Accumulation of splicing intermediates occurs in the absence of Fyv6 in in vitro splicing assays. (A) Products of the first and second steps after incubation of a radioactively labeled RP51A pre-mRNA (lane 1) in WCE from cup1Δ strains (yAAH0434 and yAAH3353, Supplemental Table 1) for 45 min (lanes 2,3). Quantitation of (B) lariat intermediates or (C) Second-step splicing efficiency from three replicates. Statistical significance was determined by unpaired Welch's two-tailed t-test (P = 0.006415 and 0.01788 for fraction of lariat intermediate and second-step efficiency, respectively). The fraction of the lariat intermediate represents the fraction of that species relative to the substrate and all splicing products while the second-step efficiency represents the fraction of mRNA relative to the sum of first- and second-step products (lariat intermediate plus mRNA). Details for calculations can be found in the Materials and Methods.
