Summary
Studies of RNA—protein interactions often require assembly of the RNA—protein complex using in vitro synthesized RNA or recombinant protein. Here, we describe a protocol to assemble a functional spliceosome in yeast extracts using transcribed or synthetic RNAs. The in vitro assembled spliceosome is stable and can be isolated by sedimentation through glycerol gradients for subsequent analysis. The protocols describe two procedures to prepare RNA: using bacte-riophage RNA polymerases or ligation of RNA oligos using T4 DNA ligase. We also describe the preparation of splicing competent yeast extracts, the assembly of the spliceosome, and the isolation of the spliceosome by glycerol gradient sedimentation. To allow exogenously added U6 RNA to be incorporated into the spliceosome, the endogenous U6 small nuclear RNA (snRNA) in the extract is eliminated by an antisense U6 DNA oligo and ribonuclease H; a “neutralizing” U6 DNA oligo was then added to protect the incoming U6 RNA. This protocol allows study of the role individual bases or the phosphate backbone of U6 plays in splicing and of the interaction between U6 snRNA and the spliceosomal proteins.
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Acknowledgments
We thank members of the Lin laboratory for sharing reagents and protocols. K.J.D. was supported in part by Ruth L. Kirschstein Predoctoral Fellowship F31 GM67579 from the National Institutes of Health (NIH). This work was supported by NIH R01 grant GM40639 and funds from City of Hope Beckman Research Institute to R.J.L.
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Dery, K.J., Yean, SL., Lin, RJ. (2008). Assembly and Glycerol Gradient Isolation of Yeast Spliceosomes Containing Transcribed or Synthetic U6 snRNA. In: Lin, RJ. (eds) RNA-Protein Interaction Protocols. Methods in Molecular Biology, vol 488. Humana Press. https://doi.org/10.1007/978-1-60327-475-3_4
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DOI: https://doi.org/10.1007/978-1-60327-475-3_4
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Print ISBN: 978-1-58829-419-7
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