mRNA capping enzyme is recruited to the transcription complex by phosphorylation of the RNA polymerase II carboxy-terminal domain

Abstract

Capping of mRNA occurs shortly after transcription initiation, preceding other mRNA processing events such as mRNA splicing and polyadenylation. To determine the mechanism of coupling between transcription and capping, we tested for a physical interaction between capping enzyme and the transcription machinery. Capping enzyme is not stably associated with basal transcription factors or the RNA polymerase II (Pol II) holoenzyme. However, capping enzyme can directly and specifically interact with the phosphorylated form of the RNA polymerase carboxy-terminal domain (CTD). This association occurs in the context of the transcription initiation complex and is blocked by the CTD-kinase inhibitor H8. Furthermore, conditional truncation mutants of the Pol II CTD are lethal when combined with a capping enzyme mutant. Our results provide in vitro and in vivo evidence that capping enzyme is recruited to the transcription complex via phosphorylation of the RNA polymerase CTD.

Figure 1

Capping enzyme binds to phosphorylated RNA Pol II. RNA Pol II was phosphorylated with TFIIH and [γ-³²P]ATP. Capping enzyme was added and the mixture was precipitated with preimmune serum (lane 1) or anti-Ceg1 antibodies (lanes 2–5). Precipitated proteins were separated by SDS-PAGE and the labeled largest subunit of polymerase (Pol IIo) was visualized by autoradiography. As competitors for binding to the capping enzyme, parallel reactions contained GST (lane 3), GST–CTD (lane 4), or phosphorylated GST–CTD (lane 5).

Figure 2

Capping enzyme binds directly to the phosphorylated CTD. Recombinant guanylyltransferase (his₇–CEG1) or purified capping enzyme was incubated with glutathione–agarose beads carrying GST, GST–CTD, or phosphorylated GST–CTD protein. The beads were pelleted and washed extensively. Capping enzyme in the pellet and supernatant (one-tenth of the total) was detected by formation of the enzyme–GMP complex followed by SDS-PAGE and autoradiography.

Figure 3

Capping enzyme is recruited to the transcription complex by phosphorylation of the Pol II CTD. (A) Capping enzyme does not associate with unphosphorylated transcription complexes. Transcription complexes were assembled by incubating hexokinase-treated yeast whole cell extract with beads containing the CYC1 promoter (lane 2) or a control fragment without a promoter (lane 1). The immobilized templates were washed extensively and tested for the presence of various transcription factors by SDS-PAGE and immunoblotting. Rpb1 is the largest subunit of Pol II, Tfb1 is a subunit of TFIIH, Tfa1 is a subunit of TFIIE, Sua7 is TFIIB, and TBP is a TBP subunit of TFIID. Before gel electrophoresis, the beads were incubated with [α-³²P]GTP and the capping enzyme–GMP intermediate (Ceg1–*pG) was detected by autoradiography. (B) Capping enzyme associates with transcription complexes containing a phosphorylated Pol II CTD. Immobilized transcription complexes were assembled in hexokinase-treated extracts. (Lane 1) A reaction with a promoter-less DNA template; (lanes 2–4) reaction with the CYC1 promoter fragment. (Lanes 1,3,4) The complexes were incubated with [γ-³²P]ATP to allow CTD phosphorylation, although the reaction in lane 4 also contained the CTD kinase inhibitor H8. All four reactions received creatine phosphate (CP) and creatine phosphate kinase (CPK) to counteract the hexokinase. The immobilized templates were precipitated and washed, and the pellet was tested for the presence of the Pol II largest subunit (Rpb1), TFIIH (Tfb1), and TFIID (TBP) by immunoblotting. In addition, capping enzyme was labeled by adding [α-³²P]GTP to form the capping enzyme–GMP complex (Ceg1–*pG). Phosphorylation of the Pol II CTD (Rpb1–*Pi) and the capping enzyme–GMP intermediate (Ceg1–*pG) was monitored by autoradiography of the blot (bottom panel).

Figure 4

Genetic interaction between capping enzyme and the Pol II CTD. Plasmid shuffling was used to introduce the wild-type (RPB1) or CTD truncation alleles (rpb1Δ101, rpb1Δ103, rpb1Δ104) of the RNA polymerase large subunit into a wild-type (strain N418, CEG1⁺) or mutant (strain YSB516, ceg1-250) capping enzyme background. Transformants were selected using the LEU2 marker on the plasmids, and the ability of the introduced alleles to support viability was tested by growth on 5-FOA, which selects against the original RPB1/URA3 plasmid. The plates were photographed after 2 days of incubation at 30°C.