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 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.
Highlights
We describe a novel approach to identify RNA-protein cross-linking sites within native small nuclear ribonucleoprotein particles from HeLa cells
We initially identified the exact crosslinking sites of the U1 70-kDa (70K) protein in stem-loop I of U1 small nuclear RNA within purified U1 small nuclear ribonucleoprotein (snRNP) and 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 show a direct interaction of the 220-kDa protein with the four adjacent uridines of the conserved loop 1 of U5 small nuclear RNA (snRNA), suggesting an extended interaction surface between these two components
Summary
Sample Preparation—U1, U2, and U5 snRNPs and U4/U6.U5 trisnRNP particles were prepared from HeLa cell nuclear extracts (Computer Cell Culture Co., Mons, Belgium) by immunoaffinity chromatography using the H20 monoclonal antibody as described previously [30]. 12 S U1 snRNPs were separated from contaminating U2 snRNPs by Mono Q anion-exchange chromatography [31] and stored at a concentration of 2 mg/ml in Mono Q elution buffer. 25 S U4/U6.U5 tri-snRNP particles were obtained from the total snRNP mixture by gradient centrifugation [30], and peak fractions were pelleted for 6 h at 70,000 rpm (TLA 100.3 rotor, Beckman Instruments). Cross-linked snRNA was extracted with phenol/chloroform, precipitated with ethanol as described above, and dissolved in 3.5 l of CE buffer. Isolation and Sequence Determination of Cross-linked Peptide-Oligonucleotide Complexes—UV-irradiated U1 snRNPs were precipitated with 3 volumes of ethanol in Corex tubes (Sorvall) and washed with 80% ethanol, and the pellet was briefly air-dried. The U1 snRNP particles were precipitated with 3 volumes of ethanol; washed with 80% ethanol; air-dried; and dissolved in an appropriate volume of 20 mM Tris-HCl (pH 7.8), 150 mM NaCl, and 5 mM EDTA containing 8 M urea. SnRNA-containing fractions were pooled and precipitated with 3 volumes of ethanol, washed with 80% ethanol, and dissolved in 25 mM Tris-HCl (pH 7.8) and 2.5 mM EDTA. MALDI-MS of the cross-linked peptide-oligonucleotide complexes was performed essentially under conditions described previously [34, 35], but without an ion-exchange procedure before measurement
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