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.
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 Tyr(112) and Leu(175) within the RNA-binding domain of the U1 70K protein to be cross-linked to G(28) and U(30) 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.
Urlaub
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Abteilung Zelluläre Biochemie, Max-Planck-Institut für Biopysikalische Chemie, Am Fassberg 11, D-37077 Göttingen, Germany.
Hartmuth
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Kostka
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Grelle
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Lührmann
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D000595:Amino Acid Sequence; D001483:Base Sequence; D006801:Humans; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D012313:RNA / Q000737:chemistry*; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000737:chemistry; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000737:chemistry; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017381:Spliceosomes / Q000737:chemistry*
D017412:Ribonucleoprotein, U1 Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D012313:RNA