FIG. 1.

Increased retention of Rna15p on polyadenylated transcripts in certain export mutants. (A) Rna15p is retained on polyadenylated GAL7 transcript in hpr1Δ, tho2Δ, sub2-201, mex67-5, mex67-6, mtr2-26, GFP-yra1-8, and sac3Δ but not in npl3-3 or rat7-1 mutant cells. Cells were grown in liquid YPGal medium at 25°C and then shifted to 37°C for 1 h before formaldehyde cross-linking. Whole-cell lysates from isogenic WT or mutant strains were incubated with Rna15p antiserum to immunoprecipitate the cross-linked protein-RNA complex. The immunoprecipitated RNA was reverse transcribed with an oligo(dT) primer in the presence of reverse transcriptase (+). To control for DNA contamination, the reverse transcriptase was also omitted from some samples (−). Each lane shows the radioactive PCR signal with a primer set within the GAL7 coding region after reverse transcription. The lanes labeled “Input” show RT-PCR amplification of input RNA from each experiment. The results for each mutant and WT control are representative of at least three independent experiments, except for hpr1Δ, which was analyzed twice. (B) Rna15p is retained on polyadenylated SSA4 transcript in the mex67-5 mutant. The RNA immunoprecipitation assay was performed as in Fig. 1A, but a primer set upstream of the SSA4 poly(A) site was used in the PCR. Immunoprecipitation with preimmune serum (pre) is shown as a control. (C) Western blotting of cell lysates with antibody against the CF IA subunit Rna15p, the CPF subunit Pta1p, or the Rpb3p subunit of RNAPII. Extract was prepared from WT, mex67-5, and mex67-6 strains after shifting to 37°C for 1 h. (D) Pta1p is retained on polyadenylated RNA in the mex67-6 mutant. The RNA immunoprecipitation assay was performed as for panel B but with Pta1p antiserum instead of Rna15p antiserum. Pta1p levels on polyadenylated RNAs were increased for both GAL7 (top) and SSA4 (bottom) transcripts. Immunoprecipitation with preimmune serum (lanes marked “pre”) is shown as a control.