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.