Volume 31, Issue 1, Pages 91-103 (July 2008) Nuclear mRNA Surveillance in THO/sub2 Mutants Is Triggered by Inefficient Polyadenylation  Cyril Saguez, Manfred Schmid, Jens Raabjerg Olesen, Mohamed Abd El-Hady Ghazy, Xiangping Qu, Mathias Bach Poulsen, Tommy Nasser, Claire Moore, Torben Heick Jensen  Molecular Cell  Volume 31, Issue 1, Pages 91-103 (July 2008) DOI: 10.1016/j.molcel.2008.04.030 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Premature Transcription Termination in THO/sub2 Mutants Is Linked to p(A) Site Position NRO analysis of nuclei harvested from WT, mft1Δ, or ppr2Δ (A); WT, hpr1Δ, sub2-201, or GFP-yra1-8 (B); WT, mft1Δ, or mft1Δ/rrp6Δ (C); and WT(HSP104::KlLEU2) or mft1Δ(HSP104::KlLEU2) (D) cells after a 15 min heat pulse at 42°C. Radioactive RNA samples were hybridized to DNA oligonucleotide NRO probes complementary to the indicated positions upstream and downstream of the HSP104 or HSP104::KlLEU2 p(A) sites. Hybridization to an 18S rRNA probe was used as an internal control. HSP104 NRO signals were quantified by normalizing to the 18S rRNA signal and setting the value of probe 2 in the WT strain to 100. At least three independent experiments were performed to calculate averages and standard deviations. To facilitate easy comparison, results for WT and mft1Δ cells from (A) are repeated in (B) and (C), respectively. Molecular Cell 2008 31, 91-103DOI: (10.1016/j.molcel.2008.04.030) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 RNA 3′ End Processing Is Impaired in THO/sub2-Deficient Extracts (A and B) Coupled in vitro pre-mRNA 3′ end processing reactions using 20 μg of extracts from the indicated strains grown at 25°C. WT strains with identical genetic backgrounds and the pcf11-9 mutant strain were used as positive and negative controls, respectively. (C) Coupled processing reactions using 20 μg of extract from WT or mft1Δ cells grown at 25°C or shifted to 37°C for 1 hr. Extracts were incubated with a precursor containing the CYC1 p(A) site and flanking sequences (tCYC1) for 30 min. Lanes 1 (marked “-”) show the unreacted tCYC1 precursor. RNA bands corresponding to the precursor (top), the upstream cleavage product (bottom), and the polyadenylated product (middle) are indicated on the right side of each image. Molecular Cell 2008 31, 91-103DOI: (10.1016/j.molcel.2008.04.030) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 RNAs Processed in tho2Δ Mutant Extract Are Polyadenylation Impaired and Unstable (A–D) Time course experiments of coupled in vitro processing of CYC1 (A) or cyc1-512 (B), CYC1 “cleavage only” (C), and “polyadenylation only” reactions (D) in WT, tho2Δ, or pap1-1 mutant extracts. The amounts of precursor, polyadenylated, and cleaved species were plotted as a percentage of input precursor added to the respective reactions. Note that some bands on the gel images are outside the linear range. Cleavage reactions (C) were carried out as for coupled reactions (A and B) except that ATP was replaced by CTP. Twenty micrograms of extract was used per reaction. Polyadenylation reactions were carried out as in (A) except that a “precleaved” precursor, which ends at the natural CYC1 p(A) site, was used. Eighty micrograms of extract was used per reaction. Molecular Cell 2008 31, 91-103DOI: (10.1016/j.molcel.2008.04.030) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Polyadenylation Defect in tho2Δ Extract Targets the CPF Complex (A and B) Extract complementation analysis of 30 min coupled 3′ end processing experiments using the tCYC1 precursor and 20 μg of the indicated extracts. In mixing experiments, 20 μg of each extract was used. Molecular Cell 2008 31, 91-103DOI: (10.1016/j.molcel.2008.04.030) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 The Polyadenylation Cofactor Fip1p Is Downregulated in THO/sub2-Deficient Extracts in a Manner Dependent on the Ubiquitin/Proteasome System (A) Western blotting analysis of extracts prepared from WT, tho2Δ, sub2-201, and pcf11-9 strains grown at 25°C using the indicated antibodies specific for components of the CFIA and CPF 3′ end processing factors. A black arrow marks the Fip1p blot. (B) Image of Pti1p blot of WT, hpr1Δ, and tho2Δ extracts prepared from strains grown at 25°C. Migrations of unmodified (Pti1p) and modified Pti1p (Pti1p#) are indicated. (C) Real-time RT-QPCR FIP1 mRNA analysis on total RNA harvested from the indicated strains grown at 25°C. FIP1 mRNA levels were normalized to ACT1 mRNA, which was unaffected by the different conditions. The FIP1/ACT1 mRNA ratio from the WT sample was set to 1. Averages and standard deviations are calculated from two experiments. (D) Fip1p and Pta1p western blotting analysis of extracts (top), or real-time RT-QPCR FIP1 mRNA analysis on total RNA (bottom), prepared from WT and mft1Δ strains grown at 25°C or temperature shifted to 37°C for 1 hr. FIP1 mRNA levels were normalized to U6 RNA, which was unaffected by the different conditions. The FIP1/U6 RNA ratio from the 25°C WT sample was set to 1. Averages and standard deviations are calculated from two experiments. (E) Coupled 3′ end processing of the tCYC1 precursor (top) and western blotting analysis of Fip1p levels (bottom) in extracts prepared from the indicated WT and tho2Δ strains or tho2Δ cells transformed with high-copy plasmids expressing the tho2Δ suppressor genes SUB2 or THO1. Image is labeled as previously. (F) (Left) Fip1p and Nop1p western blotting analysis of extracts prepared from tho2Δ/erg6Δ cells grown at 25°C in the absence (−) or presence (+) of the proteasome inhibitor MG132. (Right) Quantification of the Fip1p/Nop1p western signals. The Fip1p/Nop1p ratio from the (−) MG132 sample was set to 1. (G) Fip1p and Pap1p western blotting analysis, as indicated, of extracts prepared from WT, tho2Δ, ubp3Δ, bre5Δ, tho2Δ/ubp3Δ, and tho2Δ/bre5Δ strains grown at 25°C. Molecular Cell 2008 31, 91-103DOI: (10.1016/j.molcel.2008.04.030) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 3′ End Formation in THO-Deficient Extract Is Restored upon Codeletion of mRNA Surveillance Factors Trf4p or Rrp6p (A) Time course experiments of coupled in vitro 3′ end processing of tCYC1 in extracts from WT, hpr1Δ, hpr1Δ/trf4Δ, and hpr1Δ/rrp6Δ strains. Quantification was done as described for Figure 3. (B) Fip1p western blotting analysis of extracts shown in (A) and pap1-1 extract. Molecular Cell 2008 31, 91-103DOI: (10.1016/j.molcel.2008.04.030) Copyright © 2008 Elsevier Inc. Terms and Conditions