Together, our results indicate that the context-dependent interactions of Musashi with poly(A)-binding proteins confer a differential ability to control target mRNA translation and cell fate. Results Musashi interactome is dynamically regulated in response to progesterone stimulation To better understand the molecular components that facilitate Musashi-dependent mRNA translational control, we sought to identify interacting partner proteins in both immature oocytes (where target mRNAs are repressed) and progesterone-stimulated oocytes (where target mRNAs are translationally activated). and blocking oocyte maturation. Ectopic expression of either ePABP or PABPC1 restored Musashi-dependent mRNA translational activation and maturation of ePABP-attenuated oocytes. Consistent with these findings, PABPC1 remained associated with Musashi under conditions of Musashi target mRNA de-repression and translation during mammalian stem cell differentiation. Because association of Musashi1 with poly(A)-binding proteins has previously been implicated only in repression of Musashi target mRNAs, our findings reveal novel context-dependent roles for the interaction of Musashi with poly(A)-binding protein family members in response to extracellular cues that control cell fate. embryo development (49); and PABPC1, although the PABPC1 interaction was restricted to maturing oocytes. Contrary to the model Rabbit Polyclonal to KSR2 in which interaction with poly(A)-binding protein mediates repression of Musashi target mRNAs, we report that interaction with either ePABP or PABPC1 is necessary to promote Musashi target mRNA translational activation. Interestingly, we observed that Musashi is also associated with PABPC1 in a mammalian cancer cell line and in human stem cells under conditions of Musashi target mRNA translational activation and inhibited stem cell self-renewal. Together, our results indicate that the context-dependent interactions of Musashi with poly(A)-binding proteins confer a differential ability to control target mRNA translation and cell fate. Results Musashi interactome is dynamically regulated in response to progesterone stimulation To better understand the molecular components that facilitate Musashi-dependent mRNA translational control, we sought to identify interacting partner proteins in both immature oocytes PI4KIII beta inhibitor 3 (where target mRNAs are repressed) and progesterone-stimulated oocytes (where target mRNAs are translationally activated). In three separate experiments, we microinjected immature oocytes with RNA encoding either a GST-tagged form of Musashi1 to facilitate recovery of co-associated proteins after partial purification over GSH-Sepharose or the GST moiety alone. Oocytes were incubated overnight to allow expression of the ectopic proteins, and then the GSTCMusashi1-injected oocytes or control GST-injected oocytes were each split into two pools, one of which was left untreated (immature) and the other stimulated with progesterone. Following LC-coupled tandem MS, the spectral counts from identified proteins were normalized for protein size, giving us a normalized spectral abundance factor (50), and transformed to log2 with which to compare the relative abundance of each identified protein interacting with either GSTCMusashi1 or GST (Table S1). Fifty proteins specifically interacted with the GSTCMusashi1 protein but not the GST moiety alone (Fig. 1oocytes were injected with mRNA encoding GSTCXMsi1 or the GST moiety alone. The injected oocytes were incubated overnight to express the introduced proteins. Following incubation, one-half of the GST and one-half of the GSTCXMsi1-injected oocytes were stimulated to mature with progesterone, and the rest were left untreated (gene set enrichment analysis was performed on the 50 interacting proteins, and the top PI4KIII beta inhibitor 3 10 significant gene ontology molecular functions are shown graphically. Musashi associates with ePABP in immature and maturing oocytes The persistence of ePABP and PABP4 interactions in Musashi mRNP complexes in maturing oocytes and indeed the PI4KIII beta inhibitor 3 progesterone-dependent recruitment of PABPC1 were unexpected because Musashi target mRNAs are translationally activated, not repressed, in maturing oocytes. Here, we have sought to determine the role and contribution of ePABP and PABPC1 to Musashi-dependent mRNA translational activation and oocyte maturation. To confirm the interaction of ePABP with Musashi1 seen by MS, immature oocytes were injected with RNA encoding GST-tagged Musashi1 or the GST moiety alone, and the ability of Musashi1 to associate specifically with endogenous ePABP was determined after partial purification over GSH-Sepharose and Western blotting. To ensure that any associations observed were due to proteinCprotein interactions rather than just co-occupancy of the same mRNA, all GSH pulldown experiments included an RNase1 treatment step. Endogenous ePABP was found to co-associate in an RNA-independent manner with Musashi1 in both immature and progesterone-treated oocytes (Fig. 2oocytes were injected with mRNA encoding GSTCXMsi1 or the GST moiety alone. The injected oocytes were incubated overnight to express the introduced proteins. Following incubation, two-thirds of GSTCXMsi1-injected oocytes were stimulated to mature with progesterone, and the rest were left untreated (immature). When 50% of the oocytes reached GVBD, lysate was prepared from segregated oocytes that had not (?) or had (+) completed progesterone-stimulated GVBD as well as time-matched immature oocytes (of each panel. composite results of three independent experiments reveals increased association of ePABP with Musashi1 following progesterone stimulation. The data were normalized.