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Files in this Data Supplement:
Fig. S1. Sequence alignment of the 3′LCUs of chordates. (A) Sequence alignment of the 3′LCU among vertebrates. Schematic representation of the 3′LCU and subregion A-D is shown at the top. Pink boxes, conserved regions; numerical figures, positions of conserved regions and subregions A-D. Sequence alignment was performed using ClustalW with human (Accession number NM_032246; positions 2640-3383), mouse (Accession number XM_620516; positions 2639-3384), Xenopus, and zebrafish (Accession number BC090757; positions 756-1566). Subregions A-D are indicated in the sequence alignment. (B) Sequence alignment of amphioxus and Xenopus 3′LCUs. The amphioxus sequence was obtained from the JGI genome sequence and aligned with the Xenopus sequence using ClustalW. A possible Mex3b-responsive destabilizing element (MRDE) is underlined in pink (A,B).
Fig. S2. Destabilization of reporter mRNAs through the 3′LCU by endogenous factors is detected at stage 13, but not at stage 12. Embryos were injected with GFP reporter mRNA connected to the 3′LCU (+3′LCU), 3′LCUrev (+3′LCUrev), or subregion A (+A; 100 pg/embryo), and analyzed at indicated stages for reporter mRNA remaining in the embryos by WISH using the GFP probe.
Fig. S3. Deletion analysis of the 3′LCU for translational enhancement activity. (A-J) Embryos were injected with GFP reporter mRNA (100 pg/embryo) connected to 3′LCU, 3′LCUrev, nc3′UTR, or combinations of subregions A-D as indicated. GFP fluorescence in the injected embryos was observed at stage 12.5/13. Note that GFP fluorescence of all deletion constructs (E-J) was stronger than that of GFP alone (A) and GFP+nc3′UTR (D), but was weaker than that of GFP+3′LCU and 3′LCUrev (B,C).
Fig. S4. The effects of Mex3b on reporter mRNA. (A) Destabilization of reporter mRNA by Mex3b at stage 9.5 or later. GFP+3′LCU reporter mRNA (100 pg/embryo) was co-injected with mRNA for mex3b-HA or DKH1+2-HA (1 ng) as indicated. Injected embryos were analyzed by RT-PCR with the GFP primer set for the amount of remaining reporter mRNA at indicated stages. histone H4, the internal control. The histone H4 primer set: forward, 5′-CGGGATAACATTCAGGGTATCACT-3′; reverse, 5′-ATCCATGGCGGTAACTGTCTTCCT-3′. (B) No effect of Mex3b on translational enhancement by the 3′LCU. GFP or GFP+3′LCU reporter mRNA (100 pg/embryo) was co-injected with mex3b mRNA. Injected embryos were lysed at stage 7, and the GFP protein was detected by immunoblotting with anti-GFP antibody conjugated with Irdye800 (ROCKLAND).
Fig. S5. Deletion analysis of subregion D for Mex3b responsiveness. (A) Schematic representation of subregion D. Numbers indicate the positions of subregions D1-D5. (B) GFP reporter mRNA (100 pg/embryos) was injected with or without mex3b mRNA (63 pg/embryo) and the amount of reporter mRNA remaining in the embryo was examined by WISH using the GFP probe. Note that GFP+D2 was slightly destabilized by Mex3b. Although GFP+D1, +D3, +D4, and +D5 were not destabilized by Mex3b, D1-D4 were required for full destabilizing activity. (C) Sequence of the D2 region. A purine-rich sequence is in pink and an AU-rich sequence is underlined in blue.
Fig. S6. Time-course experiment for the effect of negative autoregulation by subregion D of the 3′LCU on the protein accumulation. Embryos were injected with the DNA construct mex3b-HA or mex3b-HA+D and lysed at indicated stages for western blotting with the anti-HA antibody to quantitate the Mex3b-HA protein. The abscissa in the graph, time (hours); the ordinate, intensity of bands (arbitrary units).
Fig. S7. Phenotypes of Mex3b-overexpressed embryos and examination of mex3bMO specificity. (A-C) Overexpression of mex3b. globin or mex3b mRNA (1 ng/embryo) was injected as indicated, and the expression of gsc, Xlim1 and fgf20 was analyzed by WISH as indicated at stage 10.5 (A,B) or stage 13 (C). (D) The specificity of mex3bMO1. mex3bMO1 (25 or 50 ng/embryo) inhibited translation of 5′UTR-myc-mex3b mRNA (lanes 2,3), but co-injection of 5mmMO (50 ng) did not (lane 4). myc-mex3b mRNA, which does not possess the MO annealing sequence, was not affected by co-injection of mex3bMO1 or 5mmMO (lanes 5−8). (E-I) Rescue of mex3bMOs phenotypes. Injection of mex3bMOs (50 ng) reduced Xbra expression (E), which was recovered by co-injection of mex3b mRNA (63-125 pg) (H). Percentages of embryos with strongly reduced (red), weakly reduced (pink), and normal-looking (white) Xbra expression are shown in a bar graph (I). (J-L) The effect of mex3bMOs on mesoderm markers. Embryos injected with 5mmMO or mex3bMOs (50 ng/embryo) as indicated were subjected at stage 10.5 to WISH with probes as indicated. (M,N) The effect of fgf20 on cdx4 expression. fgf20 mRNA (0.6 pg/embryo) was injected into the prospective neuroectoderm at the four-cell stage, and the expression of otx2 and cdx4 was analyzed by WISH at stage 13, as indicated. Frequency of the representative phenotype shown in the picture is indicated in the bottom right of each panel. Arrows and frequencies with different colours indicate ‘reduced expression’ (blue), ‘enhanced expression’ (magenta), and ‘no change’ or ‘no expansion’ (black).
Fig. S8. The effects of Mex3b, Sdc2 and Ets1 on FGF signaling. (A) The effect of Mex3b on FGF signaling pathway as assayed with cdx4-luc reporter. Luciferase reporter analysis was performed using FGF-responsive cdx4/Xcad3-luc reporter DNA. Reporter DNA with constitutively active (ca)-FGFR1 or ca-Ha-Ras mRNA was co-injected with or without mex3b mRNA and injected embryos were assayed for luciferase activity at stages 12.5-13. (B) The effect of Mex3b on ERK phosphorylation. Animal caps were dissected from embryos injected with FGF4-DNA or mRNA as indicated, and were lysed at stage 10.5. Lysates were analyzed by immunoblotting with anti-phosphorylated ERK (anti-p-ERK) or anti-ERK antibody (Cell Signaling). (C) The effect of sdc2 or ets1 on FGF signaling. Luciferase analysis was performed in the same way as in A. Amounts of injected mRNAs or DNA (pg/embryo): ca-FGFR1, 30; ca-Ha-Ras, 25; mex3b, 1000; FGF4-DNA, 10 or 5, as indicated in the panel; XFD, 1000; globin, 1000; cdx4/Xcad3-luc DNA, 100; sdc2, 100; ets1, 100. Bar graphs show the mean±s.e.m. of five samples.
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