We constructed E15E16 (wt and p.E415G) minigenes spanning COLQ exon 15 to 16 and E16E17 (wt and p.E415G) minigenes spanning COLQ exon 16 to 17 in pcDNA3.1D/V5-His-TOPO vector (Invitrogen). Amplicons were generated by PCR using pCI-COLQ (wt or p.E415G), and cloned into pcDNA3.1D/V5-His-TOPO vector.
We introduced three copies MS2-coat protein binding hairpin sequences at the 3′ end of E16E17 (wt and E415G) constructs using the megaprimer method45 (link). At first, we PCR-amplified a fragment harboring three copies MS2-coat protein binding hairpin sequences from pSP64-MS2 vector35 (link) with the primers carrying complementary sequences to E16E17 minigene where the MS2-sequences is being inserted. The PCR amplicon was used as a megaprimer for the QuikChange site-directed mutagenesis system. These vectors were used as templates to generate MS2-attached RNA substrates of E16E17 (wt and p.E415G) for isolation of early spliceosomal complex. As control, we used MS2-attached human β-globin exon 1-intron 1-exon 2 construct (pSP64-HβΔ6-MS2) as previously described35 (link).
We introduced three copies MS2-coat protein binding hairpin sequences at the 3′ end of E16E17 (wt and E415G) constructs using the megaprimer method45 (link). At first, we PCR-amplified a fragment harboring three copies MS2-coat protein binding hairpin sequences from pSP64-MS2 vector35 (link) with the primers carrying complementary sequences to E16E17 minigene where the MS2-sequences is being inserted. The PCR amplicon was used as a megaprimer for the QuikChange site-directed mutagenesis system. These vectors were used as templates to generate MS2-attached RNA substrates of E16E17 (wt and p.E415G) for isolation of early spliceosomal complex. As control, we used MS2-attached human β-globin exon 1-intron 1-exon 2 construct (pSP64-HβΔ6-MS2) as previously described35 (link).
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