Volume 13, Issue 3, Pages 377-387 (February 2004) A Function of Yeast mRNA Cap Methyltransferase, Abd1, in Transcription by RNA Polymerase II  Stephanie C. Schroeder, Diego A.R. Zorio, Beate Schwer, Stewart Shuman, David Bentley  Molecular Cell  Volume 13, Issue 3, Pages 377-387 (February 2004) DOI: 10.1016/S1097-2765(04)00007-3 Copyright © 2004 Cell Press Terms and Conditions

Figure 1 Inactivation of the Yeast Abd1 mRNA Cap 7-Methyltransferase Selectively Inhibits Pol II Binding at 5′ Ends of Genes (A) PCR analysis of anti-pol II ChIP in isogenic WT and abd1 ts mutants at 25°C (−) or 37°C for 2 hr (+) with primer pairs for the 5′ ends of indicated genes. HMR is a negative control. Anti-pol II is polyclonal anti-CTD that recognizes phosphorylated and nonphosphorylated isoforms (Schroeder et al., 2000). Lanes 1–7, titration of input DNA (in); lane 8, irrelevant antibody control, 70A (C); lanes 9–32, anti-pol II IPs with the indicated abd1 mutants. t.s., temperature shift. Signals at 37°C were reduced relative to 25°C by between 50% (for ENO2 in abd1-8 and 1-41) and 75% (for ENO2 in abd1-15 and 1-16). The maximum reduction for either ACT1 or TEF1 was 30% (for TEF1 in abd1-15). (B) Run-on transcription (lanes 1 and 2) and pol II ChIP (lanes 3 and 4) in WT and abd1-8 cells grown at 37°C (45 min) and treated with 0.5% sarkosyl. (C) Anti-pol II ChIP in isogenic WT and ceg1 mutants in guanylyltransferase as in (A). in, input. Molecular Cell 2004 13, 377-387DOI: (10.1016/S1097-2765(04)00007-3) Copyright © 2004 Cell Press Terms and Conditions

Figure 2 Inhibition of Capping in Temperative-Sensitive Mutants of the 7-Methyltransferase, ABD1, and Guanylyltransferase, CEG1 RNA from WT and mutants after 2 hr at 37°C was fractionated by binding to GST-eIF4E (McCracken et al., 1997). Rabbit globin RNA spiked into the binding reaction is a control for the cap selection. RNase protection of capped (+) and uncapped (−) fractions is shown with antisense probes for the 3′ end of the SSA1 and β-globin. Molecular Cell 2004 13, 377-387DOI: (10.1016/S1097-2765(04)00007-3) Copyright © 2004 Cell Press Terms and Conditions

Figure 3 Gene-Specific Effects of Abd1 on Pol II Binding to GAL Promoter Regions (A) Abd1 inactivation specifically inhibited pol II binding at GAL1, GAL2, and GAL10 promoters. Pol II ChIP at 5′ ends of GAL genes in WT and abd1-8 at 37°C. Inputs (in) and anti-pol II (CTD) IPs are shown. Cells were grown in selective media with 2% raffinose (R) at 25°C then shifted to 37°C for 45 min, and 2% galactose (G) was added to half the culture for 30 min. Pol II crosslinking in galactose at 37°C was reduced in abd1-8 relative to WT by 90%, 75%, 45%, and 75% for GAL1, 2, 7, and 10, respectively. (B) Abd1 inactivation inhibited binding of TFIIB, Ser5 phosphorylated pol II (CTD-P) and Ceg1 at the GAL1 5′ end. abd1-8 cells were grown in selective media with 2% raffinose, and galactose was added to 2% for 20 min at 25°C. Half the culture was then temperature shifted (t.s.) to 37°C (+), and half was kept at 25°C (−) for 45 min before crosslinking. Input (in) and ChIPs with the indicated antibodies and an irrelevant control (C) were analyzed with primer pairs from the 5′ ends of the indicated genes. Anti-CTD-P is rabbit anti-Ser5-PO4 (Schroeder et al., 2000). (C) WT Abd1 restores pol II binding at promoters in abd1-15 at 37°C. Pol II binding to the 5′ ends of indicated genes was analyzed by anti-pol II ChIP (CTD) in abd1-15 transformed with vector, pRS316 (lanes 2 and 3), or the CEN plasmid p360-ABD1 (lanes 4 and 5). Galactose induction was as in Figure 3A, and cells were crosslinked at 25°C (−) and after 2 hr at 37°C (+). Molecular Cell 2004 13, 377-387DOI: (10.1016/S1097-2765(04)00007-3) Copyright © 2004 Cell Press Terms and Conditions

Figure 4 The PGK1 Promoter Confers Abd1-Sensitive Binding of Pol II Pol II ChIP of TEF1, ACT1, ENO2, PGK1, and the PGK1-ACT1 chimera integrated at HO are shown with PCR primers specific to their 5′ ends. TEL VIR is a negative control. Lane 1, input (in); lanes 2–5, anti-pol II IPs for WT (DBY528) and abd1-15 (DBY530) at 25°C (−) and temperature shifted (t.s.) to 37°C (+). % ratios of pol II ChIP signals at 37°C/25°C from five independent PCR reactions from three separate ChIPs are quantified at right. Molecular Cell 2004 13, 377-387DOI: (10.1016/S1097-2765(04)00007-3) Copyright © 2004 Cell Press Terms and Conditions

Figure 5 Altered Pol II Distribution along TEF1 in abd1 Mutants (A) Pol II ChIP signals were quantified for the 5′ and 3′ ends of TEF1 in WT and seven abd1 mutant alleles at 25°C and 37°C (2 hr). Signals were normalized to input, and % 3′:5′ ratios of pol II density were plotted. Means and average deviations (n = 3) are shown. Map shows 5′, middle, and 3′ PCR products of TEF1 centered at +88, +652, and +1274 relative to the ATG. (B) Total pol II (top panel, rabbit anti-CTD) and Ser5-phosphorylated pol II (bottom panel, H14, anti-CTD-P) ChIP with PCR primers for the 5′, middle, and 3′ ends of TEF1 in ts mutants at 25°C (−) and after 2 hr (abd1) or 1 hr (kin28) at 37°C (+). Lane 1, input (in); lanes 2–11, isogenic WT and abd1 strains; lanes 12 and 13, kin28-3. (C) Quantification of the results in (B). Top: Total pol II ChIP signals normalized to inputs for the middle (hatched bars) and 3′ end (black bars) of TEF1 relative to the 5′ end (n = 6). Bottom: Phospho-Ser5 pol II ChIP signals normalized to inputs for the middle of TEF1 relative to the 5′ end (n = 3). Molecular Cell 2004 13, 377-387DOI: (10.1016/S1097-2765(04)00007-3) Copyright © 2004 Cell Press Terms and Conditions

Figure 6 The Transcriptional Function of Abd1 Is Independent of Methyltransferase Activity Expression of the catalytic site mutant D194A restores pol II binding to the 5′ ends of GAL2, GAL10, ENO2, PGK1, and normal 5′:3′ pol II distribution on TEF1 in abd1-15. PCR primers are for the 5′ ends of respective genes and for the middle of TEF1 as in Figure 5. Input (lane 1) and anti-pol II IPs of abd1-15 transformed with vector pRS416GAL1 (lanes 2 and 3), p360-ABD1 (lanes 4 and 5), or pRS416GAL1 abd1-D194A (lanes 6 and 7). Cultures were galactose induced for 30 min at 25°C as in Figure 3B, then half the culture was shifted to 37°C (+), and the remainder was grown at 25°C (−) for 2 hr. The average (n = 3) TEF1 mid:5′ pol II density ratios at 37°C are: lane 3, vector 47%; lane 5, ABD1 97%; lane 7, D194A 94%. Molecular Cell 2004 13, 377-387DOI: (10.1016/S1097-2765(04)00007-3) Copyright © 2004 Cell Press Terms and Conditions

Figure 7 Model for How the Capping Enzyme Abd1 Modulates Pol II Function After recruitment to the promoter, CTD phosphorylation (P) by Kin28 permits binding of Abd1 and Ceg1-Cet1. Cet1 inhibits initiation (Myers et al., 2002) whereas Abd1 exerts two positive effects denoted by open arrows: (1) enhancement of pol II binding at promoters (e.g., PGK1, ENO2, GAL1, GAL10) and (2) enhancement of pol II transit from the promoter to the middle of the gene (e.g., ACT1, TEF1). The latter process probably corresponds to promoter clearance and/or early elongation which is accompanied by dephosphorylation of the CTD on Ser5 residues. During elongation phosphorylation of the CTD occurs on Ser2 by kinases including Ctk1, and most Ceg1 is released but some Abd1 remains bound (Komarnitsky et al., 2000; Schroeder et al., 2000). Molecular Cell 2004 13, 377-387DOI: (10.1016/S1097-2765(04)00007-3) Copyright © 2004 Cell Press Terms and Conditions