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Purification and Electron Microscopy of Spliceosomal snRNPs

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Book cover RNP Particles, Splicing and Autoimmune Diseases

Part of the book series: Springer Lab Manual ((SLM))

Abstract

The main focus of this chapter is electron microscopy (EM) of RNP complexes using the negative staining technique. This technique is fast and relatively easy to perform and is adequate for imaging with a standard transmission electron microscope. The prerequisite for EM analysis is the availability of isolated intact RNP particles with a concentration not less than 10–20 μg/ml. The lower size limit of the particles that can be studied by the method depends very much on their actual shape. This chapter deals with the spliceosomal RNP subunits, called small nuclear ribonucleoproteins (snRNPs). Because of their sizes, which range from about 300 kDa (U1snRNP) to up to 1500 kDa (U4/U6.U5 tri-snRNP), the snRNPs are ideal objects for EM analysis. However, snRNP protein sub complexes as small as ~60 kDa have also been investigated successfully by negative staining EM. EM analysis is greatly facilitated if the particles studied are available as highly purified samples. Therefore, isolation procedures for snRNPs are described here in some detail.

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References

  • Ast G, Weiner AM (1996) A U1/U4/U5 snRNP complex induced by a 2’-Omethyl-oliogoribonucleotide complementary to U5 snRNA. Science 272: 881–884

    Article  PubMed  CAS  Google Scholar 

  • Bach M, Winkelmann G, Lührmann R (1989) 20S small nuclear ribonucleoprotein U5 shows a surprisingly complex protein composition. Proc Natl Acad Sci USA 86: 6038–6042

    Google Scholar 

  • Bach M, Bringmann P, Lührmann R (1990a) Purification of small nuclear ribonucleoprotein particles with antibodies against modified nucleosides of small nuclear RNAs. Methods Enzymol 181: 232–257

    Article  PubMed  CAS  Google Scholar 

  • Bach M, Krol A, Lührmann R (1990b) Structure-probing of U1 snRNPs gradually depleted of the U1-specific proteins A, C and 70k. Evidence that A interacts differentially with developmentally regulated mouse U1 snRNA variants. Nucleic Acids Res 18: 449–457

    Article  PubMed  CAS  Google Scholar 

  • Bearden JC Jr (1978) Quantitation of submicrogramm quantities of protein by an improved protein-dye binding assay. Biochim Biophys Acta 533: 525–529

    Article  PubMed  CAS  Google Scholar 

  • Behrens SE, Tyc K, Kastner B, Reichelt J, Lührmann R (1993) Small nuclear ribonucleoprotein (RNP) U2 contains numerous additional proteins and has a bipartite RNP structure under splicing conditions. Mol Cell Biol 13: 07–319

    Google Scholar 

  • Behrens SE, Kastner B, Lührmann R (1994) Preparation of U small nuclear ribonucleoprotein particles In: Celis JE (ed) Cell biology–a laboratory handbook, vol 1. Academic Press, London, pp 628–639

    Google Scholar 

  • Behrens S-E, Lührmann R(1991) Immunoaffinity purification of a [U4/ U6U5] tri-snRNP from human cells. Genes Dev 5: 1439–1452

    Google Scholar 

  • Bennett M, Michaud S, Kingston J, Reed R (1992) Protein components specifically associated with prespliceosome and spliceosome complexes. Genes Dev 6: 1986–2000

    Article  PubMed  CAS  Google Scholar 

  • Bergmann U, Wittmann-Liebold B (1990) Use of a hapten specific antidansyl antibody for the localization of ribosomal proteins by immuno electron microscopy. Biochem Int 21: 741–751

    PubMed  CAS  Google Scholar 

  • Bochnig P, Reuter R, Bringmann P, Lührmann R (1987) A monoclonal antibody against 2,2,7-trimethylguanosine that reacts with intact U1 snRNPs as well as with 7-methylguanosine-capped RNAs. Eur J Biochem 168: 461–467

    Article  PubMed  CAS  Google Scholar 

  • Boublik M (1990) Electron microscopy of ribosomes In: Spedding G (ed) Ribosomes and protein synthesis–a practical approach. IRL, Oxford, pp 273–296

    Google Scholar 

  • Bremer A, Henn C, Engel A, Baumeister W, Aebi U (1992) Has negative staining still a place in biomacromolecular electron microscopy? Ultra-microscopy 46: 85–111

    CAS  Google Scholar 

  • Bringmann P, Rinke J, Appel B, Reuter R, Lührmann R (1983) 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 2: 1129–1135

    Google Scholar 

  • Bringmann P, Lührmann R (1986) Purification of the individual snRNPs U1, U2, U5 and U4/U6 from HeLa cells and characterization of their protein constituents. EMBO J 5: 3509–3516

    CAS  Google Scholar 

  • Brosi R, Gröning K, Behrens S-E, Lührmann R, Krämer A (1993a) Interaction of mammalian splicing factor SF3a with U2 snRNP and relation of its 60-kD subunit to yeast PRP9. Science 262: 102–105

    Article  PubMed  CAS  Google Scholar 

  • Brosi R, Hauri H-P, Krämer A (1993b) Separation of splicing factor SF3 into two components and purification of SF3a activity. J Biol Chem 268: 17640–17646

    PubMed  CAS  Google Scholar 

  • Dignam JD, Lebovitz RM, Roeder RG (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11: 1475–1489

    Article  PubMed  CAS  Google Scholar 

  • Fabrizio P, Esser S, Kastner B, Lührmann R (1994) Isolation of S. cerevisiae snRNPs:comparison of U1 and U4/U6U5 to their human counterparts. Science 264: 261–265

    Article  PubMed  CAS  Google Scholar 

  • Frank J, Radermacher M, Wagenknecht T, Verschoor A (1988) Studying ribosome structure by electron microscopy and computer-image processing. In: Noller HF, Moldave K (eds) Methods in enzymology, vol 164. Academic Press, San Diego, pp 3–35

    Google Scholar 

  • Frank, J (1995) Approaches to large-scale structures. Curr Opin Struct Biol 5: 194–201

    Article  PubMed  CAS  Google Scholar 

  • Frank J, Zhu J, Penczek P, Li Y, Srivastava S, Verschoor A, Radermacher M, Grassucci R, Lata RK, Agrawal RK (1995) A model of protein synthesis based on cryo-electron microscopy of the E. coli ribosome. Nature 376: 441–444

    Article  PubMed  CAS  Google Scholar 

  • Frendeway D, Keller W (1985) Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and specific intron sequences. Cell 42: 355–367

    Article  Google Scholar 

  • Fukami A, Adachi K (1965) A new method of preparation of a self-perforated micro-plastic grid and its application. J Elec Microsc (Tokyo) 14: 112–118

    CAS  Google Scholar 

  • Furman E, Glitz DG (1995) Purification of the spliceosome A-complex and its visualization by electron microscopy. J Biol Chem 270: 15515–15522

    Article  PubMed  CAS  Google Scholar 

  • Glitz DG, Cann PA, Lasater LS, Olson HM (1988) Antibody probes of ribo-somal RNA. Methods Enzymol 164: 493–502

    Article  PubMed  CAS  Google Scholar 

  • Grabowski PJ, Seiler SR, Sharp PA (1985) A multi-component complex is involved in the splicing of messengerRNA precursor. Cell 42: 345–353

    Article  PubMed  CAS  Google Scholar 

  • Gröning K, Krämer A, Kastner B (1997) (in preparation)

    Google Scholar 

  • Guthrie C, Patterson B (1988) Spliceosomal snRNAs. Annu Rev Genet 22: 387–419

    Article  PubMed  CAS  Google Scholar 

  • Harauz G, Boekema E, Van Heel M (1988) Statistical image analysis of electron micrographs of ribosomal subunits. In: Noller HF, Moldave K (eds) Methods in enzymology, vol 164. Academic Press, San Diego, pp 35–49

    Google Scholar 

  • Hoet RM, Kastner B, Lührmann R, Van Venrooij WJ (1993) Purification and characterization of human autoantibodies directed to specific regions on U1RNA; recognition of native U1RNP complexes. Nucleic Acids Res 21: 5130–5136

    Article  PubMed  CAS  Google Scholar 

  • Jahn W (1995) Easily prepared holey films for use in cyro-electron microscopy. J Microsc 179: 333–334

    Article  Google Scholar 

  • Kastner B, Stöffler-Meilicke M, Stöffler G (1981) Arrangement of the subunits in the ribosome of Escherichia coli: demonstration by immunoelectron microscopy. Proc Natl Acad Sci USA 78: 6652–6656

    Article  PubMed  CAS  Google Scholar 

  • Kastner B, Bach M, Lührmann R (1990) Electron microscopy of small nuclear ribonucleoprotein (snRNP) particles U2 and U5: evidence for a common structure-determining principle in the major U snRNP family. Proc Natl Acad Sci USA 87: 1710–1714

    Article  PubMed  CAS  Google Scholar 

  • Kastner B, Bach M, Lührmann R (1991) Electron microscopy of U4/U6 snRNP reveals a Y-shaped U4 and U6 RNA containing domain protruding from the U4 core RNP. J Cell Biol 112: 1065–1072

    Article  PubMed  CAS  Google Scholar 

  • Kastner B, Kornstädt U, Bach M, Lührmann R (1992) Structure of the small nuclear RNP particle U1: identification of the two structural protuberances with RNP-antigens A and 70K. J Cell Biol 116: 839–849

    Article  PubMed  CAS  Google Scholar 

  • Kastner B, Lührmann R (1989) Electron microscopy of U1 small nuclear ribonucleoprotein particles: Shape of the particle and position of the 5’ RNA terminus. EMBO J 8: 277–286

    Google Scholar 

  • Krämer A (1990) Purification of small nuclear ribonucleoprotein particles active in RNA processing. Methods Enzymol 181: 215–231

    Article  PubMed  Google Scholar 

  • Krämer A (1992) Purification of splicing factor SF1, a heat-stable protein that functions in the assembly of a presplicing complex. Mol Cell Biol 12: 4545–4552

    PubMed  Google Scholar 

  • Krämer A, Utans U (1991) Three protein factors (SF1, SF3 and U2AF) function in pre-splicing complex formation in addition to snRNPs. EMBO J 10: 1503–1509

    Google Scholar 

  • Lake JA (1976) Ribosome structure determined by electron microscopy of Escherichia coli small subunits, large subunits and monomeric ribosomes. J Mol Biol 105: 131–139

    Article  PubMed  CAS  Google Scholar 

  • Lake JA (1978) Electron microscopy of specific proteins. Three dimensional mapping of ribosomal proteins using antibody labels. In: Koehler JK (ed) Advanced techniques in biological electron microscopy II. Springer, Berlin Heidelberg New York, pp 173–211

    Chapter  Google Scholar 

  • Lake JA (1982) Ribosomal subunit orientations determined in the monomeric ribosome by single and by double-labeling immune electron microscopy. J Mol Biol 161: 89–106

    Article  PubMed  CAS  Google Scholar 

  • Lamond AI, Sproat BS (1993) Isolation and characterization of ribonucleoprotein complexes In: Higgins SJ, Hames BD (eds) RNA processing–a practical approach, vol I. IRL, Oxford, pp 103–140

    Google Scholar 

  • Lomant AJ, Fairbanks G (1976) Chemical probes of extended biological structures: synthesis and properties of the cleavable protein cross-linking reagent [35S] dithiobis(succinimidyl propionat). J Mol Biol 104: 243–261

    Article  PubMed  CAS  Google Scholar 

  • Lührmann R, Appel B, Rinke J, Reuter R, Rothe S, Bald R (1982) Isolation and characterization of rabbit anti-m2’2’7 G antibodies. Nucleic Acids Res 10: 7103–7113

    Article  PubMed  Google Scholar 

  • Lührmann R, Kastner B, Bach M (1990) Structure of spliceosomal snRNPs and their role in pre-mRNA splicing. Biochim Biophys Acta 1087: 265–292

    Article  PubMed  Google Scholar 

  • Lünsdorf H, Spiess E (1986) A rapid method of preparing perforated supporting foils for thin carbon films used in high resolution transmission electron microscopy. J Microsc 144: 211–213

    Article  Google Scholar 

  • Madhani HD, Guthrie C (1994) Dynamic RNA-RNA interactions in the spliceosome. Annu Rev Genet 28: 1–26

    Article  PubMed  CAS  Google Scholar 

  • Miriami E, Angenitzki M, Sperling R, Sperling J (1995) Magnesium cations are required for the association of U small nuclear ribonucleoproteins and SR proteins with pre-mRNA in 200 S large nuclear ribonucleoprotein particles. J Mol Biol 246: 254–263

    Article  PubMed  CAS  Google Scholar 

  • Montesano-Roditis L, McWilliams R, Glitz DG, Olah TV, Perrault AR, Cooperman BS (1993) Placement of dinitrophenyl-modified ribosomal proteins in totally reconstituted Escherichia coli 30 S subunits. Localization of proteins S6, S13, S16, and S18 by immune electron microscopy. J Biol Chem 268: 18701–18709

    PubMed  CAS  Google Scholar 

  • Moore MJ, Query CC, Sharp PA (1993) Splicing of precursors to mRNA by the spliceosome In: Gesteland RF, Atkins JF (eds) The RNA world. Cold Spring Harbor Lab, Cold Spring Harbor, New York, pp 303–357

    Google Scholar 

  • Newman A (1994) Small nuclear RNAs and pre-mRNA splicing. Curr Opin Cell Biol 6: 360–367

    Article  PubMed  CAS  Google Scholar 

  • Nilsen TW (1994) RNA-RNA interactions in the spliceosome: Unraveling the ties that bind. Cell 78: 1–4

    Article  PubMed  CAS  Google Scholar 

  • Oakes MI, Clark MW, Henderson E, Lake JA (1986) DNA hybridization electron microscopy: ribosomal RNA nucleotides 1392–1407 are exposed in the cleft of the small subunit. Proc Natl Acad Sci USA 83: 275–279

    Article  PubMed  CAS  Google Scholar 

  • Olson HM, Lasater LS, Cann PA, Glitz DG (1988) Messenger RNA orientation on the ribosome. Placement by electron microscopy of antibody-complementary oligodeoxynucleotide complexes. J Biol Chem 263: 15196–15204

    Google Scholar 

  • Plessel G, Lührmann R, Kastner B (1997) Electron microscopy of assembly intermediates of the snRNP (Sm) core: morphological similarities between the RNA-free [EFG] protein heteromer and the intact snRNP core. J Mol Biol 265: 87–94

    Article  PubMed  CAS  Google Scholar 

  • Raker VA, Plessel G, Lührmann R (1996) The snRNP core assembly pathway: identification of stable core protein heteromeric complexes and an snRNP subcore particle in vitro. EMBO J 15: 2256–2269

    CAS  Google Scholar 

  • Reed R, Griffith J, Maniatis T (1988) Purification and visualization of native spliceosomes. Cell 53: 949–961

    Article  PubMed  CAS  Google Scholar 

  • Scheinman A, Atha T, Aguinaldo AM, Kahan L, Shankweiler G, Lake JA (1992) Mapping the three-dimensional locations of ribosomal RNA and proteins. Biochimie 74: 307–317

    Article  PubMed  CAS  Google Scholar 

  • Scherly D, Boelens W, Dathan NA, Van Venrooij WJ, Mattaj IW (1990) Major determinants of the specificity of interaction between small nuclear ribonucleoproteins U1A and U2B“ and their cognate RNAs. Nature 345: 502–506

    Article  PubMed  CAS  Google Scholar 

  • Ségault V, Will CL, Sproat BS, Lührmann R (1995) 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 14: 4010–4021

    Google Scholar 

  • Skoglund U, Öfverstedt L-G, Burnett RM, Bricogne G (1996) Maximum-entropy three-dimensional reconstruction with deconvolution of the contrast transfer function: a test application with adenovirus. J Struct Biol 117: 173–188

    Article  PubMed  CAS  Google Scholar 

  • Spann P, Feinerman M, Sperling J, Sperling R (1989) Isolation and visualization of large compact ribonucleoprotein particles of specific nuclear RNAs. Proc Natl Acad Sci USA 86: 466–470

    Article  PubMed  CAS  Google Scholar 

  • Spiess M, Zimmermann H-P, Lünsdorf H (1987) Negative staining of protein molecules and filaments. In: Sommerville J, Scheer V (eds) Electron microscopy in molecular biology–a practical approach. IRL, Oxford, pp 147–166

    Google Scholar 

  • Stark H, Mueller F, Orlova EV, Schatz M, Dube P, Erdemir T, Zemlin F, Brimacombe R, Van Heel M (1995) The 70S Escherichia coli ribosome at 23 A resolution: fitting the ribosomal RNA. Structure 3: 815–821

    Article  PubMed  CAS  Google Scholar 

  • Stöffler G, Stöffler-Meilicke M (1984) Immunoelectron microscopy of ribosomes. Annu Rev Biophys Bioeng 13: 303–330

    Article  PubMed  Google Scholar 

  • Stöffler-Meilicke M, Stöffler G (1988) Localization of ribosomal proteins on the surface of ribosomal subunits from Escherichia coli using immunoelectron microscopy. In: Noller HF, Moldave K (eds) Methods in enzymology, vol 164. Academic Press, San Diego, pp 503–520

    Google Scholar 

  • Sumpter V, Kahrs A, Fischer U, Kornstädt U, Lührmann R (1992) In vitro reconstitution of U1 and U2 snRNPs from isolated proteins and snRNA. Mol Biol Rep 16: 229–240

    Article  PubMed  CAS  Google Scholar 

  • Tischendorf GW, Zeichhardt H, Stöffler G (1974a) Determination of the location of proteins L14, L17, L18, L19, L22 and L23 on the surface of the 50S ribosomal subunit of Escherichia coli by immune electron microscopy. Mol Gen Genet 137: 187–208

    Article  Google Scholar 

  • Tischendorf GW, Zeichhardt H, Stöffler G (1974b) Location of proteins S5, S13, S14 on the surface of the 30S ribosomal subunits from Escherichia coli as determined by immune electron microscopy. Mol Gen Genet 134: 209–223

    Article  PubMed  CAS  Google Scholar 

  • Tischendorf GW, Zeichhardt H, Stöffler G (1975) Architecture of the Escherichia coli ribosome as determined by immune electron microscopy. Proc Natl Acad Sci USA 72: 4820–4824

    Article  PubMed  CAS  Google Scholar 

  • Van Heel M, Frank J (1981) Use of multivariate statistical statistics in analysing the images of biological macromolecules. Ultramicroscopy 6: 187–194

    PubMed  Google Scholar 

  • Walter P, Blobel G (1983) Disassembly and reconstitution of signal recognition particle. Cell 34: 525–533

    Article  PubMed  CAS  Google Scholar 

  • Will CL, Kastner B, Lührmann R (1993) Analysis of ribonucleoprotein interactions. In: Higgens SJ, Hames BD (eds) RNA processing–a practical approach, vol 1. IRL, Oxford, pp 141–177

    Google Scholar 

  • Will CL, Fabrizio P, Lührmann R (1995) Nuclear pre-mRNA splicing. In: Eckstein F, Lilley DMJ (eds) Nucleic acids and molecular biology, vol 9. Springer, Berlin Heidelberg New York, pp 342–372

    Chapter  Google Scholar 

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  1. Institute of Molecular Biology and Tumor Research, Philipps Universität, Emil-Mannkopff-Str. 2, 35037, Marburg, Germany

    Berthold Kastner

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  1. II. Physiologisches Institut, Universität Heidelberg, Im Neuenheimer Feld 326, D-69120, Heidelberg, Germany

    Johannes Schenkel

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Kastner, B. (1998). Purification and Electron Microscopy of Spliceosomal snRNPs. In: Schenkel, J. (eds) RNP Particles, Splicing and Autoimmune Diseases. Springer Lab Manual. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-80356-7_5

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  • DOI: https://doi.org/10.1007/978-3-642-80356-7_5

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