Analysis of site-specific protein–RNA cross-links in isolated RNP complexes, combining affinity selection and mass spectrometry

Preparation of site-specifically modified pre-mRNA. (A) Generation of RNA fragments. Full-length Minx pre-mRNA was prepared by transcription in vitro and 5′ and 3′ fragments were generated from it by site-directed cleavage with RNA-cleaving DNA enzymes, as shown. The 5′ fragment was dephosphorylated and the 3′ fragment was 5′ labeled with ³²P. (B) 5′ phosphorylation (nonradioactive) of [pS⁺⁹B]-nt₂₁. The 21-nt oligonucleotide contains a phosphorothioate at the intron position C9 (pS⁺⁹) and an adjacent biotin (B). (C) Three pieces of RNA, as shown, were hybridized to a splint oligonucleotide and ligated with T4 DNA Ligase, yielding [pS⁺⁹B]-pre-mRNA. The product was modified with APB at the phosphorothioate (pS), leading to [APB⁺⁹B]-pre-mRNA. After RNase T1 digest, a radioactive RNA fragment remains containing APB and biotin as indicated by the bracket. (D) As in C, but two pieces of RNA were used, resulting in the 5′ truncated product [pS⁺⁹B]-pre-mRNA or [APB⁺⁹B]-pre-mRNA-S after modification with APB.