Preparation of the pre-mRNA

The U12-type pre-mRNA for the in vitro splicing assay was modified from the MINX gene, in which the 5′SS, BPS, and 3′SS are replaced by consensus sequences of the U12-type intron. The 5′SS, BPS, and 3′SS have sequences 5′-AUAUCCUUU-3′, 5′-UCCUUAACUC-3′, and 5′-CAC-3′, respectively. The altered MINX pre-mRNA (referred to as MINX-U12) comprises a 57-nucleotide (nt) 5′ exon, a 228-nt intron, a 51-nt 3′ exon, and three tandem MS2-binding RNA aptamers at the 3′ end of the 3′ exon. To enrich the minor B^act complex in our in vitro assembly assay (37), we further modified MINX-U12 by deleting all sequences beyond nucleotide 18, counting from the 3′ end of the BPS. We then inserted three tandem MS2-binding RNA aptamers between the 5′SS and the BPS. The resulting pre-mRNA, referred to as MINX-U12∆, was used for spliceosome assembly and purification. The DNA templates for in vitro transcription were generated using polymerase chain reaction (PCR), and the RNA substrates were synthesized using the method of T7 runoff transcription.