Eukaryotic initiation factor eIF6 modulates the expression of Kermit 2/XGIPC in IGF- regulated eye development.

The eukaryotic initiation translation factor eIF6 is a highly conserved, essential protein implicated in translation. eIF6 is regulated in vivo by extracellular signals, such as IGF signaling (for a review see Miluzio et al., 2009). In Xenopus, eif6 over-expression causes a delay in eye development (De Marco et al., 2011). In this study we showed that eif6 co-immunoprecipitates with the insulin-like growth factor receptor (igfr) and may function downstream of igf in eye formation. The relationship between eif6 and gipc2, a protein partner of a variety of molecules including membrane proteins, was investigated. gipc2 is required for maintaining igf-induced akt activation on eye development (Wu et al., 2006). Significantly eif6 and gipc2 have opposite effects in eye development. While eif6 is required for eye formation below threshold levels, gipc2 knockdown impairs eye development (De Marco et al., 2011; Wu et al., 2006). In this study, it was shown that in eif6 over-expressors, the delay in eye morphogenesis is reversed by gipc2 injection, while the injection of eif6 down-regulates gipc2 expression. Real-time-PCR indicates that eif6 regulates gipc2 expression in a dose-dependent manner. In contrast, gipc2 knockdown has no significant effect on eif6 mRNA levels. These results suggest that eif6 regulation of gipc2 enables correct morphogenesis of Xenopus eye and stimulate questions on the molecular network implicated in this process.

Eukaryotic initiation factor 6 (eif6) overexpression affects eye development in Xenopus laevis.

The translation initiation factor eif6 has been implicated as a regulator of ribosome assembly, selective mRNA translation and apoptosis. Many of these activities depend upon the phosphorylation of eif6 serine 235 by PKC. Previous data showed that eif6 binds to the 60S ribosomal subunit when unphosphorylated, inhibiting assembly with the 40S subunit. Phosphorylation of Ser235 releases eif6 from the 60S subunit and allows assembly. eif6 acts as an anti-apoptotic factor via regulation of the bcl2/bax balance and acts selectively upstream of bcl2. This activity also depends upon phosphorylation of eif6 Ser235. One of the consequences of eif6 overexpression in Xenopus embryos is aberrant eye development. Here we evaluate the eye phenotype and show that it is transient. We show that the whole eye, particularly the retina layers, of the embryos injected with eif6-encoding mRNA recover by stage 42. Embryos over-expressing eif6 have normal expression of anterior- and brain-specific markers, indicating that outside the eye field, other neural regions appear unaffected by the eif6 injection. No eye defect was detected when morpholinos were used to reduce eif6 protein synthesis. We tested how two known pathways of eif6 function with respect to alteration of eye development. We found that injection of bcl2 did not produce the eye phenotype and eif6-bax co-injection did not rescue the eye defect, suggesting that the eye phenotype is not bearing on the anti-apoptotic role played by eif6 is not linked to its role as an anti-apoptotic factor. We also determined that PKC-dependant phosphorylation of Ser235 in eif6 is not required to produce defective eye development. These results indicate that the aberrant eye phenotype, produced by eif6 overexpression, is not directly linked to the PKC-regulated effects of eif6 on translation and ribosomal subunit interaction or on eif6 anti-apoptotic properties.

A transient asymmetric distribution of XNOA 36 mRNA and the associated spectrin network bisects Xenopus laevis stage I oocytes along the future A/V axis.

In Xenopus oogenesis, the mechanisms governing the localisation of molecules crucial for primary axis determination have been uncovered in recent years. In stage I oocytes, the mitochondrial cloud (MC) entraps RNAs implicated in germ line specification and other RNAs, such as Xwnt-11 and Xlsirts, that are later delivered to the vegetal pole. Microfilaments and microtubules gradually develop in the cytoplasm, sustaining organelles as well as the MC. At stage III, other mRNAs migrate to the vegetal hemisphere through a microtubule-dependent mechanism. We report here the isolation of a cDNA encoding XNOA 36, a highly conserved protein, whose function is to date not fully understood. The XNOA 36 transcript is abundantly accumulated in stage I oocytes where it decorates a filamentous network. At the end of stage I the transcript gradually segregates in a sector of the oocyte surrounding the MC and opposite the ovarian hylum. Here, XNOA 36 mRNA distributes in a gradient-like pattern extending from a peripheral network towards the interior of the oocyte. This distribution is similar to that of alpha-spectrin mRNA. Both mRNAs are segregated in one half of the 250 microm oocytes, with the MC located between the XNOA 36/alpha-spectrin mRNA-labelled and unlabelled regions. XNOA 36 mRNA localisation was uncoupled from that of alpha-spectrin mRNA by cytochalasin B or ice-nocodazole treatments, suggesting that the two transcripts rely on different mechanisms for their localisation. However, immunolocalisation experiments coupled with in situ hybridisation revealed that the XNOA 36 transcript co-localises with the protein spectrin. This observation, together with the finding that XNOA 36 mRNA co-precipitates with spectrin, indicates that these two molecules interact physically. In conclusion, our data suggest that XNOA 36 mRNA is localized and/or anchored in the oocyte through a cytoskeletal network containing spectrin. The putative implications of this finding are discussed.