Effect of Prp46p depletion on cell growth and pre-mRNA splicing. (A) Effect of Prp46p depletion on cell growth. Strains YMA151/2 and BMA64α (WT) were grown at 30°C in minimal medium lacking methionine (inducing conditions for Prp46p production in YMA151/2) or with 7 mM methionine (repressing condition) as indicated. The O.D.600 was monitored and cultures were diluted at intervals to maintain logarithmic growth. The indicated O.D.600 is therefore the calculated theoretical O.D. (B) Effect of Prp46p depletion on in vivo splicing. RNA (10 μg) extracted from aliquots of the cultures shown in A was used as template in a primer extension reaction using radiolabeled oligonucleotide primers complementary to the extreme 5′ end of exon 2 of the U3 snoRNA and to the (intronless) U1 snRNA as control. The products were resolved on a 6% (w/v) polyacrylamide gel and visualized by autoradiography. The positions of the extension products are indicated. (time [h]) Time point of sample. (Lanes 1,4,7) YMA151/2 minus methionine; (lanes 2,5,8) YMA151/2 plus 7 mM methionine; (lanes 3,6,9) BMA64α plus 7 mM methionine. (Lariat I-E2) lariat intron-exon2.
