., Dreyfuss G., Fischer U. A role for the M9 transport signal of hnRNP A1 in mRNA nuclear export. J Cell Biol. 1997 Apr 7;137(1):27–35. doi: 10.1083/jcb.137.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Izaurralde E., Kutay U., von Kobbe C., Mattaj I. W., Görlich D. The asymmetric distribution of the constituents of the Ran system is essential for transport into and out of the nucleus. EMBO J. 1997 Nov 3;16(21):6535–6547. doi: 10.1093/emboj/16.21.6535. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kutay U., Bischoff F. R., Kostka S., Kraft R., Görlich D. Export of importin alpha from the nucleus is mediated by a specific nuclear transport factor. Cell. 1997 Sep 19;90(6):1061–1071. doi: 10.1016/s0092-8674(00)80372-4. [DOI] [PubMed] [Google Scholar]
- Lehmeier T., Foulaki K., Lührmann R. Evidence for three distinct D proteins, which react differentially with anti-Sm autoantibodies, in the cores of the major snRNPs U1, U2, U4/U6 and U5. Nucleic Acids Res. 1990 Nov 25;18(22):6475–6484. doi: 10.1093/nar/18.22.6475. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Limbach P. A., Crain P. F., McCloskey J. A. Summary: the modified nucleosides of RNA. Nucleic Acids Res. 1994 Jun 25;22(12):2183–2196. doi: 10.1093/nar/22.12.2183. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Marcotrigiano J., Gingras A. C., Sonenberg N., Burley S. K. Cocrystal structure of the messenger RNA 5' cap-binding protein (eIF4E) bound to 7-methyl-GDP. Cell. 1997 Jun 13;89(6):951–961. doi: 10.1016/s0092-8674(00)80280-9. [DOI] [PubMed] [Google Scholar]
- Marshallsay C., Dickmanns A., Bischoff F. R., Ponstingl H., Fanning E., Lührmann R. In vitro and in vivo evidence that protein and U1 snRNP nuclear import in somatic cells differ in their requirement for GTP-hydrolysis, Ran/TC4 and RCC1. Nucleic Acids Res. 1996 May 15;24(10):1829–1836. doi: 10.1093/nar/24.10.1829. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Marshallsay C., Lührmann R. In vitro nuclear import of snRNPs: cytosolic factors mediate m3G-cap dependence of U1 and U2 snRNP transport. EMBO J. 1994 Jan 1;13(1):222–231. doi: 10.1002/j.1460-2075.1994.tb06252.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mattaj I. W. Cap trimethylation of U snRNA is cytoplasmic and dependent on U snRNP protein binding. Cell. 1986 Sep 12;46(6):905–911. doi: 10.1016/0092-8674(86)90072-3. [DOI] [PubMed] [Google Scholar]
- Mattaj I. W., De Robertis E. M. Nuclear segregation of U2 snRNA requires binding of specific snRNP proteins. Cell. 1985 Jan;40(1):111–118. doi: 10.1016/0092-8674(85)90314-9. [DOI] [PubMed] [Google Scholar]
- Melchior F., Paschal B., Evans J., Gerace L. Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor. J Cell Biol. 1993 Dec;123(6 Pt 2):1649–1659. doi: 10.1083/jcb.123.6.1649. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Michael W. M., Choi M., Dreyfuss G. A nuclear export signal in hnRNP A1: a signal-mediated, temperature-dependent nuclear protein export pathway. Cell. 1995 Nov 3;83(3):415–422. doi: 10.1016/0092-8674(95)90119-1. [DOI] [PubMed] [Google Scholar]
- Michael W. M., Eder P. S., Dreyfuss G. The K nuclear shuttling domain: a novel signal for nuclear import and nuclear export in the hnRNP K protein. EMBO J. 1997 Jun 16;16(12):3587–3598. doi: 10.1093/emboj/16.12.3587. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Michaud N., Goldfarb D. S. Multiple pathways in nuclear transport: the import of U2 snRNP occurs by a novel kinetic pathway. J Cell Biol. 1991 Jan;112(2):215–223. doi: 10.1083/jcb.112.2.215. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Michaud N., Goldfarb D. Microinjected U snRNAs are imported to oocyte nuclei via the nuclear pore complex by three distinguishable targeting pathways. J Cell Biol. 1992 Feb;116(4):851–861. doi: 10.1083/jcb.116.4.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Moore M. S., Blobel G. Purification of a Ran-interacting protein that is required for protein import into the nucleus. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):10212–10216. doi: 10.1073/pnas.91.21.10212. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Moore M. S., Blobel G. The GTP-binding protein Ran/TC4 is required for protein import into the nucleus. Nature. 1993 Oct 14;365(6447):661–663. doi: 10.1038/365661a0. [DOI] [PubMed] [Google Scholar]
- Moroianu J., Blobel G., Radu A. Previously identified protein of uncertain function is karyopherin alpha and together with karyopherin beta docks import substrate at nuclear pore complexes. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2008–2011. doi: 10.1073/pnas.92.6.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Moroianu J., Blobel G., Radu A. The binding site of karyopherin alpha for karyopherin beta overlaps with a nuclear localization sequence. Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6572–6576. doi: 10.1073/pnas.93.13.6572. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nakielny S., Siomi M. C., Siomi H., Michael W. M., Pollard V., Dreyfuss G. Transportin: nuclear transport receptor of a novel nuclear protein import pathway. Exp Cell Res. 1996 Dec 15;229(2):261–266. doi: 10.1006/excr.1996.0369. [DOI] [PubMed] [Google Scholar]
- Neuman de Vegvar H. E., Dahlberg J. E. Nucleocytoplasmic transport and processing of small nuclear RNA precursors. Mol Cell Biol. 1990 Jul;10(7):3365–3375. doi: 10.1128/mcb.10.7.3365. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nigg E. A. Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature. 1997 Apr 24;386(6627):779–787. doi: 10.1038/386779a0. [DOI] [PubMed] [Google Scholar]
- Palacios I., Hetzer M., Adam S. A., Mattaj I. W. Nuclear import of U snRNPs requires importin beta. EMBO J. 1997 Nov 17;16(22):6783–6792. doi: 10.1093/emboj/16.22.6783. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Palacios I., Weis K., Klebe C., Mattaj I. W., Dingwall C. RAN/TC4 mutants identify a common requirement for snRNP and protein import into the nucleus. J Cell Biol. 1996 May;133(3):485–494. doi: 10.1083/jcb.133.3.485. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Paschal B. M., Gerace L. Identification of NTF2, a cytosolic factor for nuclear import that interacts with nuclear pore complex protein p62. J Cell Biol. 1995 May;129(4):925–937. doi: 10.1083/jcb.129.4.925. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Plessel G., Fischer U., Lührmann R. m3G cap hypermethylation of U1 small nuclear ribonucleoprotein (snRNP) in vitro: evidence that the U1 small nuclear RNA-(guanosine-N2)-methyltransferase is a non-snRNP cytoplasmic protein that requires a binding site on the Sm core domain. Mol Cell Biol. 1994 Jun;14(6):4160–4172. doi: 10.1128/mcb.14.6.4160. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pollard V. W., Michael W. M., Nakielny S., Siomi M. C., Wang F., Dreyfuss G. A novel receptor-mediated nuclear protein import pathway. Cell. 1996 Sep 20;86(6):985–994. doi: 10.1016/s0092-8674(00)80173-7. [DOI] [PubMed] [Google Scholar]
- Powers M. A., Forbes D. J., Dahlberg J. E., Lund E. The vertebrate GLFG nucleoporin, Nup98, is an essential component of multiple RNA export pathways. J Cell Biol. 1997 Jan 27;136(2):241–250. doi: 10.1083/jcb.136.2.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Radu A., Blobel G., Moore M. S. Identification of a protein complex that is required for nuclear protein import and mediates docking of import substrate to distinct nucleoporins. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1769–1773. doi: 10.1073/pnas.92.5.1769. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Raker V. A., Plessel G., Lührmann R. The snRNP core assembly pathway: identification of stable core protein heteromeric complexes and an snRNP subcore particle in vitro. EMBO J. 1996 May 1;15(9):2256–2269. [PMC free article] [PubMed] [Google Scholar]
- Rout M. P., Blobel G., Aitchison J. D. A distinct nuclear import pathway used by ribosomal proteins. Cell. 1997 May 30;89(5):715–725. doi: 10.1016/s0092-8674(00)80254-8. [DOI] [PubMed] [Google Scholar]
- Schlenstedt G., Smirnova E., Deane R., Solsbacher J., Kutay U., Görlich D., Ponstingl H., Bischoff F. R. Yrb4p, a yeast ran-GTP-binding protein involved in import of ribosomal protein L25 into the nucleus. EMBO J. 1997 Oct 15;16(20):6237–6249. doi: 10.1093/emboj/16.20.6237. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sekine Mitsuo, Kadokura Michinori, Satoh Takahiko, Seio Kohji, Wada Takeshi, Fischer Utz, Sumpter Vicki, Lührmann Reinhard. Chemical Synthesis of a 5'-Terminal TMG-Capped Triribonucleotide m(3)(2,2,7)G(5)(')pppAmpUmpA of U1 RNA. J Org Chem. 1996 Jun 26;61(13):4412–4422. doi: 10.1021/jo952263v. [DOI] [PubMed] [Google Scholar]
- Siomi H., Dreyfuss G. A nuclear localization domain in the hnRNP A1 protein. J Cell Biol. 1995 May;129(3):551–560. doi: 10.1083/jcb.129.3.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sumpter V., Kahrs A., Fischer U., Kornstädt U., Lührmann R. In vitro reconstitution of U1 and U2 snRNPs from isolated proteins and snRNA. Mol Biol Rep. 1992 Sep;16(4):229–240. doi: 10.1007/BF00419662. [DOI] [PubMed] [Google Scholar]
- Wallace R. A., Jared D. W., Dumont J. N., Sega M. W. Protein incorporation by isolated amphibian oocytes. 3. Optimum incubation conditions. J Exp Zool. 1973 Jun;184(3):321–333. doi: 10.1002/jez.1401840305. [DOI] [PubMed] [Google Scholar]
- Weighardt F., Biamonti G., Riva S. Nucleo-cytoplasmic distribution of human hnRNP proteins: a search for the targeting domains in hnRNP A1. J Cell Sci. 1995 Feb;108(Pt 2):545–555. doi: 10.1242/jcs.108.2.545. [DOI] [PubMed] [Google Scholar]
- Weis K., Mattaj I. W., Lamond A. I. Identification of hSRP1 alpha as a functional receptor for nuclear localization sequences. Science. 1995 May 19;268(5213):1049–1053. doi: 10.1126/science.7754385. [DOI] [PubMed] [Google Scholar]
- Weis K., Ryder U., Lamond A. I. The conserved amino-terminal domain of hSRP1 alpha is essential for nuclear protein import. EMBO J. 1996 Apr 15;15(8):1818–1825. [PMC free article] [PubMed] [Google Scholar]
- Will C. L., Lührmann R. Protein functions in pre-mRNA splicing. Curr Opin Cell Biol. 1997 Jun;9(3):320–328. doi: 10.1016/s0955-0674(97)80003-8. [DOI] [PubMed] [Google Scholar]
- Yano R., Oakes M., Yamaghishi M., Dodd J. A., Nomura M. Cloning and characterization of SRP1, a suppressor of temperature-sensitive RNA polymerase I mutations, in Saccharomyces cerevisiae. Mol Cell Biol. 1992 Dec;12(12):5640–5651. doi: 10.1128/mcb.12.12.5640. [DOI] [PMC free article] [PubMed] [Google Scholar]