Biomedical Engineering Reference
In-Depth Information
3. If the RNA-protein complex is sticking in the wells, the addition of bovine serum
albumin may help to minimize this effect.
10.4.2
UV Cross-Linking of RNA to Proteins from Cellular
Extracts or Recombinant Sources
In addition to its use outlined above in stabilizing RNA-protein complexes, UV
cross-linking can also be used to identify RNA-protein interactions. Complexes are
allowed to form between proteins and radiolabeled RNAs. Irradiation with short
wavelength UV light forms covalent bonds between proteins and closely associated
RNA molecules. Reactions are treated with RNAse and proteins with covalently
associated short radioactive RNA tags are analyzed by conventional SDS-PAGE
and visualized by phosphorimaging. In this fashion, the approximate molecular
weight of candidate RNA binding proteins can be quickly determined and used to
predict candidate proteins from available databases. When coupled with extract-
based functional assays (e.g., splicing, polyadenylation, or RNA decay) and compe-
tition analysis, one can rapidly associate the functional relevance of RNA-protein
interactions and prioritize candidates for further study.
10.4.2.1
Specialized Materials
1. Cellular extracts (prepared as described, e.g., Sokoloski et al.
2008
) /recombinant
protein.
2. Stratalinker 2400 (Stratagene).
3. RNase OUT.
4. RNase ONE (Promega).
10.4.2.2
Method
1. Produce radiolabeled RNA through in vitro transcription using bacteriophage
polymerases (e.g., T7, SP6) and
32
P—UTP.
2. Incubate radiolabeled RNAs (~5 fmoles; 100-200 K cpm) with the extract or
recombinant protein for 5-10 min at 30 °C.
3. Transfer the sample to a well of a microtiter dish on ice.
4. Cross-link using 254 nm UV light for 1-5 min or 1,800 mJ in a Stratalinker 2400.
The light should be 1-2 cm from the dish.
5. Pipette irradiated samples back to 1.5 mL tubes containing 0.5 mL RNase ONE
(a broad spectrum ribonuclease). Incubate at 37 °C for 15 min to degrade the
input RNA.
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