Cragle C and MacNicol AM (2014), Musashi protein-directed translational activati...

XB-ART-48701

J Biol Chem 2014 May 16;28920:14239-51. doi: 10.1074/jbc.M114.548271.

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Musashi protein-directed translational activation of target mRNAs is mediated by the poly(A) polymerase, germ line development defective-2.

Cragle C , MacNicol AM .

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The mRNA-binding protein, Musashi, has been shown to regulate translation of select mRNAs and to control cellular identity in both stem cells and cancer cells. Within the mammalian cells, Musashi has traditionally been characterized as a repressor of translation. However, we have demonstrated that Musashi is an activator of translation in progesterone-stimulated oocytes of the frog Xenopus laevis, and recent evidence has revealed Musashi's capability to function as an activator of translation in mammalian systems. The molecular mechanism by which Musashi directs activation of target mRNAs has not been elucidated. Here, we report a specific association of Musashi with the noncanonical poly(A) polymerase germ line development defective-2 (GLD2) and map the association domain to 31 amino acids within the C-terminal domain of Musashi. We show that loss of GLD2 interaction through deletion of the binding domain or treatment with antisense oligonucleotides compromises Musashi function. Additionally, we demonstrate that overexpression of both Musashi and GLD2 significantly enhances Musashi function. Finally, we report a similar co-association also occurs between murine Musashi and GLD2 orthologs, suggesting that coupling of Musashi to the polyadenylation apparatus is a conserved mechanism to promote target mRNA translation.

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Species referenced: Xenopus laevis
Genes referenced: cpeb1 mos msi1 parn tent2

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	Figure 1. Musashi-1 associates specifically with the noncanonical poly(A) polymerase, GLD2. A, oocytes were co-injected with mRNA encoding HA-GLD2 and either GST-XMsi1, GST-CPEB1, or GST. The injected oocytes were incubated overnight to express the introduced proteins and then lysed. Lysates were then subjected to GST pulldown and treatment with RNase I. Associations were visualized by Western blotting. GST-XMsi1 and GST-CPEB1 associate with HA-GLD2 in an RNase I-independent manner, although the GST tag alone does not (arrowhead). UI, uninjected oocytes. B, oocytes were injected with GST-XMsi1 or GST and allowed to express the protein before lysis and pulldown. An antibody targeting endogenous GLD2 detects GLD2 associating with GST-XMsi1 but not GST (arrowhead). UI, uninjected oocytes. C, oocytes were co-injected with mRNA encoding HA-GLD2 and either GST-XMsi1 or GST. Following incubation, oocytes were stimulated to mature with progesterone. When 50% of oocytes reached GVBD, lysate was made using immature (I) and progesterone-stimulated oocytes pre-GVBD (−) and post-GVBD (+). HA-GLD2 associates with GST-XMsi1 in immature and progesterone-stimulated oocytes (arrowhead). UI, uninjected oocytes. A representative experiment is shown, and the composite results of three independent experiments are shown graphically (right panel). D, oocytes were injected with mRNA encoding GFP-XMsi1 and GST-XPARN or GST. Oocytes were then treated as in C. XMsi1 associates with PARN in immature and progesterone-stimulated oocytes (arrowhead). A representative experiment is shown, and the composite results of three independent experiments are shown graphically (right panel). E, oocytes were injected with mRNA encoding GFP-XCPEB1 and GST-XPARN or GST. Oocytes were then treated as in C and D. As described previously, cytoplasmic polyadenylation element-binding protein dissociates from PARN after progesterone addition. A representative experiment is shown.
	Figure 2. Musashi/GLD2 interaction domain lies within amino acids 190–220 of Musashi. A, oocytes were co-injected with mRNA encoding HA-GLD2 and one of the listed GST-Musashi constructs. Co-injection with HA-GLD2 and the GST moiety alone served as a negative control in all experiments. Following incubation to allow protein expression, the oocytes were lysed and subjected to GST pulldown and RNase I treatment, followed by Western blotting. Horizontal bars schematically represent the Musashi constructs, and % value denotes degree of GLD2 interaction as quantified by spot densitometry and normalized to wild-type Musashi-1 in each case. The vertical bar marks amino acids 190–220, the deduced GLD2 interaction domain. B, mapping of the domain of CPEB1 that associates with GLD2. Methodology was the same as described in A. C, representative experiments from A and B showing GLD2 association (arrowhead) with the indicated Musashi and cytoplasmic polyadenylation element-binding protein constructs.
	Figure 3. Deletion of the GLD2 binding domain in Musashi-1 compromises Musashi-mediated oocyte maturation. A, oocytes were injected with antisense oligonucleotides targeting endogenous Musashi-1 and Musashi-2. Following incubation, the oocytes were left untreated or re-injected with mRNA encoding wild-type (WT) or deletion mutant Δ190–272 of Xenopus Musashi-1 and allowed to express the protein prior to being stimulated to mature with progesterone. The Δ190–272 Musashi mutant protein is significantly compromised for rescue. The data represent three experiments. The Western panel inset demonstrates expression of the introduced Musashi rescue proteins. UI, uninjected, no rescue protein. * indicates p < 0.05. B, oocytes were injected with antisense oligonucleotides as described in A and subsequently re-injected with WT or Δ190–234 mutant mouse Musashi-1. The deletion mutant protein is significantly compromised for rescue of GVBD. * indicates p < 0.05.
	Figure 4. GLD2 is necessary for Musashi-directed polyadenylation. A, oocytes were injected with antisense oligonucleotides targeting either GLD2a/b, Musashi-1/2, or a scrambled control oligonucleotide. Following incubation, the oocytes were stimulated to mature with progesterone treatment. Maturation was scored relative to control antisense-treated oocytes. GLD2 antisense severely attenuates oocytes maturation. The combined data of four independent experiments is shown. , indicates p < 0.01; *, indicates p < 0.001. B, representative PCR for endogenous GLD2 mRNA showing loss of intact GLD2 mRNA. C, representative poly(A) length assay for the Musashi target mRNA, Mos. A robust shift in PCR product mobility, indicative of polyadenylation, is seen in both uninjected and control (scrambled oligonucleotide)-injected oocytes. Oocytes injected with GLD2 antisense show dramatically attenuated polyadenylation of the Mos mRNA pool, similar to that seen with Musashi antisense injected oocytes. I, indicates immature oocytes; − indicates progesterone-stimulated pre-GVBD; + indicates progesterone-stimulated post-GVBD. D, graphic representation of the mobility shifts seen in C. The dashed lines are the distribution of MosPCR product in immature oocytes (Imm); solid lines are the distribution of MosPCR products in progesterone-stimulated oocytes pre-GVBD (−ws). The green lines are scrambled-control oligonucleotide-injected oocytes. Red lines represent Msi1/2 antisense-injected oocytes. Black lines are GLD2a/b antisense-injected oocytes. The line peaks indicate the median of the population of mRNA lengths. The shift of the peak between immature (dashed line) and progesterone-stimulated (solid line) indicates the extent of polyadenylation. Scrambled, control oligonucleotide-injected oocytes show a polyadenylation shift of 50 adenylate residues, although both Msi1/2 and GLD2a/b oligonucleotide-injected oocytes show a polyadenylation shift of only 20 adenylates. ns, not significant.
	Figure 5. GLD2 is sufficient of promote Musashi-mediated polyadenylation and oocyte maturation. A, oocytes were injected with nuclease-free water (control) or mRNA mixtures containing GST-Musashi-1, HA-GLD2a, or GST-Musashi-1 and HA-GLD2a. The oocytes were allowed to express the injected RNA and were then stimulated to mature with progesterone. The maturation rate relative to control in five independent experiments is shown graphically. , indicates p < 0.05; , indicates p < 0.01. ns, not significant. B, representative Western blot showing protein expression from an experiment in A. C, representative poly(A) assay comparing polyadenylation of two endogenous mRNAs from Musashi/GLD2 co-injected oocytes relative to water-injected (control) oocytes. The Mos mRNA is under Musashi control and therefore polyadenylated early (prior to GVBD), whereas the cyclin B1 mRNA is under cytoplasmic polyadenylation element-binding protein control and polyadenylated late (at or after GVBD). The earliest signs of polyadenylation () appear at 3 h for all 3 conditions. − indicates pre-GVBD; + indicates post-GVBD samples. D, poly(A) length assay comparing polyadenylation of Mos mRNA from Musashi, GLD2, or water-injected (control) oocytes. The earliest signs of polyadenylation appear at 3 h for all three conditions. This indicates that the acceleration in onset of polyadenylation seen in C requires overexpression of both Musashi-1 and GLD2 together. − indicates pre-GVBD; + indicates post-GVBD. E, graphic representation of the poly(A) assay (C) for the Mos mRNA at the 3rd h. The dashed line is immature (Imm) oocytes (0 h), and the solid line is at the 3-h time point. Gray lines represent water (control) injection, and the black lines are Musashi/GLD2 co-injected oocytes. At the 3rd h, Musashi/GLD2 co-injection has promoted a polyadenylation of ∼30 adenylate residues, whereas control injected oocytes have only extended their poly(A) tails by ∼10 adenylates.
	Figure 6. Conservation of GLD2 and Musashi-1 co-association. A, oocytes were injected with GFP-tagged murine GLD2 (mGLD2) and GST-tagged Xenopus Musashi-1 (XMsi1), murine Musashi-1 (mMsi1), Musashi-2 (mMsi2), or the GST moiety alone. Co-associated proteins were assessed by Western blotting with GFP. A GFP-mGLD2-specific band was detected in both Xenopus and mouse GST-Musashi-1 pulldowns (arrowhead) but not with GST-mMusashi-2 or GST alone. A representative experiment is shown, as well as the average of three independent experiments (graph, right panel). **, indicates p < 0.01. ns, not significant. B, proposed model of Musashi regulation of polyadenylation of target mRNAs (upper panel). Our data indicate GLD2 mediates polyadenylation of Musashi target mRNAs following progesterone addition. The role and regulation of PARN within the Musashi complex are unclear (?) at the juncture. The cytoplasmic polyadenylation element-binding protein model, showing progesterone-dependent PARN expulsion, is shown for comparison (lower panel).

References [+] :

Arumugam, Ringo/cyclin-dependent kinase and mitogen-activated protein kinase signaling pathways regulate the activity of the cell fate determinant Musashi to promote cell cycle re-entry in Xenopus oocytes. 2012, Pubmed, Xenbase