Figure 5. Intron-lariat spliceosome (ILS) disassembly assays.

10–30% glycerol gradient sedimentation of purified yeast ILS (scILS) incubated in solution with ATP plus (a) no recombinant protein, (b) scPrp43 and cofactors scNtr(1•2), (c) ctPrp43 and scNtr(1•2), (d) ctPrp43-IDSB and scNtr(1•2), (e) ctPrp43-IDSB, scNtr(1•2) and 0.5 mM DTT, (f) ctPrp43-HT and scNtr(1•2), (g) ctPrp43-HL and scNtr(1•2), (h) ctPrp43-HT&HL and scNtr(1•2). U2, U5 and U6 snRNAs were visualized by Northern blotting followed by autoradiography. RNA identities are indicated on the left. Quantifications were performed with ImageQuant software (Molecular Dynamics). Numbers represent the percentage of intron-lariat RNA released in the top fractions (sum of fractions 1–11) or associated with the ILS (unreleased, sum of fractions 12–23) relative to the intron-lariat RNA distributed in all 23 fractions, the sum of which was set to 100%.

DOI: http://dx.doi.org/10.7554/eLife.21510.015

Figure 5—figure supplement 1. Isolation of intron-lariat spliceosomes (ILSs).

Activated spliceosomes (BactΔPrp2) assembled on Actin7 wild-type pre-mRNA in heat-inactivated splicing extracts from a prp2-1 yeast strain expressing a temperature-sensitive Prp2 mutant, were first purified. Purified BactΔPrp2 complexes were then incubated with recombinant Prp2 and Spp2, generating the B* spliceosome, and then Cwc25 was added to promote catalysis of step 1 of splicing and the formation of complex C. For catalysis of step 2, which generates post-catalytic spliceosomes (PCS), recombinant Prp16, Slu7 and Prp18 were added. Finally, for the purification of the ILS, the spliced mRNA was dissociated from the ILS by incubation of the PCS with Prp22 and ATP. Addition of ATP and recombinant Prp43, Ntr1 and Ntr2, leads to disassembly of the ILS into the intron-lariat, 20S U2 snRNP, 18S U5 snRNP and free U6 snRNA.

DOI: http://dx.doi.org/10.7554/eLife.21510.016