Effects of Pla1-Y86D on polyadenylation of mug8 mRNA in vivo. Total RNA (40 µg) prepared from wild-type and pla1-Y86D cells was annealed to DNA oligonucleotides complementary to the mug8 mRNA either with (+) or without (−) oligo(dT), then treated with RNase H. The reaction products were resolved by denaturing-PAGE along with an aliquot of radiolabeled DNA marker, then transferred to a nylon membrane for northern analysis. (A) Schematic of the mug8 locus and RNase H reaction products. (B) Poly(A) sites (counts/base/million) determined by 3′-end sequencing of Pla1 wild-type and Y86D cells are plotted as a function of position across the mug8 locus (shown in A) and are depicted as bars at single nucleotide resolution. (C) Northern blot analysis of the RNase H reaction products of mug8. The arrow indicates the position of a slower migrating distribution of polyadenylated mug8 RNase H fragments in Pla1 wild-type cells compared to Y86D. The blot was stripped and reprobed with a DNA oligonucleotide complementary to 7SL RNA.
