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Information stored in DNA needs to be translated into functional proteins. Ribonucleoprotein (RNP) particles play a major role in this process. In many autoimmune diseases, antibodies are directed against components of these particles. Detailed protocols for analysing the structure and function of RNPs are described in this manual.
Frank Jung, Constantin E. Sekeris, and Johannes Schenkel: Isolation and Immunochemical Characterization of hnRNP Particles.-Ruth Sperling and Joseph Sperling: The lnRNP Particle -- A Naturally Assembled Complex of Pre-mRNA and Splicing Factors.-Karl Otto Greulich: Intrinsic Fluorescence Techniques for Studies on Protein-Protein and Protein-RNA Interactions in RNP Particles.-Ulf Skoglund, Lars-Göran Öfverstedt, and Bertil Daneholt: Procedures for Three-Dimensional Reconstruction from Thin Sections with Electron Tomography.-Berthold Kastner: Purification and Electron Microscopy of Spliceosomal snRNPs.-Panayiotis G. Vlachoyiannopoulos: Detection of Autoantibodies to Extractable Cellular Antigens.-Martin Blüthner: Methods in Immunolocalization of Autoantigens.- Johannes Schenkel, Frank Jung, Apostolia Guialis, and Angela Krämer: In Vitro Splicing of Pre-mRNA in HeLa Extracts.
The maturation of the precursors of mRNA to a functional tranillegalscript in eukaryotic cells is a complex, multistep process. It includes the formation of RNP complexes and the splicing procedure. Also, the presence of snRNA packaged as snRNP particles plays an important role in this process. In addition, snRNPs are involved in various autoimmune diseases in which autoantibodies are directed against components of these particles.
Relevant techniques to investigate RNA processing, splicing, protein-RNA complex formation and protein-RNA interactions as well as the use of autoantibodies in basic and clinical research are presented in this manual.
1 Isolation and Immunochemical Characterization of hnRNP Particles.- 2 The lnRNP Particle - A Naturally Assembled Complex of Pre-mRNA and Splicing Factors.- 3 Intrinsic Fluorescence Techniques for Studies on Protein-Protein and Protein-RNA Interactions in RNP Particles.- 4 Procedures for Three-Dimensional Reconstruction from Thin Sections with Electron Tomography.- 5 Purification and Electron Microscopy of Spliceosomal snRNPs.- 6 Detection of Autoantibodies to Extractable Cellular Antigens.- 7 Methods in Immunolocalization of Autoantigens.- 8 In vitro Splicing of Pre-mRNA in HeLa Extracts.- Abbreviations.
Inhaltsverzeichnis
Frank Jung, Constantin E. Sekeris, and Johannes Schenkel: Isolation and Immunochemical Characterization of hnRNP Particles.-Ruth Sperling and Joseph Sperling: The lnRNP Particle -- A Naturally Assembled Complex of Pre-mRNA and Splicing Factors.-Karl Otto Greulich: Intrinsic Fluorescence Techniques for Studies on Protein-Protein and Protein-RNA Interactions in RNP Particles.-Ulf Skoglund, Lars-Göran Öfverstedt, and Bertil Daneholt: Procedures for Three-Dimensional Reconstruction from Thin Sections with Electron Tomography.-Berthold Kastner: Purification and Electron Microscopy of Spliceosomal snRNPs.-Panayiotis G. Vlachoyiannopoulos: Detection of Autoantibodies to Extractable Cellular Antigens.-Martin Blüthner: Methods in Immunolocalization of Autoantigens.- Johannes Schenkel, Frank Jung, Apostolia Guialis, and Angela Krämer: In Vitro Splicing of Pre-mRNA in HeLa Extracts.
Klappentext
The first insights into the site and mechanisms of RNA process ing to functional mRNA in eukaryotic cells came from the group of Georgiev (Lukanidin et al. 1972) who demonstrated the association of rapidly labelled, heterogeneous nuclear RNA (hnRNA) with a limited number of specific proteins in the cell nucleus. These "informofers", i. e. packaged precursors of mRNA (pre-mRNA or hnRNA), are in a form presumably amenable to the action of nucleases. With the availability of better analytical techniques, the considerable heterogeneity of hnRNA associated proteins was revealed (Niessing and Sekeris 1970), suggesting a role that was more composite, rather than solely structural, for these proteins. Later studies investigated the RNA binding behavior of these proteins (Schenkel et al. 1988, 1989; Wilk et al. 1983). For a long time, the small nuclear RNAs, well characterized with respect to primary structure (reviewed by Reddy and Busch 1983), were naively ignored regarding their function. Several events then set the stage for a detailed study of the intricate mechanisms of the splicing process and other steps involved in hnRNA processing: (1) The demonstration of a second class of nuclear ribonucleoproteins (RNPs), composed of small nuclear RNAs (snRNAs) and another characteristic group ofheterogene ous proteins (Lerner et al. 1980; Guialis et al. 1983); (2) the detec tion of the association of snRNPs with hnRNPs by virtue of base pairing between hnRNA and snRNA (Flytzanis et al.
Information stored in DNA needs to be translated into functional proteins. Ribonucleoprotein (RNP) particles play a major role in this process. In many autoimmune diseases, antibodies are directed against components of these particles. Detailed protocols for analysing the structure and function of RNPs are described in this manual.
