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 wi thin native small nuclear ribonucleoprotein (snRNP) particles from HeLa cel ls. It combines immunoprecipitation of the UV-irradiated particles under se mi-denaturing conditions with primer extension analysis of the cross-linked RNA moiety. In a feasibility study, we initially identified the exact cros slinking sites of the U1 70-kDa (70K) protein in stem-loop I of U1 small nu clear 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-li nked 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 immunop recipitation approach to HeLa US snRNP, as part of purified 25 S U4/U6.U5 t ri-snRNPs. Cross-linking sites between the US-specific 220-kDa protein (hum an homologue of Prp8p) and the U5 snRNA were located at multiple nucleotide s within the highly conserved loop I and at one site in internal loop 1 of U5 snRNA The cross-linking of four adjacent nucleotides indicates an extend ed interaction surface between loop 1 and the 220-kDa protein. In summary, our approach provides a rapid method for identification of RNA-protein cont act sites within native snRNP particles as well as other ribonucleoprotein particles.