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A general approach for identification of RNA-Protein cross-linking sites within native human spliceosomal small nuclear ribonucleoproteins (snRNPs) - Analysis of RNA-protein contacts in native U1 and U4/U6.U5 snRNPs

Item Type:Article
Title:A general approach for identification of RNA-Protein cross-linking sites within native human spliceosomal small nuclear ribonucleoproteins (snRNPs) - Analysis of RNA-protein contacts in native U1 and U4/U6.U5 snRNPs
Creators Name:Urlaub, H. and Hartmuth, K. and Kostka, S. and Grelle, G. and Luehrmann, R.
Abstract:We describe a novel approach to identify RNA-protein cross-linking sites within native small nuclear ribonucleoprotein (snRNP) particles from HeLa cells. It combines immunoprecipitation of the UV-irradiated particles under semi-denaturing conditions with primer extension analysis of the cross-linked RNA moiety. In a feasibility study, we initially identified the exact cross-linking sites of the U1 70-kDa (70K) protein in stem-loop I of U1 small nuclear RNA (snRNA) within purified U1 snRNPs and then confirmed the results by a large-scale preparation that allowed N-terminal sequencing and matrix-assisted laser desorption ionization mass spectrometry of purified cross-linked peptide-oligonucleotide complexes. We identified Tyr112 and Leu175 within the RNA-binding domain of the U1 70K protein to be cross-linked to G28 and U30 in stem-loop I, respectively. We further applied our immunoprecipitation approach to HeLa U5 snRNP, as part of purified 25 S U4/U6.U5 tri-snRNPs. Cross-linking sites between the U5-specific 220-kDa protein (human homologue of Prp8p) and the U5 snRNA were located at multiple nucleotides within the highly conserved loop 1 and at one site in internal loop 1 of U5 snRNA. The cross-linking of four adjacent nucleotides indicates an extended interaction surface between loop 1 and the 220-kDa protein. In summary, our approach provides a rapid method for identification of RNA-protein contact sites within native snRNP particles as well as other ribonucleoprotein particles.
Keywords:Amino Acid Sequence, Base Sequence, Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry, Molecular Sequence Data, Precipitin Tests, RNA, Small Nuclear Ribonucleoproteins, Spliceosomes, U1 Small Nuclear Ribonucleoprotein, U4-U6 Small Nuclear Ribonucleoprotein, U5 Small Nuclear Ribonucleoprotein
Source:Journal of Biological Chemistry
ISSN:0021-9258
Publisher:American Society for Biochemistry and Molecular Biology (U.S.A.)
Volume:275
Number:52
Page Range:41458-41468
Date:1 January 2000
Official Publication:https://doi.org/10.1074/jbc.M007434200
PubMed:View item in PubMed

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