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Fig. 1.
PABP1 depletion causes multiple developmental defects and embryonic lethality. (A) Western blot analysis of whole-cell extracts from stage 14�16 embryos injected with control (Ctr) or PABP1-A (PABP1) morpholino (Mo) � 10 ng of PABP1 rescue mRNA, using antibodies specific for PABP1 and ePABP. (B) Representative photographs of stage 29/30 embryos injected with control or PABP1-A morpholino � 10 ng of PABP1 rescue mRNA. PABP1 morphants show abnormal development of anterior (closed arrow) and posterior (open arrow) structures, spinal curvature (arrowhead), and developmental arrest at stage 18�20 (asterisk). (Additional photographs are shown in Fig. S2.) (C) Percentages of control, PABP1 morpholino, and PABP1 rescue embryos (stage 29/30) displaying the indicated phenotypes. Morphological defects include spinal curvature; abnormal development of eye, cement gland, tail, and fin; ventral edema; absence of posterior development; and developmental arrest at stage 18�20. Data represent the average of nine (PABP1 morpholino) or six (PABP1 rescue) independent experiments, with ∼1,200 or 900 embryos per experimental point, respectively.
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Fig. 2.
ePABP is essential for development. (A) Representative photographs of stage 30�32 embryos injected with control or ePABP-A (ePABP) morpholino � 10 ng of ePABP rescue mRNA. (Additional photographs are shown in Fig. S4.) (B) Percentages of control, ePABP morpholino, and ePABP rescue embryos (stage 30�32) displaying the indicated phenotypes. Legend is as in Fig. 1C. Data represent the average of seven (ePABP morpholino) or three (ePABP rescue) independent experiments, with ∼900 or 300 embryos per experimental point, respectively. Control and (C) PABP1 or (D) ePABP morphants were labeled metabolically with [35S]methionine, and newly synthesized proteins were TCA precipitated. Data are shown as percentage of [35S]methionine incorporation relative to control (set to 100%) and represent the average of two independent experiments, with ∼60 embryos per experimental point. Error bars indicate SEM. *P < 0.05 and **P < 0.01 versus control. P values were determined by t test.
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Fig. 4.
PABP4 and ePABP cannot rescue PABP1-depleted embryos efficiently. Embryos were injected with control or PABP1-A (PABP1) morpholino � (A) 1 ng of PABP1-FLAG or PABP4-FLAG rescue mRNA or (B) 10 ng of PABP1 or ePABP rescue mRNA. Percentages of stage 29/30 embryos displaying the indicated phenotypes are shown; numbers indicate the percentage of PABP1-specific phenotypes in each case (morphological plus movement defects). Data represent the average of approximately (A) 350 embryos or (B) 500 embryos per experimental point, in three or four independent experiments, respectively. ***P < 0.0001, as determined by Fisher's exact test. (Photographs are shown in Fig. S8
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Fig. S8. PABP4 and ePABP cannot rescue PABP1-depleted embryos efficiently. (A) Representative photographs of stage 29/30 embryos injected with control or PABP1-A (PABP1) morpholino � 1 ng of PABP1-FLAG or PABP4-FLAG rescue mRNA. (B) Representative photographs of stage 29/30 embryos injected with control or PABP1-A morpholino � 10 ng of PABP1 or ePABP rescue mRNA.
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Fig. 5.
Multiple regions of PABP1 determine specificity. Embryos were injected with control or PABP1-A (PABP1) morpholino � 10 ng of PABP1, ePABP, or either (A) eRRM/1Ct or (C) 1RRM/eCt rescue mRNA. Percentages of stage 26�31 embryos displaying the indicated phenotypes are given. Numbers indicate the percentage of PABP1-specific phenotypes in each case (morphological plus movement defects). Data represent the average of approximately (A) 160 embryos or (C) 135 embryos per experimental point, in four or three independent experiments, respectively. ***P < 0.0001, as determined by Fisher's exact test. (B and D) Western blots of embryos from A and C using an anti-PABP1 antibody, which does not recognize 1RRMs/eCt (1/e) because it is raised against a C-terminal PABP1 peptide. 1, PABP1; e, ePABP; e/1, eRRM/1Ct.
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Fig. S1. Translation of PABPs is inhibited efficiently by specific morpholinos. (A) PABP1, (B) ePABP, or (C) PABP4 mRNA was translated in vitro in the presence of [35S]methionine and morpholino antisense oligos (Mo): Control, PABP1-A (PABP1), ePABP-A (ePABP), or PABP4.
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Fig. S2. PABP1 depletion causes multiple developmental defects. (A) Representative photographs of stage 29/30 embryos injected with control or PABP1-A (PABP1) morpholino � 10 ng of PABP1 rescue mRNA. PABP1 morphants display the following defects: spinal curvature (embryos B, C, G, H, K, O, R, T, U, V, W, Z); abnormal development of tail and/or fin (embryos G, H, L, O, AA); absence of posterior elongation (embryos D, E, F, J, K, M, N, P, V); abnormal development of anterior structures (embryos J, V, Z, AA); ventral edema (embryos A, E, K, S, Y); and developmental arrest (embryos I, Q). Most PABP1-rescued embryos do not show morphological or movement defects. (B) An alternative PABP1 morpholino (PABP1-B) causes developmental defects and lethality similar to those caused by PABP1-A. Embryos were injected with control or PABP1-B morpholino; percentages of stage 26 embryos displaying the indicated phenotypes are shown. Data represent the average of three independent experiments with ∼300 embryos per experimental point.
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Fig. S3. PABP overexpression does not result in early developmental defects. Embryos were injected with (A) 1 ng or 10 ng of mRNA encoding PABP1, ePABP, or PABP4 or (B) 1 ng or 10 ng of mRNA encoding PABP1-FLAG or PABP4-FLAG. Embryos were allowed to develop until stage 47�50; the percentages of normal and defective embryos are shown. In A phenotypic defects were not observed at a significant level over control, permitting their use in rescue experiments. In B the 10-ng dose of PABP1-FLAG and PABP4-FLAG induced embryonic defects, possibly associated with the FLAG-tag. Therefore, the 1-ng dose was used in all FLAG rescue experiments. (C) PABP1-FLAG and PABP4-FLAG proteins are expressed at similar levels. Western blot analysis of whole-cell extracts from stage 16�18 embryos injected with control or PABP1-A (PABP1) morpholino � 1 ng of PABP1-FLAG or PABP4-FLAG rescue mRNA, using an anti-FLAG antibody. Asterisk indicates a nonspecific band.
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Fig. S4. ePABP depletion causes widespread developmental defects. (A) Western blot analysis of whole-cell extracts from stage VI oocytes coinjected with an ePABP-expressing mRNA � control (Ctr) or ePABP-A (ePABP) morpholino, using an ePABP-specific antibody. This shows that ePABP expression is inhibited efficiently in vivo by the morpholino. (B) Representative photographs of stage 30�32 embryos injected with control or ePABP-A morpholino � 10 ng of ePABP rescue mRNA. ePABP morphants display the following defects: spinal curvature (embryos A, B, C, H, V, W, Y); abnormal development of tail (embryos D, J); absence of posterior elongation (embryos A, F, G, K, S, Z); abnormal development of anterior structures (embryos K, L, M, Q, R, S, T, U, X, Y); and ventral edema (embryos E, F, I, K, L, M, N, S, Y). Embryo O appears morphologically normal. The majority of ePABP-rescued embryos do not show morphological defects. (C) An alternative ePABP morpholino (ePABP-B) causes the same developmental defects and lethality as ePABP-A. Embryos were injected with control or ePABP-B morpholino; percentages of stage 26�29 embryos displaying the indicated phenotypes are shown. Data represent the average of four independent experi- ments, with ∼500 embryos per experimental point.
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Fig. S5. PABP4 is conserved in vertebrates. (A) Neighbor-joining phylogenetic tree of vertebrate PABP family members rooted with Drosophila melanogaster (dm) PABP. Bootstrap (1,000 replicates) values are shown at the nodes. PABPs from Homo sapiens (hs), Mus musculus (mm), Gallus gallus (gg), X. laevis (xl), and Xenopus tropicalis (xt) are compared. (B) Table showing the percentage of amino acid identity between X. laevis and human PABP1, PABP4, and ePABP. (C and D) RT-PCR analysis of total RNA extracted from the indicated X. laevis adult tissues (C) or developmental stages (D) using primers specific for the indicated PABPs. β-actin serves as an internal control. In adults, ePABP mRNA is present only in the gonads, consistent with the protein expression (1, 2) whereas PABP1 and PABP4 mRNAs are widespread. During development, PABP4 mRNA is most abundant in early oocytes and after the midblastula transition (stage 7/8 embryos). Although ePABP protein levels begin to diminish after approximately stage 35 (2, 3), ePABP mRNA is absent after midblastula transition. Whilst PABP1 mRNA is present in all stages analyzed, PABP1 protein levels start to rise significantly only after stage 15, continuing to increase until approximately stage 41 (2, 3).
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Fig. S6. X. laevis PABP4 stimulates translation. (A) A proportion of PABP4 associates with polysomes. Stage VI oocytes were injected with PABP4-FLAG� expressing mRNA, and the resulting whole-cell extracts were treated with cycloheximide or EDTA, fractionated on 10�50% sucrose gradients, and Western blotted with an anti-FLAG or anti-ePABP antibody. The positions of messenger ribonucleoprotein complexes (mRNPs), 80S (or 40S and 60S), and polysomes are indicated. Release of PABP4 and endogenous ePABP by EDTA is indicative of sedimentation into the heavier fractions resulting from association with actively translating polysomes. (B) Tethering of PABP4 activates reporter mRNA translation to a similar extent as PABP1 and ePABP. Stage VI oocytes expressing the indicated MS2-fusion protein were coinjected with an unadenylated luciferase reporter mRNA containing 3′ UTR MS2 binding sites (Luc-MS2) and a β-galac- tosidase control mRNA. Luciferase was normalized to β-galactosidase activity, and the fold stimulation relative to MS2 (set to 1) is shown. Changes in β-galactosidase activity normally are <15%. (C) PABP4 stimulates only the translation of reporter mRNAs to which it is tethered. Oocytes were injected as in B, with Luc-MS2 or with an identical reporter lacking the MS2 binding sites (Luc-ΔMS2) and β-galactosidase control mRNA. Data in B and C represent the average stimulations of at least three independent experiments; error bars indicate SEM. (D) Tethering of PABP4 does not affect mRNA stability. Oocytes were injected as in B, with Luc-MS2 and β-galactosidase mRNAs. Total RNA was extracted either immediately or after 16 h and analyzed by quantitative RT-PCR with primers specific for the reporter mRNAs. Normalized luciferase/β-galactosidase values are shown. Data represent the average of two independent experiments; error bars indicate SEM. Thus, the effects of PABP4 are the direct result of changes in mRNA translation and not changes in mRNA stability, as previously shown for PABP1 and ePABP (1, 2).
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Fig. S7. PABP1, ePABP, and PABP4 activate translation through a similar mechanism. (A and B) PABP4, like PABP1 and ePABP, binds poly(A) but not poly(C) RNA efficiently. A range of concentrations (4, 2, 1, 0.5, and 0.25 nM) of GST-PABP1 and (A) GST-PABP4 or (B) GST-ePABP were immobilized on glutathione beads and incubated with 1 nM poly(A) RNA (pA). To control for specificity, 4 nM GST protein (G) was incubated with poly(A) RNA; and 4 nM PABP1 (1), PABP4 (4), and ePABP (e) were incubated with 1 nM poly(C) RNA (pC). After elution, RNA and proteins were detected by fluorescence (Top) and Western blotting with an anti-GST antibody (Bottom), respectively. (C) PABP4 interacts with initiation factors. Yeast two-hybrid analysis with the indicated PABP N termini (Nt) (LexA DNA-binding domain fusions) against eukaryotic initiation factor 4G (eIF4G) and poly(A)-binding protein interacting protein 1 (PAIP1) (Gal4 activation domain fusions) shows that PABP4 maintains these interactions. (D) The translation termination factor eukaryotic release factor 3 (eRF3) also is a PABP4 partner, as shown by yeast two-hybrid analysis with the indicated PABP C termini (Ct) (Gal4 activation domain fusions) against eRF3 (Gal4 DNA-binding domain fusion). (E) PABP4 can interact with itself and other PABP proteins. Yeast two-hybrid analysis with the indicated PABP C termini (LexA DNA-binding domain fusions) against the PABP4 C terminus (4Ct) or PAIP1 (Gal4 activation domain fusions) reveals PABP�PABP interactions. In C�E, iron regulatory protein-1 (IRP1) and MS2 are negative controls. Expression of β-galactosidase (blue) indicates protein�protein interaction.
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