1. Regulation of Yeast mRNA 3′ End Processing by Phosphorylation
Xiaoyuan He, Claire Moore 
Molecular Cell 
Volume 19, Issue 5, Pages (September 2005)
DOI: /j.molcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1
Depletion of Glc7 Is Detrimental to Poly(A) Addition, but Not Cleavage
(A) Levels of the CPF subunits Fip1 and Pta1 decline upon Glc7 depletion. Extracts from glc7-td cells grown at 25°C or incubated at 37°C for 30 or 60 min were examined by Western blot using antibodies against the indicated proteins.
(B) In vitro cleavage of substrate containing the GAL7 poly(A) site. Extract was prepared from glc7-td (lanes 2–4) or wild-type (wt) FY23 (lanes 5–7) cells grown at 25°C or shifted to 37°C for 30 or 60 min. Reactions were performed in the presence of 2′ dATP to block polyadenylation.
(C) In vitro poly(A) addition of substrate ending at the GAL7 poly(A) site. Reactions were conducted as in (A) but with ATP instead of 2′ dATP.
(D) Depletion of Glc7 causes shorter poly(A) tails in vivo. Poly(A) tail length was analyzed by 3′ end labeling of total RNA from the indicated cultures, followed by RNase degradation that leaves only the poly(A) tails intact, as described in the Experimental Procedures. After electrophoresis through a 18% polyacrylamide-8.3 M urea gel, the poly(A) tails were visualized with a phosphorimager.
For (B) and (C), the positions of precursor and cleaved or polyadenylated product are indicated on sides, with lane 1 showing unreacted precursor RNA.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Rescue of Glc7-Depleted Extract Requires the Action of Glc7 as a Phosphatase
(A) Rescue of polyadenylation activity by addition of Glc7-His6, Fip1-His6, GST-Pta1, or purified CPF. Extract from glc7-td cells depleted at 37°C for 30 min was supplemented with the indicated proteins before initiating poly(A) addition. In lanes 18–20, 150 ng of Fip1 or 75 ng of Pta1 was used. Lanes 10 and 11 show that CPF alone has no activity on the RNA substrate.
(B) Poly(A) addition is rescued by treatment with other phosphatases. Extracts from glc7-td cells shifted to 37°C for 30 min (lanes 3–7) or from rna14-2 cells (lanes 8–11) were incubated with rabbit PP1 (2.5 U/ml, lanes 4, 5, and 10) or λ protein phosphatase (400 U/ml, lanes 6, 7, and 11) before initiating poly(A) addition.
(C) Ypi1-His6 inhibits Glc7. GST-Pta1 phosphorylated by Cka1 with [γ-32P]ATP was treated with Glc7-His6 in the absence (lanes 2–5) or presence (lanes 6 and 7) of Ypi1-His6. Lane 1, untreated phosphorylated GST-Pta1.
(D) Ypi1 inhibits poly(A) addition. Extracts from glc7-td cells grown at 25°C (lanes 2 and 4–6) or shifted to 37°C (lanes 3 and 7–10) were incubated with GAL7 RNA and ATP. Extract was supplemented with Ypi1-His6 (lanes 4–6 and 8–10) and Glc7-His6 (lanes 7–10).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Phosphorylated Pta1 Accumulates In Vivo upon Glc7 Depletion and Cannot Rescue Polyadenylation in Glc7-Depleted Extract
(A) Pta1 was immunoprecipitated from the indicated extracts and immunoblotted with anti-phosphoserine antibody (top). The membrane was stripped and probed with anti-phosphothreonine antibody (middle) or anti-Pta1 antibody (bottom). Lane 1 shows a control immunoprecipitation in the absence of extract.
(B) Phosphorylated or mock-phosphorylated GST-Pta1 was added to Glc7-depleted extract, and poly(A) addition assays were conducted as described for Figure 1C.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Phosphorylation and Dephosphorylation of CPF
(A) CPF was phosphorylated by CKI or the Cka1 subunit of CKII, but not by Cdc2. Kinases were incubated with [γ-32P]ATP (lanes 2, 3, and 6) or with [γ-32P]ATP and CPF (lanes 4, 5, and 7). Proteins were resolved by SDS-PAGE and detected by phosphorimager analysis. For comparison, a silver stain of CPF is shown in lane 1.
(B) Identification of CPF subunits phosphorylated by CKI. CPF phosphorylated by CKI using [γ-32P]ATP (lane 1) was incubated with the indicated antibodies in the absence (lanes 2, 4, 6, and 8) or presence (lanes 3, 5, 7, and 9) of 0.1% SDS. Pull-down of phosphorylated CPF subunits was also performed with calmodulin beads, with lane 10 showing input and lane 11 material bound to beads. Proteins were resolved by SDS-PAGE and detected by phosphorimager analysis.
(C) Phosphorylated CPF subunits are dephosphorylated by Glc7. CPF phosphorylated by CKI (lane 2) was treated with Glc7-His6 (lanes 3–5) or GST-Ssu72 (lanes 6–8). Lane 1 shows CKI alone with [γ-32P]ATP.
(D) Western blot analysis of proteins in CPF with and without phosphorylation. CPF was treated with Cka1 and ATP or mock-treated with ATP alone, isolated on calmodulin beads, and eluted proteins were examined by Western blot for the presence of the indicated proteins.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Cka1, but Not Ctk1, Affects 3′ End Processing In Vitro
(A) Cka1 inhibits poly(A) addition in vitro when incubated with wt (FY23) extract before initiating poly(A) addition.
(B) Mutations in CKA1 affect both steps of 3′ end processing. Extracts prepared from wt and mutant cells grown at 25°C were used in cleavage (lanes 1–7) or poly(A) addition assays (lanes 8–13) as described in Figure 1. Precursor RNA is shown in lanes 1 and 8.
(C) Deletion of CTK1 has no effect on 3′ end processing in vitro. Processing assays were conducted as in (B). Lanes 1 and 4 show precursor RNA.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Interactions of Glc7 with CPF Subunits
(A) Radioactively labeled Cft1, Cft2, Brr5, Fip1, Yth1, and Pap1 were synthesized by in vitro translation and incubated with Glc7-His6. Complexes were immunoprecipitated with antibody against the V5 epitope on Glc7-His6, resolved by SDS-PAGE, and detected by autoradiography. 10% of the input of each in vitro translated protein is shown.
(B) GST pull-downs with Glc7-His6 and GST, GST-Pti1, GST-Ssu72, or GST-Pta1. Complexes were isolated on glutathione Sepharose beads. Glc7 was detected by Western blot using antibody against the His6 epitope. 10% of the Glc7-His6 input is shown in lane 1.
(C) GST pull-downs with truncations of Pta1 and Glc7-His6 as described in (B).
(D) GST pull-downs with truncations of Pti1.
(E) Genetic interactions of PTA1 and GLC7. GLC7 was overexpressed in wt, pta1-2, or pta1-3 strains cultured at 25°C or 37°C.
(F) Genetic interactions of PTI1 and GLC7. GLC7 was overexpressed in wt, pti1-7, or pti1-14 strains grown at 25°C or 37°C.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Model Depicting the Role of Phosphorylation in mRNA 3′ End Processing
Phosphorylation of Pta1 and perhaps other CPF proteins by a kinase such as CKII prevents poly(A) addition until Glc7 activates the complex by dephosphorylation.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions