Fig 2: Tau interacts with factors involved mRNA 3′ end processing to form (a) complex(es). (A–F) Tau can form (a) complex(es) with Pin1, p53 and poly(A)-specific ribonuclease (PARN) in NEs of HCT116 cells independently of DNA damaging conditions. Endogenous-immunoprecipitation (e-IP) assays with antibodies against tau (A), PARN (B,E), p53 (C,F) or Pin1 (D) were performed using NEs of HCT116 cells treated with juglone (5 μM) for 2 h, and/or exposed to UV irradiation (40 Jm−2) and allowed to recover for 2 h. NEs were treated with RNase A. Equivalent amounts of the pellets (IP) were resolved by SDS-PAGE, and proteins were detected by Western blot using the indicated antibodies. (A–D) Tau-13 mouse monoclonal antibody was used for Western blot detection. (E,F) Nitrocellulose membranes were dephosphorylated before tau-1 use in Western blot detection. Ten percentage of the NEs used in the e-IP assays are shown as input. Representative e-IP reactions from three independent biological assays are shown.

Fig 3: Pin1 is part of a deadenylation activator complex that includes PARN, tau and p53 under damaging conditions. (A,B) Levels of nuclear deadenylation correlate with Pin1 activity and expression in HCT116 cells. NEs from cells incubated with either (A) 5 µM juglone for 2 h or (B) Pin1/control siRNAs for 48 h were used in deadenylation assays. Indicated samples were also treated with UV (40 Jm-2) and allowed to recover for 2 h. The deadenylation reactions were incubated for 90 min. RNAs were purified and analyzed by denaturing PAGE. Representative deadenylation reactions from three independent biological assays are shown. Positions of the polyadenylated RNA L3(A30) and the L3 deadenylated product are indicated. On the right, relative deadenylation (RD) levels, calculated as {L3 fragment/[L3 fragment + L3(A30)]} ×100, were plotted for each condition. The RD levels for control conditions were arbitrarily set at 1.0. The means ± standard deviation of RD values are indicated. The P-values are indicated as *(=0.05) or **(=0.005). Quantifications were done with ImageJ software (http://rsb.info.nih.gov/ij/). Right panels show Western blot analysis of indicated proteins from NEs tested for deadenylation. (C) Pin1-mediated activation of nuclear deadenylation is dependent on p53 expression. NEs from HCT116 or HCT116 p53-/- cells incubated with 5 µM juglone and/or treated with UV (40 Jm-2) and allowed to recover for 2 h were used in deadenylation assays. Deadenylation reactions were performed as described in (A,B). RD levels in samples from HCT116 cells were arbitrarily set at 1.0. Right panels show Western blot analysis of indicated proteins from NEs used in deadenylation assays. (D) Levels of nuclear deadenylation were affected by Pin1-inhibitor all-trans retinoic acid (ATRA) in HCT116 cells. NEs from cells incubated with 1 µM ATRA for 72 h were used in deadenylation assays. Indicated samples were also treated with UV (40 Jm-2) and allowed to recover for 2 h. Deadenylation reactions were performed as described in (A). RD level for untreated HCT116 cells was arbitrarily set at 1.0. Right panels show Western blot analysis of indicated proteins from NEs used in deadenylation assays.

Fig 4: PIN1 is required for maintaining FAAP20 stability.(A) U2OS cells transfected with siRNA control (CTL) or PIN1 were treated with 50 µg/mL CHX for the indicated times, and degradation kinetics of endogenous FAAP20 was monitored by WB. (B) U2OS cells were serially transfected with indicated siRNA oligos and Flag-FAAP20 encoding plasmid, and cell lysates were analyzed by WB. Where indicated, 10 µM of proteasome inhibitor MG132 was incubated for 6 h before harvest. (C) (Top) a schematic for the PIN1 knockout strategy using CRISPR/Cas9. The 20-nucleotide sgRNA target loci on the bottom strand of exon 1 are marked in blue line along with a PAM sequence in red. The cleavage site for the Cas9 nuclease is indicated by the red triangle, which is 29 nucleotides upstream from the ATG start codon. (Bottom) confirmation of knockout by the WB analysis of U2OS WT (vector-transfected clone) or independent PIN1-/- clones. (D) (Left) U2OS WT or PIN1-/- #6 clones expressing Flag-FAAP20 were treated with 50 µg/mL CHX for the indicated times, and degradation of Flag-FAAP20 was analyzed by WB. (Right) quantification of Flag-FAAP20 levels by ImageJ. A dotted line indicates the half-life of protein degradation. Data shown are the mean ± SD from two independent experiments. * P <0.001 compared to WT, Student’s t-test. (E) U2OS WT or PIN1-/- cells transiently expressing Flag-FAAP20 were left untreated or treated with 10 µM MG132 for 6 h, and cell lysates were analyzed by WB. (F, G) U2OS cells expressing Flag-FAAP20 WT or isomerization-defective mutants were treated with 50 µg/mL CHX for the indicated times and analyzed by WB. (H) U2OS cells stably expressing PIN1 WT or catalytically dead mutant (K63A/R68A/R69A) were transfected with Flag-FAAP20-encoding plasmid, and cell lysates were analyzed by WB. (I) 293T cells transfected with the indicated plasmids were treated with 20 µM MG132 for 4 h, lysed under denaturing conditions, and incubated with Ni-NTA agarose to capture polyubiquitinated Flag-FAAP20. Flag-FAAP20 in Figure 4 was transiently expressed by plasmid transfection.

Fig 5: Protein phosphatase 2A dephosphorylates FAAP20 at the CPD motif.(A) U2OS cells expressing Flag-FAAP20 were left untreated or treated with 100 nM okadaic acid (OA) for 2 h, and FAAP20 pS113 levels were analyzed by WB using anti-pS113 antibody. (B) (Left) U2OS cells were serially transfected with siRNA control or PP2Ac and Flag-FAAP20-encoding plasmid, and FAAP20 pS113 levels were analyzed by WB. (Right) confirmation of PP2Ac knockdown by RT-qPCR. mRNA expression was normalized by GAPDH mRNA (mean ± SD; n = 3 independent experiments). * P <0.001, Student’s t-test. (C) U2OS cells were transfected with indicated plasmids, and FAAP20 pS113 levels were analyzed by WB. (D) 293T cells transfected with HA-B56a and Flag-FAAP20 WT or N-terminal deletion mutants (?N30: a.a.1-30 deletion, ?N48: a.a.1-48 deletion, and ?N63: a.a.1-63 deletion) were subjected to anti-Flag IP and WB. * IgG heavy chain. (E) Summary of the binding regions for B56a, FANCA, and PIN1 revealed in this study.