FIG. 1.

Cwc22 is not a component of the NTC but is essential for pre-mRNA splicing both in vivo and in vitro. (A) Extracts prepared from PRP19-HA (lane 1), CWC22-HA (lane 2), and nontagged (lane 3) strains were immunoprecipitated with anti-HA antibody, followed by Western blotting using antibodies against the HA epitope and components of the NTC. (B) Growth curves of GAL-CWC22 cells in yeast extract-peptone-dextrose (YPD) and yeast extract-peptone galactose (YPG). Cells were grown in galactose-containing synthetic minimum medium to mid-log phase and then shifted to YPD or YPG medium. Cells were collected at 0, 4, 8, 12, 16, 20, 24, 28, 32, and 36 h after the shift for measurements of the optical density at 600 nm (OD₆₀₀). (C) Total RNA extracted from collected cells was analyzed by primer extension using a U3 primer, R13. The prp2 mutant was grown at 37°C for 2 h before harvesting. Gal, galactose; Glu, glucose; WT, wild type. (D) Splicing was carried out in mock-treated (lanes 1 and 4) or Cwc22-depleted (lanes 2, 3, 5, and 6) extracts using wild-type (lanes 1 to 3) or ACAC (lanes 4 to 6) actin pre-mRNA with (lanes 3 and 6) or without (lanes 1, 2, 4, and 5) the addition of 100 ng recombinant Cwc22. M, mock; D, depletion.