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. 1997 Nov 25;94(24):12898-903.
doi: 10.1073/pnas.94.24.12898.

Mammalian capping enzyme complements mutant Saccharomyces cerevisiae lacking mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase II

Z Yue  1 , E Maldonado, R Pillutla, H Cho, D Reinberg, A J Shatkin
Affiliations

Mammalian capping enzyme complements mutant Saccharomyces cerevisiae lacking mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase II

Z Yue et al. Proc Natl Acad Sci U S A. .

Abstract

5'-Capping is an early mRNA modification that has important consequences for downstream events in gene expression. We have isolated mammalian cDNAs encoding capping enzyme. They contain the sequence motifs characteristic of the nucleotidyl transferase superfamily. The predicted mouse and human enzymes consist of 597 amino acids and are 95% identical. Mouse cDNA directed synthesis of a guanylylated 68-kDa polypeptide that also contained RNA 5'-triphosphatase activity and catalyzed formation of RNA 5'-terminal GpppG. A haploid strain of Saccharomyces cerevisiae lacking mRNA guanylyltransferase was complemented for growth by the mouse cDNA. Conversion of Lys-294 in the KXDG-conserved motif eliminated both guanylylation and complementation, identifying it as the active site. The K294A mutant retained RNA 5'-triphosphatase activity, which was eliminated by N-terminal truncation. Full-length capping enzyme and an active C-terminal fragment bound to the elongating form and not to the initiating form of polymerase. The results document functional conservation of eukaryotic mRNA guanylyltransferases from yeast to mammals and indicate that the phosphorylated C-terminal domain of RNA polymerase II couples capping to transcription elongation. These results also explain the selective capping of RNA polymerase II transcripts.

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Figures

Figure 1
Figure 1
MCE and human capping enzyme cDNA clones. (A) Schematic diagram of the ORFs (1,794 bp, solid bars), relative sizes of the untranslated regions, and the sequences and positions of initiation sites and polyadenylation signals. (B) Predicted amino acid sequences of the mouse and human enzymes. Residues that are identical (95% of total) are omitted from the human sequence.
Figure 2
Figure 2
Sequence alignments of cellular mRNA guanylyltransferases. The deduced amino sequences of the C-terminal regions of the mouse and human enzymes (residues 232–597) are compared with C. elegans CEL-1 (residues 238–573; ref. 19) and to the nearly full-length S. pombe (13) and S. cerevisiae (9) guanylyltransferase subunits of capping enzyme. Dark background indicates regions of sequence identity; the active site motif, including Lys-294, in the mammalian enzymes is bracketed.
Figure 3
Figure 3
Expression of capping enzyme transcripts in vivo. (A) Poly(A)+ RNAs prepared from mouse adult tissues (Left) and embryos at the indicated days of gestation (Right) were probed with a mouse EST as described in Materials and Methods. (B) Human adult tissue poly(A)+ RNAs were probed with the same EST and a human β-actin cDNA. Positions of marker RNAs and hybridized transcripts (arrows) are indicated.
Figure 4
Figure 4
In vitro synthesis and specific immunoprecipitation of MCEs. (A) 35S-methionine-labeled proteins were synthesized in vitro by using luciferase (Luc) cDNA (lane 1) or cDNAs for wild type (lane 2), mutant (lane 3), and C-terminal fragment (Δ210, lane 4) of MCE. Products were analyzed by SDS/PAGE and autoradiography. (B) Products shown in A were immunoprecipitated with αMCE and analyzed by SDS/PAGE and autoradiography.
Figure 5
Figure 5
Guanylyltransferase activity of MCEs. (A) In vitro translated and immunoprecipitated proteins were incubated with α[32P]GTP as described in Materials and Methods and analyzed by SDS/PAGE and autoradiography. Lane 1, no added DNA; lanes 2–5, products synthesized with cDNA templates for full-length MCE, C-terminal fragment (Δ210), and mutants K294A and K290A, respectively. Mouse L cell nuclear extract (lane 6) and partially purified HeLa cell capping enzyme (lane 7) were immunoprecipitated with αMCE and then incubated with α[32P]GTP. Proteins were analyzed by SDS/PAGE and autoradiography. (B) 5′-Terminal guanylylation of T7 transcripts was assayed by incubating the RNA with α[32P]GTP and immunoprecipitates of in vitro synthesized wild-type (lane 2) or K294A mutant (lane 3) MCE or vaccinia virus capping enzyme (VCE, lane 4). (C) Cap formation on the radiolabeled RNAs shown in B was verified by digestion with P1 nuclease followed by alkaline phosphatase and TLC analysis of the digests with authentic GpppG and GppppG as markers.
Figure 6
Figure 6
MCE contains RNA 5′-triphosphatase. RNA containing 5′-terminal γ[32P]GTP, prepared as described in Materials and Methods, was incubated with in vitro-synthesized, immunoprecipitated MCE wild type (lane 2), mutant K294A (lane 3), N-terminal truncation mutants Δ210 (lane 4), and Δ144 (lane 5) or vaccinia virus capping enzyme (lane 1). Samples were analyzed for release of labeled Pi by TLC and autoradiography.
Figure 7
Figure 7
Complementation of S. cerevisiae lacking mRNA guanylyltransferase. Growth was measured on selective media using CEG1− cells transformed with empty parental pG-1 (vector), pG-1 containing wild-type (MCE) or mutant (K294A) MCE or with a CEG1-containing URA3 plasmid (CEG1) as detailed in Materials and Methods.
Figure 8
Figure 8
Capping enzyme directly interacts with the phosphorylated form of RNA pol II. (A) A HeLa cell protein fraction containing approximately equal amounts of the phosphorylated (pol IIo) and unphosphorylated (pol IIa) forms of pol II (lane 1) was mixed with αMCE-protein A beads in the presence or absence of affinity-purified HeLa capping enzyme (HCE, lane 2). Immunoprecipitates (lane 3 and 4) were extensively washed and loaded onto a 5–20% gradient SDS-polyacrylamide gel with the input (10%, lanes 1 and 2) for Western blot analysis. Immunoblots were developed for the CTD of the largest subunit of pol II (Upper) or capping enzyme (Lower). (B) The carboxy-terminal domain of capping enzyme is sufficient to mediate direct and selective interaction with the phosphorylated form of pol II. The HeLa protein fraction containing the two forms of pol II (lane 1) was mixed with antihexahistidine tag antibody (α6xHis)-protein G beads in the presence or absence of purified, recombinant hexa-histidine tagged carboxy-terminal domain of MCE Δ210. Immunoprecipitates (lanes 2 and 3) were extensively washed and loaded onto a 5–20% gradient SDS-polyacrylamide gel with the input (10%, lane 1) for Western blot analysis. Immunoblots were developed for the CTD of the largest subunit of pol II (Upper) or MCE (Lower).
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