GST-tagged Pab1 or Sbp1, immobilized on a glutathione–sepharose resin, was incubated with the target proteins or their mutants containing individual domains with or without 5′UTRPab1 at 4°C for 30 min in the binding buffer (50 mM Tris–HCl, pH 7.5, 120 mM KCl, 2 mM DTT, 2 mM MgCl2, 0.5% Triton-X-100). The resin was then washed four times with cold binding buffer and subsequently boiled in 1× SDS-loading buffer (50 mM Tris–HCl pH 6.8, 2% SDS, 10% Glycerol, 1% β-mercaptoethanol, 0.02% bromophenol blue) to elute bound proteins. RNase treatment was carried out after the formation of RNP by incubating RNase A with the RNP at 25°C for 30 min. The proteins were resolved by SDS–PAGE and detected by either Coomassie Brilliant Blue or SYPRO Ruby staining.