Purification of protein–RNA cross-links by biotin–streptavidin selection. Pre-mRNA was prepared as shown in Figure 1 and incubated with nuclear extract for 15 min at 30°C. Subsequently, complex A was purified by glycerol gradient centrifugation. U1 snRNP was not included in the figure, as it is not investigated whether U1 was base-paired with the 5′ splice site in this complex (Das et al. 2000). The fractions containing complex A were pooled, irradiated, and digested with RNase T1. Cross-linked proteins that were covalently bound to the nucleotide next to the biotin-containing residue were affinity purified with streptavidin beads. After stringent washing, the proteins were eluted from the beads for analysis by mass spectrometry.
