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J. Biol. Chem., Vol. 275, Issue 52, 41458-41468, December 29, 2000

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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^*

Henning Urlaub, Klaus Hartmuth, Susanne Kostka^§, Gerlinde Grelle^§, and Reinhard Lührmann^¶

From the  Abteilung Zelluläre Biochemie, Max-Planck-Institut für Biopysikalische Chemie, Am Faßberg 11, D-37077 Göttingen, Germany and the ^§ Abteilung Proteinchemie, Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Straße 10, D-13125 Berlin, Germany

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.

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