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. 1989 Jan;8(1):277–286. doi: 10.1002/j.1460-2075.1989.tb03374.x

Electron microscopy of U1 small nuclear ribonucleoprotein particles: shape of the particle and position of the 5' RNA terminus.

B Kastner 1, R Lührmann 1
PMCID: PMC400800  PMID: 2469573

Abstract

Small nuclear ribonucleoprotein particles U1 (U1 snRNPs) from HeLa cells were purified to homogeneity and examined in the electron microscope by negative staining with the double carbon film technique. A survey of several hundred views of the U1 snRNP particles revealed a main body about 8 nm in diameter with two characteristic protuberances 4-7 nm long and 3-4 nm wide. Anti-m3G antibodies were then used to define the site of the m3G base in the 5' cap of the U1 snRNA, close to the site where base-pairing occurs with the 5' splice sequence of pre-mRNA. The m3G base is located on the body of the U1 snRNP, close to one of the protuberances and approximately opposite the other.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bindereif A., Green M. R. Ribonucleoprotein complex formation during pre-mRNA splicing in vitro. Mol Cell Biol. 1986 Jul;6(7):2582–2592. doi: 10.1128/mcb.6.7.2582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Black D. L., Chabot B., Steitz J. A. U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing. Cell. 1985 Oct;42(3):737–750. doi: 10.1016/0092-8674(85)90270-3. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Bozzoni I., Annesi F., Beccari E., Fragapane P., Pierandrei-Amaldi P., Amaldi F. Splicing of Xenopus laevis ribosomal protein RNAs is inhibited in vivo by antisera to ribonucleoproteins containing U1 small nuclear RNA. J Mol Biol. 1984 Dec 25;180(4):1173–1178. doi: 10.1016/0022-2836(84)90276-6. [DOI] [PubMed] [Google Scholar]
  5. Branlant C., Krol A., Ebel J. P., Lazar E., Haendler B., Jacob M. U2 RNA shares a structural domain with U1, U4, and U5 RNAs. EMBO J. 1982;1(10):1259–1265. doi: 10.1002/j.1460-2075.1982.tb00022.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bringmann P., Rinke J., Appel B., Reuter R., Lührmann R. Purification of snRNPs U1, U2, U4, U5 and U6 with 2,2,7-trimethylguanosine-specific antibody and definition of their constituent proteins reacting with anti-Sm and anti-(U1)RNP antisera. EMBO J. 1983;2(7):1129–1135. doi: 10.1002/j.1460-2075.1983.tb01557.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brunel C., Widada J. S., Lelay M. N., Jeanteur P., Liautard J. P. Purification and characterization of a simple ribonucleoprotein particle containing small nucleoplasmic RNAs (snRNP) as a subset of RNP containing heterogenous nuclear RNA (hnRNP) from HeLa cells. Nucleic Acids Res. 1981 Feb 25;9(4):815–830. doi: 10.1093/nar/9.4.815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Calvet J. P., Pederson T. Base-pairing interactions between small nuclear RNAs and nuclear RNA precursors as revealed by psoralen cross-linking in vivo. Cell. 1981 Nov;26(3 Pt 1):363–370. doi: 10.1016/0092-8674(81)90205-1. [DOI] [PubMed] [Google Scholar]
  9. Chabot B., Steitz J. A. Multiple interactions between the splicing substrate and small nuclear ribonucleoproteins in spliceosomes. Mol Cell Biol. 1987 Jan;7(1):281–293. doi: 10.1128/mcb.7.1.281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Fisher D. E., Conner G. E., Reeves W. H., Wisniewolski R., Blobel G. Small nuclear ribonucleoprotein particle assembly in vivo: demonstration of a 6S RNA-free core precursor and posttranslational modification. Cell. 1985 Oct;42(3):751–758. doi: 10.1016/0092-8674(85)90271-5. [DOI] [PubMed] [Google Scholar]
  12. Fradin A., Jove R., Hemenway C., Keiser H. D., Manley J. L., Prives C. Splicing pathways of SV40 mRNAs in X. laevis oocytes differ in their requirements for snRNPs. Cell. 1984 Jul;37(3):927–936. doi: 10.1016/0092-8674(84)90427-6. [DOI] [PubMed] [Google Scholar]
  13. Hamm J., Kazmaier M., Mattaj I. W. In vitro assembly of U1 snRNPs. EMBO J. 1987 Nov;6(11):3479–3485. doi: 10.1002/j.1460-2075.1987.tb02672.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Krainer A. R., Maniatis T. Multiple factors including the small nuclear ribonucleoproteins U1 and U2 are necessary for pre-mRNA splicing in vitro. Cell. 1985 Oct;42(3):725–736. doi: 10.1016/0092-8674(85)90269-7. [DOI] [PubMed] [Google Scholar]
  15. Krämer A. Analysis of RNase-A-resistant regions of adenovirus 2 major late precursor-mRNA in splicing extracts reveals an ordered interaction of nuclear components with the substrate RNA. J Mol Biol. 1987 Aug 5;196(3):559–573. doi: 10.1016/0022-2836(87)90032-5. [DOI] [PubMed] [Google Scholar]
  16. Krämer A., Keller W., Appel B., Lührmann R. The 5' terminus of the RNA moiety of U1 small nuclear ribonucleoprotein particles is required for the splicing of messenger RNA precursors. Cell. 1984 Aug;38(1):299–307. doi: 10.1016/0092-8674(84)90551-8. [DOI] [PubMed] [Google Scholar]
  17. Lake J. A. Ribosome structure determined by electron microscopy of Escherichia coli small subunits, large subunits and monomeric ribosomes. J Mol Biol. 1976 Jul 25;105(1):131–139. doi: 10.1016/0022-2836(76)90200-x. [DOI] [PubMed] [Google Scholar]
  18. Lerner M. R., Boyle J. A., Mount S. M., Wolin S. L., Steitz J. A. Are snRNPs involved in splicing? Nature. 1980 Jan 10;283(5743):220–224. doi: 10.1038/283220a0. [DOI] [PubMed] [Google Scholar]
  19. Liautard J. P., Sri-Widada J., Brunel C., Jeanteur P. Structural organization of ribonucleoproteins containing small nuclear RNAs from HeLa cells. Proteins interact closely with a similar structural domain of U1, U2, U4 and U5 small nuclear RNAs. J Mol Biol. 1982 Dec 15;162(3):623–643. doi: 10.1016/0022-2836(82)90392-8. [DOI] [PubMed] [Google Scholar]
  20. Maniatis T., Reed R. The role of small nuclear ribonucleoprotein particles in pre-mRNA splicing. Nature. 1987 Feb 19;325(6106):673–678. doi: 10.1038/325673a0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Mount S. M., Pettersson I., Hinterberger M., Karmas A., Steitz J. A. The U1 small nuclear RNA-protein complex selectively binds a 5' splice site in vitro. Cell. 1983 Jun;33(2):509–518. doi: 10.1016/0092-8674(83)90432-4. [DOI] [PubMed] [Google Scholar]
  23. Padgett R. A., Mount S. M., Steitz J. A., Sharp P. A. Splicing of messenger RNA precursors is inhibited by antisera to small nuclear ribonucleoprotein. Cell. 1983 Nov;35(1):101–107. doi: 10.1016/0092-8674(83)90212-x. [DOI] [PubMed] [Google Scholar]
  24. Patton J. R., Pederson T. The Mr 70,000 protein of the U1 small nuclear ribonucleoprotein particle binds to the 5' stem-loop of U1 RNA and interacts with Sm domain proteins. Proc Natl Acad Sci U S A. 1988 Feb;85(3):747–751. doi: 10.1073/pnas.85.3.747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Reed R., Griffith J., Maniatis T. Purification and visualization of native spliceosomes. Cell. 1988 Jun 17;53(6):949–961. doi: 10.1016/s0092-8674(88)90489-8. [DOI] [PubMed] [Google Scholar]
  26. Rinke J., Appel B., Blöcker H., Frank R., Lührmann R. The 5'-terminal sequence of U1 RNA complementary to the consensus 5' splice site of hnRNA is single-stranded in intact U1 snRNP particles. Nucleic Acids Res. 1984 May 25;12(10):4111–4126. doi: 10.1093/nar/12.10.4111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rogers J., Wall R. A mechanism for RNA splicing. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1877–1879. doi: 10.1073/pnas.77.4.1877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sarma V. R., Silverton E. W., Davies D. R., Terry W. D. The three-dimensional structure at 6 A resolution of a human gamma Gl immunoglobulin molecule. J Biol Chem. 1971 Jun 10;246(11):3753–3759. [PubMed] [Google Scholar]
  29. Sharp P. A. Splicing of messenger RNA precursors. Science. 1987 Feb 13;235(4790):766–771. doi: 10.1126/science.3544217. [DOI] [PubMed] [Google Scholar]
  30. Spector D. L., Schrier W. H., Busch H. Immunoelectron microscopic localization of snRNPs. Biol Cell. 1983;49(1):1–10. doi: 10.1111/j.1768-322x.1984.tb00215.x. [DOI] [PubMed] [Google Scholar]
  31. Strycharz W. A., Nomura M., Lake J. A. Ribosomal proteins L7/L12 localized at a single region of the large subunit by immune electron microscopy. J Mol Biol. 1978 Dec 5;126(2):123–140. doi: 10.1016/0022-2836(78)90355-8. [DOI] [PubMed] [Google Scholar]
  32. Stöffler G., Stöffler-Meilicke M. Immunoelectron microscopy of ribosomes. Annu Rev Biophys Bioeng. 1984;13:303–330. doi: 10.1146/annurev.bb.13.060184.001511. [DOI] [PubMed] [Google Scholar]
  33. Tischendorf G. W., Zeichhardt H., Stöffler G. Determination of the location of proteins L14, L17, L18, L19, L22, L23 on the surface of the 5oS ribosomal subunit of Escherichia coli by immune electron microscopy. Mol Gen Genet. 1974;134(3):187–208. doi: 10.1007/BF00267715. [DOI] [PubMed] [Google Scholar]
  34. Zhuang Y., Weiner A. M. A compensatory base change in U1 snRNA suppresses a 5' splice site mutation. Cell. 1986 Sep 12;46(6):827–835. doi: 10.1016/0092-8674(86)90064-4. [DOI] [PubMed] [Google Scholar]
  35. Zillmann M., Rose S. D., Berget S. M. U1 small nuclear ribonucleoproteins are required early during spliceosome assembly. Mol Cell Biol. 1987 Aug;7(8):2877–2883. doi: 10.1128/mcb.7.8.2877. [DOI] [PMC free article] [PubMed] [Google Scholar]

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