Functional aspects of nucleic acid helicases can be interrogated by various in vitro methods, using purified components, including nucleic acid binding and unwinding assays. Here we describe detailed protocols for the production and purification of the spliceosomal Ski2-like RNA helicase, Brr2, and one of its regulatory factors, the Jab1 domain of the Prp8 protein from yeast. Furthermore, we include a production protocol for radioactively labeled yeast U4/U6 di-snRNA substrate. We describe polyacrylamide gel-based assays to investigate Brr2's RNA binding and unwinding activities. The purification protocols and activity assays can be easily adapted for the purification and functional interrogation of other helicases, cofactors, and RNA substrates.

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Reference Type

Journal Article

Authors

Absmeier E, Wahl MC

Primary Lit For

RRI1 | BRR2 | U4/U6 small nuclear ribonucleoprotein complex

Additional Lit For

Review For