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.1998 Nov 15;12(22):3482-7.
doi: 10.1101/gad.12.22.3482.

Allosteric interactions between capping enzyme subunits and the RNA polymerase II carboxy-terminal domain

Affiliations

Allosteric interactions between capping enzyme subunits and the RNA polymerase II carboxy-terminal domain

E J Cho et al. Genes Dev..

Abstract

mRNA capping is a cotranscriptional event mediated by the association of capping enzyme with the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. In the yeast Saccharomyces cerevisiae, capping enzyme is composed of two subunits, the mRNA 5'-triphosphatase (Cet1) and the mRNA guanylyltransferase (Ceg1). Here we map interactions between Ceg1, Cet1, and the CTD. Although the guanylyltransferase subunit can bind alone to the CTD, it cannot be guanylylated unless the triphosphatase subunit is also present. Therefore, the yeast mRNA guanylyltransferase is regulated by allosteric interactions with both the triphosphatase and CTD.

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Figures

Figure 1
Figure 1
CET1 overexpression suppressesCEG1 temperature-sensitive mutants in an allele-specific manner. (A) Growth of wild-type and conditionalceg1 strains. Strains harboring the indicated plasmids andCEG1 alleles were assayed for growth at 30°C and 37°C. Low copy indicates a centromeric plasmid; high copy indicates a 2μ plasmid. All strains contain a plasmid-borneceg1 allele covering a deletion of the chromosomalCEG1 locus, with the exception of *ceg1-250, in which the mutant allele is integrated at the chromosomal locus. (B) Whole-cell extracts were prepared from wild-type orceg1 mutant strains grown for 4.5 hr at 30°C or 37°C. Extracts were assayed by immunoblotting with anti-Ceg1 antibody. (Lane 1) 100 ng Ceg1; (lane 2) no extract; (lanes 3–8) 40 μg of extract from the indicated strain. (C) Schematic representation of Ceg1 as modeled by theChlorella virus PBCV-1 guanylyltransferase structure (Hakansson et al. 1997). Relative locations of mutant residues are denoted by * and the allele number; (K) active site lysine.
Figure 1
Figure 1
CET1 overexpression suppressesCEG1 temperature-sensitive mutants in an allele-specific manner. (A) Growth of wild-type and conditionalceg1 strains. Strains harboring the indicated plasmids andCEG1 alleles were assayed for growth at 30°C and 37°C. Low copy indicates a centromeric plasmid; high copy indicates a 2μ plasmid. All strains contain a plasmid-borneceg1 allele covering a deletion of the chromosomalCEG1 locus, with the exception of *ceg1-250, in which the mutant allele is integrated at the chromosomal locus. (B) Whole-cell extracts were prepared from wild-type orceg1 mutant strains grown for 4.5 hr at 30°C or 37°C. Extracts were assayed by immunoblotting with anti-Ceg1 antibody. (Lane 1) 100 ng Ceg1; (lane 2) no extract; (lanes 3–8) 40 μg of extract from the indicated strain. (C) Schematic representation of Ceg1 as modeled by theChlorella virus PBCV-1 guanylyltransferase structure (Hakansson et al. 1997). Relative locations of mutant residues are denoted by * and the allele number; (K) active site lysine.
Figure 1
Figure 1
CET1 overexpression suppressesCEG1 temperature-sensitive mutants in an allele-specific manner. (A) Growth of wild-type and conditionalceg1 strains. Strains harboring the indicated plasmids andCEG1 alleles were assayed for growth at 30°C and 37°C. Low copy indicates a centromeric plasmid; high copy indicates a 2μ plasmid. All strains contain a plasmid-borneceg1 allele covering a deletion of the chromosomalCEG1 locus, with the exception of *ceg1-250, in which the mutant allele is integrated at the chromosomal locus. (B) Whole-cell extracts were prepared from wild-type orceg1 mutant strains grown for 4.5 hr at 30°C or 37°C. Extracts were assayed by immunoblotting with anti-Ceg1 antibody. (Lane 1) 100 ng Ceg1; (lane 2) no extract; (lanes 3–8) 40 μg of extract from the indicated strain. (C) Schematic representation of Ceg1 as modeled by theChlorella virus PBCV-1 guanylyltransferase structure (Hakansson et al. 1997). Relative locations of mutant residues are denoted by * and the allele number; (K) active site lysine.
Figure 2
Figure 2
Ceg1 guanylylation is inhibited by binding to phosphorylated CTD and stimulated by Cet1. (A) Recombinant guanylyltransferase (Ceg1), RNA triphosphatase (Cet1) or both (Cet1 + Ceg1) were incubated with glutathione agarose beads carrying GST, GST–CTD, or phosphorylated GST–CTD protein [GST–CTD(P)]. Beads were pelleted, and bound proteins were detected by immunoblotting (top,middle). The ability of Ceg1 to be guanylylated was assayed by the addition of [α-32P]GTP (Ceg1-*pG,bottom). (B) 150 ng Ceg1 was incubated with GST–CTD(P) in the absence (lane9) or presence (lane10) of 200 ng of Cet1 and precipitated as inA. A standard curve was generated in the presence of GST–CTD with 1.4 ng (lanes1,5), 7.0 ng (lanes2,6), 14 ng (lanes3,7), or 42 ng (lanes4,8) of Ceg1. Lanes 5–8 also contained 100 ng of Cet1. Ceg1 used in this experiment was completely deguanylylated (see Materials and Methods). Ceg1 protein (top) and enzyme-GMP complex (bottom) were visualized as inA and quantitated by densitometry.
Figure 3
Figure 3
Amino acids 205–265 of Cet1 are required for interaction with and stimulation of Ceg1. (A) Coimmunoprecipitation of Ceg1 with various deletions of Cet1. Ceg1 was incubated with Cet1, Cet1(1–265), Cet1(265–549), Cet1(205–549), or Cel1(1–236) and the mixture precipitated with either preimmune serum (P) or anti-Ceg1 immune serum (I) as described in Materials and Methods. Proteins were visualized by anti-6× His monoclonal antibody. (+) A positive control lane containing the indicated triphosphatase derivative. The signal of Ceg1 protein was masked by the heavy chain of rabbit IgG which migrates with Ceg1. (*) The Cet1 derivatives coprecipitated with Ceg1. (B) Activation of guanylyltransferase by Cet1 derivatives. Ceg1 was incubated with Cet1, Cet1(1–265), Cet1(265–549), Cet1(205–549), or Cel1(1–236). The mixture was allowed to interact with GST, GST–CTD, or phosphorylated GST–CTD [GST–CTD(P) was radioactively labeled as described in Fig. 2]. Formation of the enzyme-GMP intermediate (Ceg1-p*G) was assayed on the precipitated beads. (C) Summary of Cet1 domains required for RNA 5′-triphosphatase activity, Ceg1 subunit interaction, and guanylyltransferase activity stimulation.
Figure 4
Figure 4
Amino acids 205–265 of Cet1 are sufficient for interaction with Ceg1. GST (lane1) or GST fused to amino acids 205–265 of Cet1 (lane4) were incubated alone (lanes2,4) or with Ceg1 (lanes3,5). Reactions were precipitated with anti-Ceg1 antibody and pellets were washed and then immunoblotted with anti-GST monoclonal antibody.
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References

    1. Cho E-J, Takagi T, Moore CR, Buratowski S. mRNA capping enzyme is recruited to the transcription complex by phosphorylation of the RNA polymerase II carboxy-terminal domain. Genes & Dev. 1997;11:3319–3326. - PMC - PubMed
    1. Eisenmann DM, Arndt KM, Ricupero SL, Rooney JW, Winston F. SPT3 interacts with TFIID to allow normal transcription in Saccharomyces cerevisiae. Genes & Dev. 1992;6:1319–1331. - PubMed
    1. Fresco LD, Buratowski S. Active site of the mRNA capping enzyme guanylyltransferase from Saccharomyces cerevisiae: Similarity to the nucleotidyl attachment motif of DNA and RNA ligases. Proc Natl Acad Sci. 1994;91:6624–6628. - PMC - PubMed
    1. ————— Conditional mutants in the yeast mRNA capping enzyme show that the cap enhances, but is not required for, mRNA splicing. RNA. 1996;2:584–595. - PMC - PubMed
    1. Hakansson K, Wigley DB. Structure of a complex between a cap analogue and mRNA guanylyltransferase demonstrates the structural chemistry of RNA capping. Proc Natl Acad Sci. 1998;95:1505–1510. - PMC - PubMed

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