Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription

Abstract

The activities of several mRNA processing factors are coupled to transcription through binding to RNA polymerase II (Pol II). The largest subunit of Pol II contains a repetitive carboxy-terminal domain (CTD) that becomes highly phosphorylated during transcription. mRNA-capping enzyme binds only to phosphorylated CTD, whereas other processing factors may bind to both phosphorylated and unphosphorylated forms. Capping occurs soon after transcription initiation and before other processing events, raising the question of whether capping components remain associated with the transcription complex after they have modified the 5' end of the mRNA. Chromatin immunoprecipitation in Saccharomyces cerevisiae shows that capping enzyme cross-links to promoters but not coding regions. In contrast, the mRNA cap methyltransferase and the Hrp1/CFIB polyadenylation factor cross-link to both promoter and coding regions. Remarkably, the phosphorylation pattern of the CTD changes during transcription. Ser 5 phosphorylation is detected primarily at promoter regions dependent on TFIIH. In contrast, Ser 2 phosphorylation is seen only in coding regions. These results suggest a dynamic association of mRNA processing factors with differently modified forms of the polymerase throughout the transcription cycle.

Figure 1

Capping enzyme is localized to promoter regions. Cross-linked and sheared chromatin was immunoprecipitated with the indicated antibodies. After reversal of cross-linking and purification of the DNA, PCR was used to test for the presence of promoter or coding sequences (CDS) from the indicated genes. Each PCR reaction contained a second primer pair that amplifies a region of chromosome VII devoid of ORFs, thus providing an internal background control (indicated by *). Input (bottom) shows the signal from the chromatin before immunoprecipitation. HA–Rpb3 is an epitope-tagged subunit of RNA Pol II, Ceg1 is the guanylyltransferase subunit of capping enzyme, and TFIIE was immunoprecipitated with a polyclonal antibody directed against the small subunit Tfa2. Primer pairs are as described in Materials and Methods. The numbers immediately below each lane are quantitated PCR signals in arbitrary units after normalization for amplification efficiency and subtraction of background.

Figure 2

Capping enzyme and cap methyltransferase show different patterns of association during transcription. (A) Chromatin IP/PCR was carried out as in Fig. 1 except that the Intergenic region control primers (last lane) were not included in the other PCR reactions. The immunoprecipitating antibodies are listed to the left of the autoradiographs. Cet1 is the RNA triphosphatase subunit of capping enzyme, and Abd1 is the mRNA guanine N7-methyltransferase. The small subunit of TFIIE (Tfa2) was used to monitor the transcription initiation complex and a triple HA-tagged polymerase subunit (HA–Rpb3) was used to monitor Pol II. (B) Schematic diagram of PCR primer pairs for PMA1, PDR5, and ADH1. Open bar represents the gene ORF. The gray bars represent PCR products with coordinates relative to the initiation codon of the ORF. Mapped features of PMA1, ADH1, and PDR5 promoters are also indicated.

Figure 3

Differences between capping enzyme and cap methyltransferase are not due to the specific antibodies. Chromatin IP/PCR reactions were carried out on strains containing HA-tagged Ceg1 (capping enzyme guanylyltransferase), Abd1 (methyltransferase), TATA-binding protein (TBP), or Rpb3 (Pol II). Signals were normalized to the input DNA signal (right panels) and background (Intergenic primer pair on chromosome V, denoted by asterisk) subtracted. The ratio of cross-linking in coding (CDS) to promoter regions was calculated for each factor (see table at bottom). A zero indicates that the ratio was <0.05.

Figure 4

mRNA cleavage factor IB (Hrp1) cross-links throughout the gene. Chromatin IPs were performed using antibodies indicated to the left of the panels. Hrp1 is an RNA-binding protein implicated in polyadenylation and mRNA degradation (Kessler et al. 1997; Chen and Hyman 1998; Minvielle-Sebastia et al. 1998). For comparison, cross-linking patterns of the capping enzyme guanylyltrasferase Ceg1, the Rbp3 subunit of RNA Pol II, and the Input chromatin are shown.

Figure 5

Phosphorylation of CTD Ser 5 is localized to promoters. Chromatin IP/PCR was performed with monoclonal H14, an antibody that specifically recognizes Ser 5 phosphorylation of the CTD heptamer repeat (Bregman et al. 1995; Patturajan et al. 1998). Primer pairs for promoter or coding sequences (CDS) of the indicated genes were used. In addition, PCR reactions contained a primer pair recognizing an Intergenic region of chromosome V as an internal negative control.

Figure 6

The TFIIH kinase Kin28 is required for CTD Ser 5 phosphorylation and capping enzyme recruitment to promoters in vivo. Chromatin IP/PCR reactions were carried out with wild-type and Kin28 mutant yeast strains. Kin28 is the catalytic kinase subunit of basal transcription factor TFIIH. The Kin28 (T17D) allele produces a stable protein with dramatically reduced kinase activity (M. Keogh and S. Buratowski, unpubl.). Immunoprecipitating antibodies are indicated to the left of the autoradiographs. The CTD signal is with monoclonal 8WG16, which recognizes the unphosphorylated CTD, whereas CTD phosphorylated at Ser 5 (CTD-S5-P) is recognized by the monoclonal H14. TFIIE was monitored using a polyclonal antiserum against the small subunit Tfa2.

Figure 7

Ser 2 phosphorylation of the CTD is localized to coding regions of the gene. Chromatin IP/PCR reactions were carried out using the indicated antibodies. The presence of the CTD is probed with monoclonal antibody 8WG16, the phosphorylated Ser 5 (CTD-S5-P) is recognized by monoclonal H14, and the phosphorylated Ser 2 (CTD-S2-P) is recognized by monoclonal H5. Primer pairs are as in previous figures. Asterisks in B designate the product from the Intergenic primer pair (chromosome VII), which was used as an internal negative control in this experiment.

Figure 8

Schematic diagram of the RNA Pol II CTD cycle. The white circle represents Pol II, the black line represents the gene with the arrow denoting the promoter, and the smaller shapes represent capping enzyme and other RNA-processing factors. See discussion for details.