1. Volume 9, Issue 5, Pages 1101-1111 (May 2002)
Functional Interaction of Yeast Pre-mRNA 3′ End Processing Factors with RNA Polymerase II 
Donny D Licatalosi, Gabrielle Geiger, Michelle Minet, Stephanie Schroeder, Kate Cilli, J.Bryan McNeil, David L Bentley 
Molecular Cell 
Volume 9, Issue 5, Pages (May 2002)
DOI: /S (02)00518-X

2. Figure 1 Genetic Interaction between CTD Truncation and pcf11 Mutants
Growth of isogenic strains with mutations in PCF11 (pcf11-2) or RPB1 (CTD truncation Δ104, or rpo21-18). Note that when pcf11-2 is combined with the CTD truncation (row 6), the cells grow slower than either single mutant (rows 2 and 4) or the pcf11-2, rpo21-18 double mutant (row 5) at 25°C, 30°C, and 32°C. Cells were grown under selection for Leu+ and Ura+ and serial dilutions were plated on YPD plates incubated at the indicated temperatures.
Molecular Cell 2002 9, DOI: ( /S (02)00518-X)

3. Figure 2
Accurate but Less Efficient Poly(A) Site Cleavage in Transcripts Made by RNA pol IIΔCTD
(A) Anti-pol II ChIP in DBY154 (rpb1-1, HA-Rpb3) at 25°C and after 45 min at 37°C. Cells were transformed with the indicated pFL38 plasmids. 32P-labeled PCR products for SSA1, ACT1, and ENO2 5′ ends, and HMR-E (negative control) are shown for input and IPs with anti-CTD, -HA, and 70A control antibody. Note that a significant amount of HA-Rpb3 cross-links to SSA1 (but not ACT1 or ENO2) in rpb1ΔCTD cells at 37°C (lane 22) relative to the vector control (lane 6).
(B) SSA1 transcripts made by RNA pol IIΔCTD are cleaved at the poly(A) site. Total RNA from DBY121 (rpb1-1, Δxrn1) transformed with the indicated pFL38 plasmids was harvested at intervals after shifting to 37°C and analyzed by RNase protection with 3′ SSA1 and 5S (loading control) probes. Cleaved (C) and uncleaved precursors (U) are indicated.
Molecular Cell 2002 9, DOI: ( /S (02)00518-X)

4. Figure 3
The RNA Pol II CTD Affects Poly(A) Site Selection but Not Splicing in Yeast
(A) Diagram of the pDL16 reporter gene. The five ACT1 poly(A) sites and RNase protection probes I (pBS RP51A-lacZ) and II (pBS zeoACT1; see Experimental Procedures) are indicated. Protection products from ACT1 and pDL16 transcripts do not overlap.
(B) RNase protection of total RNA from DBY120 transformed with pDL16 and the indicated pFL38 plasmids. RNA was harvested at intervals after shifting to 37°C and analyzed with probe II. pDL16 transcripts cleaved at each poly(A) site (C), uncleaved precursors (U), and the 5S loading control are indicated. Chromosomal ACT1 transcripts cleaved at sites 1 and 4 are shown in the bottom panel.
(C) RNase protection of pDL16 transcripts with probe I. The gel was quantified by Phophorimager and % splicing calculated. Note that most transcripts made by RNA pol IIΔCTD are accurately spliced.
Molecular Cell 2002 9, DOI: ( /S (02)00518-X)

5. Figure 4 RNA Pol IIΔCTD Transcripts Have Short Poly(A) Tails
(A) Poly(A)+ and poly(A)− RNA from DBY276 transformed with pFL38, pFL38-RPB1, or pFL38-rpb1ΔCTD at 25°C and after 180 min. at 37°C was assayed by RNase protection for cleaved SSA1 transcripts as in Figure 2B. Poly(A)+:poly(A)− was calculated after normalizing to the globin control (see Experimental Procedures). For the 180 min. samples, the background in lanes 3 and 4 was subtracted. Lanes 7 and 8 were exposed for a shorter time than the other lanes.
(B) Decay of CYH2 mRNA is unaffected by RNA pol IIΔCTD. RNase protection of poly(A)-fractionated RNA from DBY276 as in (A).
(C) LM-PAT of SSA1 poly(A) tails from DBY121 cells transformed with the indicated plasmids. Poly(A) tail lengths are indicated at right.
(D) cRT-PCR of SSA1 mRNA 3′ ends. RNA was from DBY120 transformed with the indicated plasmids harvested after 60 min at 37°C. Poly(A) tail lengths are at right. Remaining SSA1 RNA in the vector control strain (lane 1) is mostly deadenylated. The distributions of SSA1 PCR products in lanes 2 and 3 determined by Phosphorimager are shown in the graph.
Molecular Cell 2002 9, DOI: ( /S (02)00518-X)

6. Figure 5
Cross-Linking of 3′ End Processing Factors to Transcribed Genes In Vivo Requires Functional RNA Pol II
(A) ChIP of DBY154 (rpb1-1) at 25°C and after 1 hr at 37°C. 32P-labeled PCR products corresponding to the 3′ ends of the indicated genes are shown for input and IPs with anti-CTD, -Pcf11, -Rna14, and 70A. HMR-E is a negative control.
(B) ChIP of DBY153 (rpb1-1) containing vector pFL38 (top panel) or pFL38-RPB1 (bottom panel) at 25°C and after 1 hr at 37°C is shown. RNA pol II (anti-CTD), Pcf11, and Fip1 cross-linking at 37°C is rescued by RPB1.
(C) ChIP of DBY333 (rpb1-1) with GFP-tagged Hrp1 and HA-tagged Rpb3 at 25°C and after 1 hr at 37°C. Lanes 7 and 8 show the isogenic strain DBY154 without the GFP tag as a negative control. Lanes with IPs (3–5, and 8) were exposed longer than the inputs (1, 2, and 7).
(D) Western blots of total protein extracted from equal numbers of cells of DBY154 or DBY333 (for GFP-Hrp1) at 25°C and after 1 hr at 37°C.
Molecular Cell 2002 9, DOI: ( /S (02)00518-X)

7. Figure 6
The 3′ End Processing Factors Pcf11, Fip1, and Hrp1 Localize to the 5′ and 3′ Ends of Transcribed Genes
(A) ChIP of DBY153 at 25°C with anti-CTD, -Pcf11 and -Fip1. PCR products are centered at −640, +77, and relative to the ENO2 ATG. The −640 product corresponds to the poorly transcribed SPC97 gene. Background from the control antibody 70A was subtracted, and the values for each PCR product were normalized to the input.
(B) ChIP of DBY333 (lanes 1–3) at 25°C with anti-HA and anti-GFP to detect epitope-tagged Rpb3 and Hrp1, respectively, are shown. Lanes 4 and 5 show DBY154 lacking the GFP epitope tag at 25°C as a negative control.
(C) ChIP of DBY153 at 25°C as in (A). PCR products are centered at −551, +88, and relative to the TEF1 ATG. The −551 product overlaps the poorly transcribed YPR079W gene. TELVIR is a negative control. Note that the 70A background (lane 5) is relatively high for the −551 region.
(D) ChIP of DBY333 (lanes 1–3) at 25°C, as in (B). Lanes 4 and 5 show DBY154 lacking the GFP tag at 25°C as a negative control.
Means and standard deviations from 4 PCR reactions are shown in the bar charts. These normalized signals are expressed relative to values at the 5′ ends of ENO2 (+77) and TEF1 (+88).
Molecular Cell 2002 9, DOI: ( /S (02)00518-X)

8. Figure 7 Pcf11 Binds Preferentially to the CTD Phosphorylated on Ser2
(A) Recombinant Pcf11-His6 was bound to GST-yCTD or GST mutant CTD (mut-CTD) immobilized on glutathione sepharose beads. GST-yCTD was phosphorylated (lanes 2 and 3) in HeLa nuclear extract. Input (0.5%), unbound (U, 0.5%), and bound (B, 20%) fractions were immunoblotted with anti-Pcf11. Similar results were obtained with anti-His6.
(B) Recombinant Pcf11 was bound to biotinylated peptides with four heptad repeats immobilized on streptavidin magnetic beads. Input (1%), unbound (U, 1%), and bound (B, 20%) fractions were immunoblotted with anti-Pcf11.
(C) H5, H14, and rabbit anti-CTD antibodies were bound to the immobilized peptides as in (B). Bound antibody was eluted and immunoblotted with anti-mouse IgM (H5, H14) or anti-rabbit IgG (anti-CTD).
(D) ChIP analysis of YJJ662 at 25°C with anti-CTD, -Pcf11, H5 (Ser2-PO4), H14 (Ser5-PO4), Pcf11, and 70A. Data from four PCR reactions in the bar chart were normalized to the 5′ end of TEF1 as in Figure 6.
Molecular Cell 2002 9, DOI: ( /S (02)00518-X)