In vitro splicing assay and RT-PCR

The splicing extract was thawed on ice and heated at 35°C for 30 min to inactivate mutant Prp2. The splicing reaction was assembled in a 20-μl volume on ice. The reaction buffer contains 60 mM KH₂PO₄/K₂HPO₄, pH 7.0, 3% (w/v) PEG8000, 2 mM ATP, 2 mM spermidine, 2.5 mM MgCl₂, 0.5 nM pre-mRNA substrate, 40% splicing extract in the presence or absence of 1 ng/μL recombinant Prp2. To examine the impact of Spp2 truncations on splicing, each of the various truncated Spp2 variants was added to the extract first at a concentration of about 40 nM. The reaction mixture was incubated at 23°C for 1 hour, followed by proteinase K digestion. RNA from the in vitro splicing assay was extracted using phenol:chloroform:isopentanol at a volume ratio of 25:24:1 (Coolaber Science & Technology). Reverse transcription was performed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and random hexamers. The reverse transcription (RT)–PCR products were resolved on 2% (w/v) agarose gel and stained by GoldView (Beijing SBS Genetech).