Assembly and Glycerol Gradient Isolation of Yeast Spliceosomes Containing Transcribed or Synthetic U6 snRNA

Abstract

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 bacteriophage 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.

RNA catalyses nuclear pre-mRNA splicing

“…After depletion of U6, to promote degradation of the U6 d1 oligonucleotide before addition of the synthetic U6, DNase I (Ambion, 0.05 U/ μL final) was added together with the glucose used to deplete ATP. Additionally, to ensure complete inactivation of the U6 d1 oligonucleotide, an oligonucleotide antisense to U6 d1 (U6 αd1, 0.3 μM final) 57 was added immediately prior to reconstitution and incubated on ice for 5 minutes. For reconstitution U6 was added back to a final concentration of 0.2 – 0.3 μM.…”