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Comparative Study
. 2001 Jan 16;98(2):445-50.
doi: 10.1073/pnas.98.2.445. Epub 2001 Jan 9.

Cleavage/polyadenylation factor IA associates with the carboxyl-terminal domain of RNA polymerase II in Saccharomyces cerevisiae

D Barillà  1 , B A Lee, N J Proudfoot
Affiliations
Comparative Study

Cleavage/polyadenylation factor IA associates with the carboxyl-terminal domain of RNA polymerase II in Saccharomyces cerevisiae

D Barillà et al. Proc Natl Acad Sci U S A. .

Abstract

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II plays an important role in transcription and processing of the nascent transcript by interacting with both transcription and RNA processing factors. We show here that the cleavage/polyadenylation factor IA of Saccharomyces cerevisiae directly contacts CTD. First by affinity chromatography experiments with yeast extracts we demonstrate that the Rna15p, Rna14p, and Pcf11p subunits of this complex are associated with phosphorylated CTD. This interaction is confirmed for Rna15p by yeast two-hybrid analysis. Second, Pcf11p, but not Rna15p, is shown to directly contact phosphorylated CTD based on in vitro binding studies with recombinant proteins. These findings establish a direct interaction of cleavage/polyadenylation factor IA with the CTD. Furthermore, a quantitative analysis of transcription run-on performed on temperature-sensitive mutant strains reveals that the lack of either functional Rna14p or Pcf11p affects transcription termination more severely than the absence of a functional Rna15p. Moreover, these data reinforce the concept that CTD phosphorylation acts as a regulatory mechanism in the maturation of the primary transcript.

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Figures

Figure 1
Figure 1
CF IA associates with phosphorylated CTD. A whole-cell extract from strain W303-1B was chromatographed onto GST, GST-CTD, and phosphorylated GST-CTD columns. After binding, the beads were pelleted and washed, and bound proteins were detected by immunoblotting with α-Rna15p (A), α-Rna14p (B), and α-Fip1p (C) antibodies. Lane 1, extract (2% of total input); lanes 2–4, pellet fractions (100%); lanes 5–7, supernatant fractions (8%). (D) Affinity chromatography performed with the use of a crude extract from pcf11-9 mutant transformed with pGBD-Pcf11. The protein was detected by α-Gal4 DNA binding domain antibody. Lane 1, extract (10% of total input); lanes 2–4, pellet fractions (60%); lanes 5–7, supernatant fractions (4%).
Figure 2
Figure 2
Pcf11p contains a CTD-binding domain and interacts directly with the CTD. (A) Schematic representation of the domain organization of Pcf11p. The CTD-binding domain is indicated as CTD-BD. (B) Alignment of conserved CTD-binding domains. The N-terminal CTD-binding domain of S. cerevisiae Pcf11p is compared with domains of homologous proteins from S. pombe (SPAC4G9.04C gene product; GenBank accession no. Z69727), Neurospora crassa (B1D1.390 gene product; no. CAB91288), C. elegans (CELR144.2 gene product; no. U23515), Drosophila melanogaster (CG10228 gene product; no. AAF58192), Homo sapiens (Pcf11 homolog; no. AF046935), Arabidopsis thaliana (T4B21.1 gene product; no. AAD03447), S. cerevisiae Nrd1p (no. CAA96158), Rattus norvegicus rA4 (no. U49058), and R. norvegicus SCAF8 (no. U49055). Bold residues indicated with asterisks are identical among at least five of the sequences. In addition to these residues, there are many similarities that are not highlighted. The gaps introduced to maximize the homology are represented by dashed lines. (C) Pcf11p interacts directly with the CTD, whereas Rna15p does not. Recombinant His-tagged Pcf11p and Rna15p were chromatographed on GST, GST-CTD, and phosphorylated GST-CTD resins. The bound fraction was eluted with SDS sample buffer and detected by Western blot with antihistidine monoclonal antibodies. Load (100% of input), pellet (100%), and supernatant (10%) fractions are shown. Lanes 1–3, Rna15p chromatography; lanes 4–6, Pcf11p chromatography. The lower bands detectable in lanes 5 and 6 are degradation products of full-length Pcf11p.
Figure 3
Figure 3
Two-hybrid analysis for Rna15p. Quantitative liquid β-galactosidase assays for the interaction between the CTD and Rna15p (full-length), Rna15p (), and Rna15p () were performed in duplicate for at least two transformants. Mean values are shown in the graph.
Figure 4
Figure 4
Transcription termination in pcf11 and rna14 mutants is more severely disrupted than in rna15. (A) Run-on analysis performed under permissive conditions (24°C) and after a shift to restrictive temperature (37°C) for 30 min in the strains indicated. The numbers at the base of the panels indicate the probes, and M indicates the M13 probe used as a background hybridization control. (B) Schematic diagram of plasmid pGCYC1 showing the arrangement of M13 probes used in the run-on experiments relative to the CYC1 poly(A) site (position 502). (C) Quantitative analysis of run-on profiles. PhosphorImager quantitation was performed by using imagequant software; the transcription levels of probes 1–6 is normalized according to the U content and corrected for the M13 background signal.
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