1. Opposing Polymerase-Deadenylase Activities Regulate Cytoplasmic Polyadenylation 
Jong Heon Kim, Joel D. Richter 
Molecular Cell 
Volume 24, Issue 2, Pages (October 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 The Nuclear Cyclin B1 Pre-mRNA Has a Long Poly(A) Tail
(A) Schematic diagram of the cyclin B1 transcript 3′ end structure and experimental strategy.
(B) Total RNA from oocytes was reverse transcribed with oligo d(T)18-linker primer (e) and amplified with the indicated primer pairs. The products were analyzed by electrophoresis on a 1.2% agarose gel.
(C) The amplified products from lanes 6 and 7 in (B) were analyzed by electrophoresis through a 5% polyacrylamide gel.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 The CPE Is Required for Cytoplasmic Deadenylation of mRNA
(A) Sequences of the cyclin B1 and A1 3′ UTRs containing or lacking CPEs; the histone-like B4 3′ UTR sequence is also shown.
(B–D) 32P-labeled RNAs were adenylated in vitro with poly(A) polymerase to contain about 150 nt; they were then injected into oocytes and collected immediately or 12 hr later. Some oocytes were incubated with progesterone for an additional 6 hr, at which time they matured (M). Total RNA was extracted and analyzed on a denaturing 5% polyacrylamide gel.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 PARN Interacts with Cytoplasmic Polyadenylation Machinery
(A) Extracts from oocytes, some of which were incubated with progesterone to induce maturation, were immunoprecipitated (IP) with symplekin antibody or control mouse IgG. Immunoblots of the precipitates were probed for symplekin, CPSF100, CPSF73, PARN, CPEB, Gld2, and, as negative controls, actin and mos (N.B., all CoIP reactions contained 50 μg/ml RNase A). p74 and p62 refer to two forms of PARN. P-CPEB refers to phospho-CPEB.
(B) Nuclear, cytoplasmic, and total protein was probed on immunoblots for symplekin, CPSF100, CPSF73, PARN, CPEB, and tubulin.
(C) Oocytes were lysed in IP buffer containing ribonuclease inhibitor and then subjected to IP with IgG or antibody against symplekin or PARN. RNA was then isolated from precipitates and analyzed by RT-PCR using primers for cyclin B1, mos, and β-actin.
(D) Oocytes were injected with IgG or affinity-purified PARN antibody and in vitro-polyadenylated CPE-containing RNA. Following 8 hr incubation, total RNA was extracted and analyzed by denaturing 5% polyacrylamide gel.
(E) Oocytes were injected with IgG or affinity-purified PARN antibody; 16 hr later, total RNA was extracted, reverse transcribed with an oligo d(T)18-linker primer, and amplified with a cyclin B1-specific primer in the presence of trace amounts of 32P-dATP. The products were analyzed by 5% polyacrylamide gel electrophoresis.
(F) m7GpppG capped or ApppG pseudocapped cyclin B1 32P-labeled RNAs were adenylated in vitro with E. coli poly(A) polymerase to contain about 150 nt; they were injected into oocytes and collected at the indicated times. Total RNA was extracted and analyzed on a denaturing 5% polyacrylamide gel.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 PARN Interacts with CPEB and Gld2
(A) mRNAs encoding CPSF160, maskin, symplekin, CPEB, and Gld2 were translated in a rabbit reticulocyte lysate in the presence of 35S-methionine and applied to glutathione columns containing GST or GST-PARN (WT). The columns were washed, and the bound material was analyzed by SDS-10% PAGE. Note that two proteins are produced by in vitro translation of symplekin mRNA (Barnard et al., 2004).
(B) Extracts from oocytes were subjected to IP with PARN antibody or control rabbit IgG, which was followed by immunoblotting for symplekin, CPSF100, CPSF73, PARN, and CPEB, and for actin as a negative control.
(C) mRNA encoding myc-tagged PARN was injected into the oocytes; 18 hr later, they were lysed and subjected to IP with myc antibody.
(D and E) (D) mRNAs encoding myc-CPEB and (E) myc-Gld2 were injected into oocytes; following an 18 hr incubation, the oocytes were lysed and subjected to IP with myc antibody. The coprecipitating proteins were analyzed on immunoblots.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
CPEB S174 Phosphorylation by Aurora A Abrogates Binding of PARN
(A) Interaction between PARN and CPEB when the Aurora A phosphorylation site on CPEB S174 has been mutated either to alanine (A) or aspartic acid (D) (phosphomimetic). Note that residues S174 and S180 were mutated; see text for details. GST-PARN (WT) bound to glutathione resin was incubated with 35S-methionine-labeled WT CPEB and CPEB proteins containing S174A/S180A (AA) or S174D/S180D (DD) double mutations. Note that CPEB-WT and CPEB-AA (arrow), but not CPEB-DD, interacted with PARN.
(B) mRNAs encoding myc-CPEB(AA) and myc-CPEB(DD) were injected into oocytes that were subjected to myc antibody IP and immunoblotting for the indicated proteins. Actin served as a negative control; p74 and p62 refer to two forms of PARN.
(C) Extracts from oocytes, some of which were incubated with progesterone to induce maturation, were immunoprecipitated with symplekin antibody or control mouse IgG. Immunoblots of the precipitates were probed for symplekin, CPSF100, CPSF73, and PARN, and, as a negative control, actin. p74 and p62 refer to two forms of PARN.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 PARN Controls the Polyadenylation of CPE-Containing RNA
(A) Oocytes injected with mRNA encoding WT or catalytically inactive (D28A) PARN were also injected with CPE-containing RNA. After incubation, RNA from the oocytes was extracted and resolved by 5% denaturing PAGE. Note that injection of the catalytically inactive PARN was sufficient to induce polyadenylation. The bottom panel shows the levels of myc-PARN synthesized in oocytes.
(B) Expression levels of myc-tagged PARN were analyzed with anti-PARN antibody in comparison with endogenous PARN from mock-injected oocytes.
(C) mRNAs encoding myc-PARN(WT) and myc-PARN(D28A) were injected into oocytes and subjected to IP with myc antibody and immunoblotting for components of the polyadenylation machinery as well as actin.
(D) mRNAs encoding myc-Gld2(WT) or myc-Gld2(D242A) alone, myc-Gld2(WT) and myc-PARN(WT), or myc-Gld2(WT) and myc-PARN(D28A) were injected into oocytes. After 16 hr incubation, 32P-labeled cyclin B1 3′ UTR was injected and the oocytes were incubated an additional 8 hr. Total RNA was then extracted and analyzed by denaturing 5% polyacrylamide gel. The bottom panel shows the expression levels of the myc-tagged proteins.
(E) Aurora A promotes polyadenylation of endogenous mRNA. The left panel shows a flow chart of the experimental design. Oocytes were homogenized, and symplekin was immunoprecipitated. To the antibody beads was added Aurora A kinase followed by α-32P-ATP. Total RNA from the immunoprecipitates was then extracted and digested with RNase A and T1, which leaves poly(A) intact. The products were then analyzed on 5% sequencing polyacrylamide gel and visualized by a phosphorimager. The arrow head denotes the poly(A) tail generated by Aurora A phosphorylation of CPEB (right).
(F) Poly(A) tail status of coimmunoprecipitated endogenous cyclin B1 RNA. Prior to treatment with RNases A and T1, an aliquot of the extracted RNAs was subjected to a PAT assay with a cyclin B1-specific primer. The products were analyzed on a 5% polyacrylamide gel.
(G) Oocytes injected with mRNA encoding myc-PARN were subjected to an identical symplekin CoIP procedure as described above. Following incubation in buffer containing or lacking Aurora A, the beads were collected by a magnetic separator and washed, and the symplekin and PARN in the supernatants and pellets were detected by immunoblotting. Note that Aurora A caused the expulsion of the 62 kDa form of PARN, the only one detected on the symplekin beads, from the cytoplasmic polyadenylation complex.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 Model of Poly(A) Tail Dynamics of CPE-Containing mRNA
In the nucleus, CPE-containing pre-mRNA acquires a long poly(A) tail, which is then exported to the cytoplasm, where it is bound by the CPEB-containing cytoplasmic polyadenylation machinery. This machinery includes PARN, whose activity overrides the polyadenylating activity of the poly(A) polymerase Gld2, which results in a short poly(A) tail. Progesterone stimulates the activation of Aurora A, which phosphorylates CPEB S174. This event causes the dissociation of PARN from the RNP complex, resulting in Gld2-catalyzed default polyadenylation. The circled P refers to phosphorylated CPEB S174.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions