Takada H et al. (2009), The RNA-binding protein Mex3b has a fine-tuning...

XB-ART-39965

Development 2009 Jul 01;13614:2413-22. doi: 10.1242/dev.029165.

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The RNA-binding protein Mex3b has a fine-tuning system for mRNA regulation in early Xenopus development.

Takada H , Kawana T , Ito Y , Kikuno RF , Mamada H , Araki T , Koga H , Asashima M , Taira M .

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Post-transcriptional control by RNA-binding proteins is a precise way to assure appropriate levels of gene expression. Here, we identify a novel mRNA regulatory system involving Mex3b (RKHD3) and demonstrate its role in FGF signaling. mex3b mRNA has a 3' long conserved UTR, named 3'LCU, which contains multiple elements for both mRNA destabilization and translational enhancement. Notably, Mex3b promotes destabilization of its own mRNA by binding to the 3'LCU, thereby forming a negative autoregulatory loop. The combination of positive regulation and negative autoregulation constitutes a fine-tuning system for post-transcriptional control. In early embryogenesis, Mex3b is involved in anteroposterior patterning of the neural plate. Consistent with this, Mex3b can attenuate FGF signaling and destabilize mRNAs for the FGF signaling components Syndecan 2 and Ets1b through their 3' UTRs. These data suggest that the 3'LCU-mediated fine-tuning system determines the appropriate level of mex3b expression, which in turn contributes to neural patterning through regulating FGF signaling.

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Species referenced: Xenopus
Genes referenced: cdx4 ets1 etv2 fgf2 fgf20 fgf4 fgf8 gsc hoxd1 mex3b myc otx2 rax sdc2 sdcbp sox2 tbxt
GO keywords: RNA binding [+]

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	Fig. 1. Characterization of mex3b. (A,B) Schematic representations of Xenopus and human Mex3b/MEX3B proteins (A) and mex3b/MEX3B mRNAs (B). Sequence identities are indicated. Note that the identity of the 3' long conserved UTR (3'LCU) is higher than that of the CDS. (C) Sequence alignment of the 3'LCUs. (D-J) Expression patterns of mex3b in Xenopus embryos at the four-cell (D,E), early gastrula (F,G), late gastrula (H), early neurula (I) and tailbud (J) stages. (G) Sagittal-hemisection of F. Arrowheads indicate the dorsal blastopore. A, animal view; D, dorsal view; L, lateral view; V, vegetal view.
	mex3b (mex-3 RNA binding family member B ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 29, dorsal view, anterior up.
	Fig. 2. Reporter mRNA assay for the 3â²LCU. (A) Schematic representations of GFP mRNA reporter constructs. The GFP or GFP-PEST (not shown) CDS was connected to the 3â²LCU, the reverse 3â²LCU (3â²LCUrev), and a non-conserved region of mex3b 3â²-UTR (nc3â²UTR; positions 1787-2527) as indicated. (B-H) Destabilization of reporter mRNA by the 3â²LCU and subregion A. The amount of remaining reporter mRNA in injected embryos was examined at stage 13 by WISH using the GFP probe (B-G) or at stages 12 and 13 by qRT-PCR (H). *P<0.05. (I-P) Translational enhancement by the 3â²LCU and 3â²LCUrev using GFP (I-L) or GFP-PEST (M-P). GFP fluorescence were observed at stage 13 (I-L) or 14 (M-P). (Q) Western blot analysis. Reporter mRNA was injected into two dorsal-animal blastomeres at the four-cell stage. All injected embryos were viewed dorsally at the neurula stage. Injected embryos were collected at indicated stages. Amounts of injected reporter mRNA (pg/embryo): 100 (B-P); 500 (Q).
	mex3b (mex-3 RNA binding family member B) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior up.
	Fig. 3. Destabilization of reporter mRNA by Mex3b through the 3â²LCU. (A-D) Destabilization by Mex3b through the 3â²LCU and subregion D. Injection and WISH were performed as described in Fig. 2. mex3b mRNA (63 pg/embryo) was co-injected with reporter mRNA (right panels). (E) Co-immunoprecipitation of GFP+3â²LCU mRNA with Mex3b. GFP+3â²LCU or GFP reporter mRNA (100 pg) was co-injected with mRNA for mex3b-HA,Î KH1+2-HA or HA-globin (1 ng). Reporter mRNA was co-immunoprecipitated with the anti-HA antibody and analyzed by RT-PCR.
	Fig. 4. A negative autoregulatory loop of Mex3b. (A-C) myc-mex3b and the 3â²LCU. (D-F) mex3b-HA and subregion D. Schematic representations of expression constructs are shown (A,D). Myc-Mex3b and Mex3b-HA exhibited basically the same activity. Embryos injected with each DNA construct were lysed at stage 13. Transcripts were quantitated by qRT-PCR using the SV40 primer set (B,E). Proteins were quantitated by immunoblotting with the anti-Myc (C) or anti-HA (F) antibody. The abscissa, amounts of injected DNA (pg/embryo); the ordinate, relative expression (arbitrary units).
	Fig. 5. Functional analysis of Mex3b in early embryogenesis. (A-J) Overexpression of mex3b by mRNA injection. (K-Qâ€²) Knockdown of mex3b by MO injection. mRNA (1 ng) or MO (50 ng) was co-injected with nÎ²-gal mRNA as a tracer into one blastomere in the dorsal equatorial region (A,I,K,P; indicated by DER) or the neuroectoderm (the rest). nÎ²-gal staining (red) indicates the injected side. Arrows in blue or magenta indicate reduced or increased expression, respectively. Frequency of specimen with the indicated phenotype is shown in the right bottom of each panel in blue (reduction), magenta (enhancement) or black (no change). Embryos were injected with globin (upper panels of A-F,H), mex3b (lower panels of A-F,I,J) or Myc-Î´KH1+2 (G) mRNA as indicated, and were subjected to WISH for genes as indicated at stage 10.5 (A) or stage 13 (B-G), or to immunostaining of somites with the 12/101 antibody followed by clearing with benzyl benzoate/benzyl alcohol (H-J). 5mmMO- or mex3bMO-injected embryos were subjected to WISH at stage 10.5 (K) and stage 13 (L-O), or observed for morphological appearances at stage 35 (P,Q). Transverse section (Qâ€²) of Q indicates small eye (arrowheads in Q,Qâ€²) (R-T) Effects of the 3â€²LCU on mex3b expression. Embryos were injected with GFP, GFP+3â€²LCU or GFP+3â€²LCUrev mRNA (3 ng/embryo), and subjected to WISH for endogenous mex3b mRNA at stage 12.5. Arrows in S and T indicate increased expression.
	Fig. 6. Inhibition of FGF signaling by Mex3b. (A,B) Animal cap assay. Animal caps were dissected from embryos injected with globin or mex3b or XFD mRNA (1 ng/embryo) and treated with FGF2 (100 ng/ml) or activin (20 ng/ml). Xbra or gsc expression was examined by WISH at stage 10.5 equivalent. (C,D) FGF reporter assay. Reporter DNA cdx4/Xcad3-luc (100 pg) with or without fgf8 mRNA (100 pg) or fgf4 DNA (10 pg) was co-injected with mex3b mRNA (1 ng; C) or mex3bMOs (50 ng; D), and injected embryos were assayed for luciferase activity at stage 12.5/13. Bars represent the meanÂ±s.e.m.
	Fig. 7. Analysis of sdc2 and ets1b for Mex3b targets and FGF signaling. (A-E) Response of the 3â€² UTRs of sdc2 and ets1b to Mex3b-mediated destabilization. Injection, WISH (A-D) and qRT-PCR (E) were performed as described in Fig. 2. GFP reporter mRNA containing the 3â€² UTR of indicated genes (100 pg/embryo) was co-injected with (right panel) or without (left panel) mex3b mRNA (63 pg). Reporter mRNA remaining in the embryos were examined at stage 12.5/13. Note that the 3â€² UTR of fgf20 (A), syntenin (B), sdc2 (C) or ets1 (D) destabilized reporter mRNA to some extent in respond to endogenous factors (left panels), but only the 3â€² UTR of sdc2 (C) or ets1 (D) responded to Mex3b for mRNA destabilization (right panels). (F) FGF reporter assay. Reporter DNA cdx4/Xcad3-luc (100 pg) was co-injected with fgf4 DNA (10 pg), sdc2 mRNA (100 pg) and ets1 (100 pg) as indicated, and injected embryos were assayed for luciferase activity at stage 12.5/13.
	Fig. 8. A model for post-transcriptional regulation by Mex3b. Mex3b protein levels are determined by fine-tuning mechanisms involving TLEEs and the LGDE, and a negative autoregulatory loop through the MRDE. Mex3b in turn controls the stability of target mRNAs such as ets1b and sdc2, which may contribute to regulate FGF responsiveness. An, poly(A) tail.