Growth properties of pta1 N-terminus mutants. (A) Pta1 deletion mutants. Amino acid sequences encoded by the pta1 alleles are indicated by bars. (B) In vivo expression of truncated forms of Pta1. Whole-cell extracts were prepared from strains expressing either full-length Pta1 or the indicated deletion derivatives and analyzed by Western blotting using a Pta1 monoclonal antibody. The nonviable truncations (top) were expressed as fusions to the GAL4 activation domain two-hybrid tag. (* indicates wild-type [WT] Pta1 of strain XH5.) (C) Plasmid shuffle complementation assay. The XH5 [YCpURA3-PTA1] plasmid shuffle strain expressing epitope-tagged versions of wild-type Pta1 or the indicated deletion derivatives were streaked onto 5-FOA medium to counterselect the YCpURA3-PTA1 plasmid. Plates were photographed following incubation for 4 days at 24°C. (D) Relative growth rates at different temperatures. Strains were grown in liquid YPD medium, and 10-fold serial dilutions were spotted onto YPD plates and incubated for 2 to 3 days at the indicated temperatures.

Different regions are required for Pta1 interactions with Ssu72, Ysh1, and Pti1. (A) Yeast two-hybrid analysis. Plasmids carrying SSU72 fused to the GAL4 activation domain (pGAD-Ssu72) and plasmids carrying pta1 variants fused to the GAL4 DNA binding domain were cotransformed into strain PJ69-4A. Tenfold serial dilutions of the resulting strains were spotted onto CM−LT, as indicated. Activation of HIS3 expression was tested by spotting the same strains onto CM-LTH and incubation for 2 to 3 days at the indicated temperatures. As a control, vector pGAD-Ssu72 was tested in the presence of empty pGBD vector (none). (B) Western blot analysis. Whole-cell extracts were prepared from the different two-hybrid strains grown at 30°C and analyzed by Western blotting using a monoclonal antibody recognizing the GAL4 DNA binding domain. (C) GST-pull down assays. Full-length recombinant Pta1 (GST-Pta1), truncated pta1 [GST-pta1(1-300)], or GST alone was bound to glutathione-Sepharose beads and then incubated with recombinant Ssu72. Proteins bound to the beads and 10% of the recombinant Ssu72 input were separated on a 10% polyacrylamide-SDS gel and detected by Western blot analysis using antibodies directed against GST and Ssu72. “*” indicates full-length GST-Pta1. (D) Pta1 regions. Schematic diagram of Pta1 summarizing the regions needed for wild-type interactions with Ssu72, Ysh1, and Pti1.

The first 300 amino acids of Pta1 inhibit pre-mRNA 3′-end processing in vitro. (A) In vitro cleavage and poly(A) addition assay. Extract prepared from wild-type cells was supplemented with equal amounts of GST alone or GST-Pta1(1-300) and incubated with ATP and ³²P-labeled full-length GAL7-1 RNA (Pre, unreacted precursor) for 20 min at 30°C. Products were resolved on a denaturing 5% polyacrylamide gel and visualized with a PhosphorImager apparatus. Positions of substrate and product are indicated on the left. (B) Model for the role of the first 300 amino acids of Pta1 and Ssu72 in modulating Pta1 activity during mRNA 3′-end processing.

In vivo depletion of Pta1 affects pre-mRNA 3′-end processing in vitro. (A) Western blot analysis. Whole-cell extracts were prepared from pta1-td cells grown at 25°C or shifted to 37°C for the indicated times or from cells expressing normal Pta1 (wild type [WT]). The blot was probed with antibodies against the indicated proteins. (B) In vitro cleavage and poly(A) addition assays. For coupled cleavage-polyadenylation assays (left), the extracts used in A were incubated with ATP and ³²P-labeled full-length GAL7-1 RNA (Pre, unreacted precursor) for 20 min at 30°C. The same conditions were used for poly(A) addition assays (right) except that the precleaved RNA GAL7-9 was used as a precursor. Products were resolved on a denaturing 5% polyacrylamide gel and visualized with a PhosphorImager apparatus. Positions of substrate and product are indicated on the left and right. (C) Pta1 interacts with different CF I and CPF subunits. ³⁵S-labeled in vitro-translated subunits of 3′-end processing factors were incubated with GST or GST-Pta1 bound to glutathione-Sepharose beads. The proteins bound to GST or GST-Pta1, and 10% of the input was separated on a 10% polyacrylamide-SDS gel and detected by autoradiography. (D) Model summarizing interactions between Pta1 and different CF I and CPF subunits.

The Pta1 N-terminal 75 amino acids are required for snoRNA termination, CTD-serine-5 phosphorylation, and gene looping but not for mRNA 3′-end formation. (A) In vitro 3′-end processing assay. Extracts prepared from the strains containing the wild type (WT) and the indicated pta1 mutants, grown at 30°C or shifted to 39°C for 60 min, were incubated with ATP and ³²P-labeled full-length GAL7-1 RNA (Pre, unreacted precursor) for 20 min at 30°C. Products were resolved on a denaturing 5% polyacrylamide gel and visualized with a PhosphorImager apparatus. Positions of substrate and product are indicated on the left. (B) In vivo 3′-end processing and termination assay. Quantitative measurements of β-galactosidase activity were performed using wild-type or mutant strains transformed with reporter vectors lacking or containing an efficient polyadenylation signal upstream of the lacZ gene, as illustrated at the bottom. The amount of readthrough is expressed as a percentage of that obtained from vector lacking the poly(A) site. The data are the averages of data for 48 independent assays of each strain. (C) Analysis of RNAP II CTD serine-5-P levels. Western blots of whole-cell extracts from wild-type (PTA1) or pta1 Δ1-75 strains grown at 30°C or shifted to 39°C for 60 min were probed using antibodies directed against the unphosphorylated form of RNAP II (8WG16), RNAP II CTD serine-5-P (H14), the RNAP II Rpb3 subunit, Pta1, Ssu72, or Rpa1 (loading control). The ssu72-td degron strain grown at 24°C in the presence of 0.1 mM CuSO₄, or shifted to 37°C in the absence of CuSO₄ for 60 min, was included as a control for the accumulation of CTD serine-5-P (25). No Ssu72 protein was detected in the ssu72-td strain at 24°C because the degron-encoded hemagglutinin (HA) epitope tag causes Ssu72-HA to comigrate with Rpb3; however, previous experiments established that Ssu72-HA accumulates in the ssu72-td strain at 24°C but is depleted at 37°C (25). (D) Detection of extended transcripts from snoRNA loci. Total RNA was prepared from strains containing the wild type (PTA1) or the pta1Δ1-75 mutant grown at 30°C or shifted to 39°C for 60 min or the ref2Δ strain grown at 30°C. Extended transcripts were detected by reverse transcription with 5′-end-labeled primers complementary to regions downstream of each snoRNA 3′ end, as depicted at the bottom. Primer extension of U6 RNA was used as a normalization control. (E) Effects of pta1-Δ75 on gene looping at the SEN1, BLM10, GAL10, and HEM3 genes. (Right) Schematic representation of the genes used in the 3C analysis indicating positions of the HindIII sites, drawn approximately to scale, and the divergent P1 and T1 primer pairs. Open reading frame lengths are indicated under each gene. (Left) Gene looping was assayed in the wild-type or pta1 Δ1-75 strains grown at 30°C or shifted to 39°C for 60 min. PCR products derived from the P1-T1 primer pair by 3C analysis were resolved by gel electrophoresis. Control PCR represents an intergenic region of chromosome V generated using convergent primers. P1-T1 PCR products were quantified by dividing P1-T1 PCR signals by control PCR signals for each sample; these ratios were then divided by the ratio of the wild-type 24°C sample for each gene to yield the number depicted beneath each lane.

Pta1 interaction with Ssu72 is not required for cleavage and polyadenylation when the first 300 amino acids of Pta1 are missing. (A) Rescue of degron-mediated Pta1 depletion by pGBD-Pta1. Tenfold serial dilutions of the pta1-td strain transformed with either pGBD-PTA1 or pGBD-pta1 Δ1-300 were spotted onto complete medium lacking uracil and tryptophan in the presence or absence of 0.1 mM CuSO₄ and incubated for 2 or 3 days at 24°C or 37°C. (B) Western blot analysis. Whole-cell extracts were prepared from pta1-td strains transformed with either the wild type (WT) or the pta1 Δ1-300 plasmid grown at 24°C or shifted to 37°C for 40 min and analyzed by Western blotting using antibodies against the indicated proteins. (C) In vitro cleavage and poly(A) addition assays. The extracts used in B were incubated with ATP and ³²P-labeled full-length GAL7-1 RNA (Pre, unreacted precursor) for 5, 10, and 20 min at 30°C. Products were resolved on a denaturing 5% polyacrylamide gel and visualized with a PhosphorImager apparatus. Positions of substrate and product are indicated on the left.