Protein interactions in Xenopus germ plasm RNP particles

Hermes is an RNA-binding protein that we have previously reported to be found in the ribonucleoprotein (RNP) particles of Xenopus germ plasm, where it is associated with various RNAs, including that encoding the germ line determinant Nanos1. To further define the composition of these RNPs, we performed a screen for Hermes-binding partners using the yeast two-hybrid system. We have identified and validated four proteins that interact with Hermes in germ plasm: two isoforms of Xvelo1 (a homologue of zebrafish Bucky ball) and Rbm24b and Rbm42b, both RNA-binding proteins containing the RRM motif. GFP-Xvelo fusion proteins and their endogenous counterparts, identified with antisera, were found to localize with Hermes in the germ plasm particles of large oocytes and eggs. Only the larger Xvelo isoform was naturally found in the Balbiani body of previtellogenic oocytes. Bimolecular fluorescence complementation (BiFC) experiments confirmed that Hermes and the Xvelo variants interact in germ plasm, as do Rbm24b and 42b. Depletion of the shorter Xvelo variant with antisense oligonucleotides caused a decrease in the size of germ plasm aggregates and loosening of associated mitochondria from these structures. This suggests that the short Xvelo variant, or less likely its RNA, has a role in organizing and maintaining the integrity of germ plasm in Xenopus oocytes. While GFP fusion proteins for Rbm24b and 42b did not localize into germ plasm as specifically as Hermes or Xvelo, BiFC analysis indicated that both interact with Hermes in germ plasm RNPs. They are very stable in the face of RNA depletion, but additive effects of combinations of antisense oligos suggest they may have a role in germ plasm structure and may influence the ability of Hermes protein to effectively enter RNP particles

Localisation of RNAs into the germ plasm of vitellogenic xenopus oocytes

We have studied the localisation of mRNAs in full-grown Xenopus laevis oocytes by injecting fluorescent RNAs, followed by confocal microscopy of the oocyte cortex. Concentrating on RNA encoding the Xenopus Nanos homologue, nanos1 (formerly Xcat2), we find that it consistently localised into aggregated germ plasm ribonucleoprotein (RNP) particles, independently of cytoskeletal integrity. This implies that a diffusion/entrapment-mediated mechanism is active, as previously reported for previtellogenic oocytes. Sometimes this was accompanied by localisation into scattered particles of the “late”, Vg1/VegT pathway; occasionally only late pathway localisation was seen. The Xpat RNA behaved in an identical fashion and for neither RNA was the localisation changed by any culture conditions tested. The identity of the labelled RNP aggregates as definitive germ plasm was confirmed by their inclusion of abundant mitochondria and co-localisation with the germ plasm protein Hermes. Further, the nanos1/Hermes RNP particles are interspersed with those containing the germ plasm protein Xpat. These aggregates may be followed into the germ plasm of unfertilized eggs, but with a notable reduction in its quantity, both in terms of injected molecules and endogenous structures. Our results conflict with previous reports that there is no RNA localisation in large oocytes, and that during mid-oogenesis even germ plasm RNAs localise exclusively by the late pathway. We find that in mid oogenesis nanos1 RNA also localises to germ plasm but also by the late pathway. Late pathway RNAs, Vg1 and VegT, also may localise into germ plasm. Our results support the view that mechanistically the two modes of localisation are extremely similar, and that in an injection experiment RNAs might utilise either pathway, the distinction in fates being very subtle and subject to variation. We discuss these results in relation to their biological significance and the results of others

A Highly Conserved Poc1 Protein Characterized in Embryos of the Hydrozoan Clytia hemisphaerica: Localization and Functional Studies

Poc1 (Protein of Centriole 1) proteins are highly conserved WD40 domain-containing centriole components, well characterized in the alga Chlamydomonas, the ciliated protazoan Tetrahymena, the insect Drosophila and in vertebrate cells including Xenopus and zebrafish embryos. Functions and localizations related to the centriole and ciliary axoneme have been demonstrated for Poc1 in a range of species. The vertebrate Poc1 protein has also been reported to show an additional association with mitochondria, including enrichment in the specialized “germ plasm” region of Xenopus oocytes. We have identified and characterized a highly conserved Poc1 protein in the cnidarian Clytia hemisphaerica. Clytia Poc1 mRNA was found to be strongly expressed in eggs and early embryos, showing a punctate perinuclear localization in young oocytes. Fluorescence-tagged Poc1 proteins expressed in developing embryos showed strong localization to centrioles, including basal bodies. Anti-human Poc1 antibodies decorated mitochondria in Clytia, as reported in human cells, but failed to recognise endogenous or fluorescent-tagged Clytia Poc1. Injection of specific morpholino oligonucleotides into Clytia eggs prior to fertilization to repress Poc1 mRNA translation interfered with cell division from the blastula stage, likely corresponding to when neosynthesis normally takes over from maternally supplied protein. Cell cycle lengthening and arrest were observed, phenotypes consistent with an impaired centriolar biogenesis or function. The specificity of the defects could be demonstrated by injection of synthetic Poc1 mRNA, which restored normal development. We conclude that in Clytia embryos, Poc1 has an essentially centriolar localization and function

Polo-Like Kinase-1 Controls Aurora A Destruction by Activating APC/C-Cdh1

Polo-like kinase-1 (Plk1) is activated before mitosis by Aurora A and its cofactor Bora. In mitosis, Bora is degraded in a manner dependent on Plk1 kinase activity and the E3 ubiquitin ligase SCF-βTrCP. Here, we show that Plk1 is also required for the timely destruction of its activator Aurora A in late anaphase. It has been shown that Aurora A destruction is controlled by the auxiliary subunit Cdh1 of the Anaphase-Promoting Complex/Cyclosome (APC/C). Remarkably, we found that Plk1-depletion prevented the efficient dephosphorylation of Cdh1 during mitotic exit. Plk1 mediated its effect on Cdh1, at least in part, through direct phosphorylation of the human phosphatase Cdc14A, controlling the phosphorylation state of Cdh1. We conclude that Plk1 facilitates efficient Aurora A degradation through APC/C-Cdh1 activation after mitosis, with a potential role for hCdc14A