Effect of Prp45p depletion on pre-mRNA splicing in vivo. (A) Northern analysis of splicing. RNA was extracted from aliquots of the cultures grown for the indicated times under permissive or nonpermissive conditions. RNA (10 μg) was fractionated on a 1% (w/v) formaldehyde gel, blotted to Hybond-N membrane (Amersham), probed with a radiolabeled DNA fragment complementary to exon 1 of the RP28 gene, and analyzed by autoradiography. The positions of the RP28 pre-mRNA and mRNA are indicated. The blot was stripped and reprobed for the intronless PGK gene as a control for loading. (B) Primer extension analysis of splicing of U3 precursor RNA. RNA (10 μg) was used in a primer extension reaction with a radiolabeled oligonucleotide primer complementary to the extreme 5′ end of exon 2 of the U3 snoRNA. As a control, a primer for the intronless U1snRNA was used in the same reaction. The products were resolved on a 6% (w/v) polyacrylamide gel and visualized by autoradiography. The positions of the extension products are indicated.
