Figure 3.

The 3′ end of U6 is required in vitro for Prp43p-dependent discard of a suboptimal substrate. (A) As with the rPrp43p-Q423E mutant (Mayas et al. 2010), 3′-truncated U6 impedes discard of a suboptimal lariat intermediate. Radiolabeled UBC4 pre-mRNA having a suboptimal UgG 3′ splice site was spliced in mutant dbr1Δ (yJPS799) extracts with buffer, with rPrp43p-Q423E, or after RNase H cleavage of U6 in extract with DNA oligo AS 95–112 (see Fig. 1A). Splicing reactions were fractionated on a glycerol gradient; input (i) and fraction numbers are indicated above the top panel. The migration of spliceosome-bound and discarded lariat intermediate is indicated. See also Supplemental Figure S2. (B) Quantitation of lariat intermediate levels from glycerol gradients in A. Data are normalized to input levels of lariat intermediate.