A two-tracked approach to analyze RNA-protein crosslinking sites in native, nonlabeled small nuclear ribonucleoprotein particles.
Much attention is currently being devoted to questions of protein and RNA tertiary structures and to the quaternary arrangement of the individual macromolecules in ribonucleoprotein (RNP) particles. In this article we describe two complementary strategies that allow the identification of RNA-protein contact sites in assembled, nonlabeled RNP particles after UV crosslinking. The first combines immunoprecipitation of UV-irradiated RNP particles under mildly denaturing conditions followed by primer-extension analysis of the crosslinked (and thus coprecipitated) RNA. The second involves the purification of crosslinked peptide-oligonucleotide from RNP particles and the subsequent analysis of the crosslinked peptide and RNA by Edman degradation and matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS), respectively. Although the first approach provides a rapid method for the exact identification of RNA-protein contact sites in purified nonlabeled RNP particles, the latter adds valuable information about potential RNA binding domains within proteins and, thus, about the arrangement of these proteins within the quaternary structures of complex RNP assemblies. Recently, we applied both these strategies successfully to native purified spliceosomal RNP. These methods may be generally applicable to the analysis of RNP complexes, especially as they avoid labeling and reconstitution, both of which risk introducing artifacts.
Urlaub
Henning
H
Max-Planck-Institute for Biophysical Chemistry, Department of Cellular Biochemistry, Am Fassberg 11, D-37077 Göttingen, Germany. henning.urlaub@mpi-bpc.mpg.de
Hartmuth
Klaus
K
Lührmann
Reinhard
R
D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D005821:Genetic Techniques; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011233:Precipitin Tests; D011485:Protein Binding; D012313:RNA / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D017411:Ribonucleoproteins, Small Nuclear; D012313:RNA