Effects of Pla1-Y86D on polyadenylation of mRNAs in vivo. Total RNA (10 μg) prepared from pla1 wild-type and pla1-Y86D cells was treated with RNase H in the presence of DNA oligonucleotides complementary to mRNAs either with (+) or without (−) oligo(dT). The reaction products were resolved by denaturing-PAGE along with an aliquot of radiolabeled DNA marker and transferred to a nylon membrane. Northern blot analyses of the RNase H reaction products for (A) act1, (B) adh1, (C) pgk1, (D) fba1, (E) ssa2, (F) eno101, and (G) rps13 are shown. The positions and sizes (nt) of the DNA markers are indicated in each panel. A schematic of the genomic locus and detected fragments is shown above each blot (drawn to scale). The end of the coding sequence is depicted by a yellow arrow and the 3′-UTR is shown as a blue extension in the direction of synthesis. The location of the complementary DNA oligonucleotide used to generate RNase H cleavage products and the 5′-32P labeled oligonucleotide probe used for detection are shown. The gene-specific RNase H products downstream from the cleavage sites are shown as yellow wavy lines with poly(A) tails, or without poly(A) tails when treated with RNase H in the presence of oligo(dT). The poly(A) sites determined by 3′-end sequencing of wild-type cells are plotted as a function of position across the gene and are depicted as bars at single nucleotide resolution.
