Cooke A , Park S , Dewey CN , Wickens M , Sheets MD .

GM31892 NIGMS NIH HHS , GM50942 NIGMS NIH HHS , HG005232 NHGRI NIH HHS , R01 GM031892 NIGMS NIH HHS , R01 GM050942 NIGMS NIH HHS , T32GM07215 NIGMS NIH HHS , R01 HG005232 NHGRI NIH HHS , T32 GM007215 NIGMS NIH HHS , R37 GM031892 NIGMS NIH HHS

	FIGURE 1. xCR1 mRNA translation can be repressed by Bicaudal-C. (A) Diagram of repression assay. Luciferase reporter mRNAs Luc/xCR1 (contains the 3′ UTR of the xCR1 mRNA) or Luc/Cyclin-BI (contains the 3′ UTR of the cyclinB1 mRNA) were injected into Xenopus embryos with or without the mRNA encoding candidate repressors (HA-Bic-C or MYC-Nanos1). Injected embryos were assayed for luciferase (Sheets et al. 1994; Fritz and Sheets 2001; Zhang et al. 2009), and the ratio of luciferase with and without a putative repressor was calculated as a measure of repression. (B) Bic-C specifically repressed the Luc/xCR1 reporter mRNA. (C,D) Regions of the xCR1 3′ UTR used in luciferase reporter mRNAs. The TCE (translational control element) was previously referred to as the Mut2 region (Zhang et al. 2009) and shown to be sufficient for repression in vegetal cells. Reporter mRNAs were analyzed for repression by Bic-C as described in A and B. (E) Repression by Bicaudal-C required a 5′ cap. The CSFV-Luc/xCR1-TCE reporter mRNAs contain the IRES from the CSFV 5′ of the luciferase coding region and the TCE of the xCR1 3′ UTR. The reporter mRNAs were analyzed for repression by Bic-C as described in A and B.
	FIGURE 2. The C-terminal region of Bic-C contains translational repression activity. (A) The regions of Bic-C fused to MS2. (B) Diagram of the tethered translation assay (Coller et al. 1998; Coller and Wickens 2002, 2007). mRNAs encoding different Bic-C MS2 fusions were injected into oocytes, followed by injection of two reporter mRNAs; the 3′ UTR of the firefly luciferase reporter mRNA contained three MS2-binding sites and a 39-nt poly(A) tail; the Renilla luciferase reporter mRNA lacked MS2 sites. (C) The C-terminal half of Bic-C repressed translation independent of poly(A). Top panel: relative repression of firefly luciferase poly A39 mRNA translation by each protein. Middle panel: translational repression of firefly luciferase mRNA lacking a poly(A) tail by each protein. Bottom panel: analysis of fusion protein expression by immunoblotting with α–HA and α–actin antibodies.
	FIGURE 3. The N-terminal region of Bic-C specifically binds the TCE region of the xCR1 3′ UTR that directs translational repression. (A) Radiolabeled RNAs were mixed with 0, 50, and 200 nM GST-N-term Bic-C protein, and binding was analyzed by native gel electrophoresis. The 1–308, TCE (286–637), and 615–941 radiolabeled RNAs were derived from the xCR1 3′ UTR (see Fig. 2). The negative control RNA was derived from the pSTBlue plasmid. (B) Xenopus embryos injected with mRNAs encoding HA-tagged versions of Bic-C, the N-terminal half of Bic-C, or the C-terminal half of Bic-C. The Bic-C proteins were immunoprecipitated from blastula stage injected embryos with αHA antibodies, and the mRNAs present were analyzed using RT-PCR and RNA-seq. (C) The presence of the xCR1 and cyclinB1 mRNAs in the input and pellet fractions as assayed with RT-PCR. The input samples were unfractionated extract, and the pellet samples were the HA immunoprecipitates. Proteins from the input and pellet samples were analyzed by immunoblotting and the HA antibody (lower panel). The low molecular weight species detected in the uninjected sample (lane 5) is a cross-reacting species with the HA antibody.
	FIGURE 4. Identification of Bicaudal-C-regulated mRNAs. (A) Reporter mRNAs created with the 3′ UTRs of 14 different Bic-C-associated mRNAs identified by RIP-seq were assayed as described in Figure 1A. Reporter mRNAs containing the xCR1 TCE and the cyclin B1 3′ UTR serve as positive and negative controls, respectively. (B,C) Diagram of vegetal cell-specific repression assay (Zhang et al. 2009). Reporter mRNAs are injected into animal cells or vegetal cells of separate eight-cell embryos. When embryos reach stage 7, luciferase activity is assayed and the ratio in vegetal cells vs. animal cells calculated.

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