Approaches for the Identification and Characterization of RNA–Protein Interactions - Biophysical Approaches to Translational Control of Gene Expression - page 207

10.4.3.2 Methods

1. In separate tubes, add 5 mg of biotinylated specific or nonspeci fi c RNA oligomers
to ~3 mg of HeLa cytoplasmic extract (Sokoloski et al. 2008 ) . Adjust reaction
mixtures to 2.5 mM EDTA to help stabilize the RNA oligomers by blocking
exonuclease activities commonly found in extracts.
2. Incubate for 1 h at 4 °C with rotation. Centrifuge at 16,000 × g at 4 °C to remove
proteins that have precipitated during handling and incubations. Transfer the
supernatants to new tubes.
3. Add 20 mL streptavidin agarose beads (that have been equilibrated in buffer D)
to each tube; incubate 15 min at 4 °C with rotation.
4. Centrifuge at 4,000 × g for 5 min at 4 °C to pellet streptavidin beads bound with
RNA oligomers. Remove supernatant and discard.
5. Wash beads five times with buffer D at 4 °C.
6. Add 300 mL of HSCB solution to the beads and incubate for 5 min at room
temperature to release proteins from the bound RNA. If necessary, 1 mM DTT
can be added and the mixture heated to 100 °C to release tightly associated
proteins. Remove the supernatant to a fresh microcentrifuge tube.
7. Add 100 mL water, 400 mL of methanol, and 100 mL of chloroform; vortex.
8. Centrifuge at 16,000 × g for 2 min.
9. Remove the upper aqueous layer and add another 400 mL of methanol.
10. Centrifuge at 16,000 × g for 2 min.
11. Remove all the supernatant carefully, dry the pellet.
12. Add 2× protein loading dye to the pellet, heat the sample at 65 °C for 5 min, and
spin at maximum speed in a microcentrifuge for 15 min.
13. Resolve proteins on a 10% SDS-PAGE gel and visualize bands by silver
staining.
14. Excise protein bands of interest and analyze via tandem mass spectrometry fol-
lowing trypsin digestion.

10.4.3.3 Notes

1. For maximal ef fi ciency, titrate the speci fi c RNA oligo to identify the amount
needed to provide a sufficient number of binding sites to saturate the available
protein of interest. This could be done, for example, by competition analysis
using a radiolabeled RNA substrate in a UV cross-linking assay as described in
Sect. 10.4.2 above.
2. By altering salt conditions, etc., one may create selective conditions to maximize
the amount of specific RNA-protein interactions while minimizing the amount
of nonspecific interactions. The stability of the RNA-protein interaction of inter-
est to a variety of monovalent and divalent cations, etc., can readily be assayed
by UV cross-linking as described in Sect. 10.4.2 above.