A General Approach for Identification of RNA-Protein Cross-linking Sites within Native Human Spliceosomal Small Nuclear Ribonucleoproteins (snRNPs)

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 crosslinking 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.The spliceosome catalyzes the two-step trans-esterification reaction that occurs during splicing of nuclear pre-mRNA, i.e. excision of the introns and ligation of the exons. Assembly of the spliceosome is an ordered process and involves the interaction of U1, U2, and U4/U6.U5 small nuclear ribonucleoprotein (snRNP) 1 particles with the intron-containing pre-mRNA and a multitude of additional spliceosomal factors (for review, see Refs. 1-5). Although catalysis in the spliceosome appears to be RNA-based, both protein-protein and RNA-protein interactions contribute to the formation of its catalytic core (1-5).The structures of the spliceosomal components are largely solved at the levels of primary structure and RNA secondary structures (3, 5). Much attention is currently being devoted to the issues of protein and RNP tertiary structures (6) and to the quaternary arrangement both of the individual macromolecules in the U snRNP particles and of these particles in the functioning spliceosome (7).The U1 snRNP particle from HeLa cells has been extensively investigated in terms of RNA-protein and protein-protein interactions, as it is only of moderate complexity. Thus, a relatively detailed picture of its morphology and tertiary structure (in comparison with other spliceosomal RNPs) has emerged (3, 7, 8). The seven common spliceosomal Sm proteins (G, E, F, D1, D2, D3, and B) assemble on the Sm site of the U1 snRNA to form a doughnut-like structure (9, 10). The U1-specific proteins U1 70K and A specifically interact via their N-terminal RNAbin...

Cooperative structure of the heterotrimeric pre-mRNA retention and splicing complex

“…After 1:20 dilution with NET buffer, the RNA was digested with a final concentration of 6 units/µl RNAse T1 (Ambion) at 37 °C for 1 h. Snu17p-TAP was immunoprecipitated directly from the digestion reaction, essentially as described in ref. 76. Proteins were eluted from the IgG beads (GE Healthcare) by addition of 1× NuPAGE LDS sample buffer (Invitrogen) and incubation at 70 °C for 10 min, and were separated on Novex NuPAGE gels (Invitrogen).…”

Regulation of non-AU-rich element containing c-fms proto-oncogene expression by HuR in breast cancer

“…Ultraviolet cross-linking and label transfer of HuR with 3 0 UTR c-fms RNA Ultraviolet cross-linking of HuR was performed as described elsewhere (Stolow and Berget, 1990;Gott et al, 1991;Urlaub et al, 2000), with modifications. RNAs of 3 0 UTR c-fms labeled with 32 P-UTP to the same specific activity were incubated with 0.5 mg HuR-GST or 0.5 mg GST.…”

Impact of proline and aspartic acid residues on the dissociation of intermolecularly crosslinked peptides

“…Crosslinked proteins are typically subjected to enzymatic digestion to create more easily interpreted peptide segments upon subsequent tandem mass spectrometric analysis. Crosslinking has also been utilized to identify protein folds [6], conformational changes of proteins upon activation [14,15], and RNA-protein interactions [16]. While the chemical crosslinking experiment is often rather straightforward, the technique is restricted by several limitations which must be overcome to obtain the desired structural information.…”