Assembly and glycerol gradient isolation of yeast spliceosomes containing transcribed or synthetic U6 snRNA.

摘要: Studies of RNA-protein interactions often require assembly the complex using in vitro synthesized RNA or recombinant protein. Here, we describe a protocol to assemble functional spliceosome yeast extracts transcribed synthetic RNAs. The assembled is stable and can be isolated by sedimentation through glycerol gradients for subsequent analysis. protocols two procedures prepare RNA: bacteriophage polymerases ligation oligos T4 DNA ligase. We also preparation splicing competent extracts, spliceosome, isolation gradient sedimentation. To allow exogenously added U6 incorporated into endogenous small nuclear (snRNA) extract eliminated an antisense oligo ribonuclease H; "neutralizing" was then protect incoming RNA. This allows study role individual bases phosphate backbone plays interaction between snRNA spliceosomal proteins.