The RNA-binding protein XSeb4R: a positive regulator of VegT mRNA stability and translation that is required for germ layer formation in Xenopus

Abstract

VegT represents a localized maternal determinant essentially required for endoderm formation in Xenopus. Here, we report on the identification of the RNA-binding protein XSeb4R as a positive regulator of VegT. XSeb4R interacts directly with the 3'-untranslated region of VegT mRNA, stabilizes it, and stimulates translation. Ablation of XSeb4R activity results in impairment of endoderm and mesoderm formation, while ectopic expression of XSeb4R in ectodermal cells induces endodermal and mesodermal gene expression. These observations unravel a novel mode of VegT regulation at the post-transcriptional level that is essential for germ layer formation in Xenopus.

Figure 1.

XSeb4R overexpression activates endo- and mesodermal marker genes via VegT in animal cap explants. (A) Activation of the expression of markers in animal cap explants derived from embryos injected at four- to eight-cell stages with 150 pg of XSeb4R-GR mRNA per blastomere and analyzed by RT–PCR. (CE) Control embryos; (CC) control uninjected caps. (B) Whole-mount in situ hybridization using VegT/Apod antisense probe demonstrate that XSeb4R overexpression causes strong VegT activation on the injected side (IS) of stage 15 embryos in the area corresponding to mesoderm formation (100%, n = 150). (C) RT–PCR analysis of the expression markers in animal cap explants injected with XSeb4R mRNA alone (250 pg), or coinjected with different MOs expected to inhibit translation of their target mRNAs; these were either VegT-MO (40 ng), Apod-MO (40 ng), or a combination of both MOs. The Apod mismatch MO (Apod-MM) (40 ng) was used as a control. (D) Real-time RT–PCR quantification of VegT and XSeb4R mRNA levels in animal (an), marginal (m), and vegetal (vg) explants from blastula stage embryo. Expression in the ectodermal (animal) explants is arbitrarily set to 1. Values for marginal and vegetal zone explants are thus fold increase in respect to the level of expression detected in the ectodermal explants.

Figure 2.

XSeb4R protein binds directly to the 3′-UTR of VegT mRNA. (A) Bacterially purified GST-XSeb4R and GST-XSeb4RΔRRM proteins were tested by UV-cross-linking assays for their ability to interact with full-length ³²P-VegT mRNA. Unlabeled VegT 3′-UTR, ORF, or LacZ mRNAs were used as competitors. (B) Schematic representation of the VegT 3′-UTR subfragments tested by UV-cross-linking. The corresponding base pairs of the cDNA sequence (Acc. number: U59483) are indicated. (C) UV-cross-linking experiments of bacterially expressed, purified GST-XSeb4R or MT-GST-XSeb4R produced in embryos. Note that XSeb4R binds to multiple sites within the 3′-UTR region of VegT mRNA.

Figure 3.

XSeb4R stimulates translation. (A) A given protein can be targeted to a reporter mRNA harboring MS2-binding sites in its 3′-UTR by fusion to the RNA-binding viral MS2 coat protein. Coinjection of synthetic mRNA encoding either MS2 fused to the full-length XSeb4R (MS2-XSeb4R), or a mutant version lacking the N-terminal RNA recognition motif (MS2-XSeb4RΔRRM) together with a firefly luciferase reporter mRNA harboring three MS2-binding sites for (Luc-MS2) leads to robust luciferase activity in Xenopus oocytes. XSeb4R-MS2 lacking the C terminus, untethered XSeb4R, or MS2 alone fails to induce luciferase activity. Translational activation by MS2-PAB1P serves as positive control. MS2-XSeb4R does not activate a reporter construct lacking the MS2 recognition sites (Luc-ΔMS2). (B) Delineation of the VegT 3′-UTR region required for translational activation through XSeb4R. The VegT 3′-UTR and the indicated fragments were tested for conferring translational activation via XSeb4R in Xenopus oocytes. All firefly luciferase values were normalized to renilla luciferase; shown is the fold-activation relative to the Luc-MS2 or the Luc-VegT 3′-UTR reporter activity alone

Figure 4.

XSeb4R stabilizes its target mRNAs. (A) Real-time RT–PCR analysis of VegT transcripts in animal cap explants from embryos injected with XSeb4R mRNA, XSeb4R-MO, or control uninjected embryos collected at different time points after dissection. An average of 50 was employed per batch of animal cap explants. Note the significant increase or decrease of the VegT mRNA level in caps overexpressing XSeb4R or depleted of XSeb4R, respectively. Data are expressed in arbitrary units (A.U.). (B) XSeb4R overexpression causes persistent staining of a VegT 3′-UTR fragment that strongly binds XSeb4R (VegT F8), but not of a fragment that does not do so (VegT F7). The indicated combinations of mRNAs were injected into the animal pole of one blastomere in two-cell stage albino embyos and batches of 20–25 embryos were fixed at various stages. The mRNA fragments of interest were traced by whole-mount in situ hybridization, using VegT antisense mRNA as a probe.

Figure 5.

XSeb4R is required for endoderm formation and mesoderm induction. (A) XSeb4R knockdown decreases VegT mRNA and inhibits the expression of endoderm and mesodermal markers (100% inhibited, n = 41 for VegT, n = 28 for Wnt8, n = 31 for Xbra, n = 36 for GATA4, and n = 42 for Sox17β). XSeb4R-MO was injected into the marginal or the vegetal region of two blastomeres (50 ng each) from four-cell-stage embryos together with lacZ mRNA. Embryos were fixed at stage 11 and stained with Xgal (blue). For rescue, 150 pg of XSeb4R mRNA were used. Total rescue was obtained in 27 of 42 embryos for VegT, whereas only partial rescue was observed for Wnt8 (13 of 22) Xbra (20 of 34) Sox17β (36 of 42) and GATA4 (27 of 40). (B) A competing Seb4R-binding RNA fragment derived from the VegT 3′-UTR (F8) inhibits mesodermal and endodermal gene expression. Embryos injected at the marginal or vegetal regions with 3 ng of VegT F8 or 4.5 ng of VegT F7 mRNA were fixed at gastrula stage 11 and analyzed by in situ hybridization for the indicated probes. The VegT probe detects the injected UTR fragments and the endogeneous transcripts. VegT F8 but not VegT F7 UTR injection causes significant suppression of the tested markers (Wnt8: VegT F8, 36 of 52; VegT F7, 19 of 57; Xbra: VegT F8, 34 of 46; VegT F7, 15 of 51; Sox17β: VegT F8, 32 of 40; VegT F7, eight of 34).