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

The 40th anniversary issue of Differentiation—Cilia in development, differentiation and disease

This special issue on cilia/flagella has been compiled to celebrate the 40th anniversary of the International Society of Differentiation, Inc. (ISD) and its journal Differentiation. The ISD was founded as a result of the First International Conference on Differentiation held in Nice in 1971. The purpose of the Society, as stated in the bylaws, is “to encourage and develop research and communication in the fields of cell and developmental biology, molecular biology and oncology through meetings and publications”. ISD has held onto this commitment over 40 years by arranging major international conferences on a regular basis, every second year for more than the last two decades, by supporting selected smaller meetings, and by publishing the journal Differentiation. The journal Differentiation was launched in 1972, with the first issue published – delayed a little, as usual – by Macmillan Journals Limited in February 1973 (Fig. 1, Table 1). The first editor of the journal, Dimitri Viza starts his editorial by saying that “paucity is not a syndrome from which biological literature can be said to suffer” and then, almost apologetically, justifies the launching of a new journal by discussing a gap he sees in the existing literature, i.e., the need for a synthetic view on cell differentiation as a special problem. The ideas in this first editorial, including the notion of cancer as an aspect of cell differentiation, still characterize the journal: Differentiation intends to maintain its broad focus, but with its special interest in the overlap between developmental biology and disease, the traditional niche of the Journal of ISD. Accordingly, and unlike many other long-lived journals, Differentiation has retained its name over the decades, although the cover included a subtitle during the first few years of publication and later again from the late 1980s till the late 1990s. These subtitles, “Differentiation; research in biological diversity” and later “Differentiation, ontogeny and neoplasia” followed by “Differentiation, ontogeny, neoplasia and differentiation therapy” also perfectly reflected the timely scope of the journal. Today, the key areas of Differentiation's scope include cancer, morphogenesis and stem cells, as well as the more obvious cell differentiation in vivo and in vitro

Localisation of ‘early pathway’ mRNAs and Hermes protein into the germ plasm of stage VI oocytes.

<p>A. The procedure for examining the cortical distribution of labelled RNAs. After injection and incubation of oocytes in OCM (with or without vitellogenin-containing serum) for 24 to 72 h, oocytes were held in an inverted position between a slide and a coverslip, in a chamber made with a latex spacer. They were then examined by confocal microscopy using a 40× oil-immersion lens. B. Full-length Cy5-labelled <i>nanos1</i> and <i>Xpat</i> RNAs localise in islands of particles at the vegetal pole 48 h after injection. C. Low power stereo microscope view from the side of the vegetal pole of a whole stage VI oocyte, 24 h after injection with mRNA encoding YFP-Hermes. YFP-Hermes protein is clearly localised to a field of fluorescent islands at the vegetal pole (white arrows), typical of germ plasm markers. In the stereo microscope the depth of focus is large so that a much larger area than that occupied by the germ plasm is in focus. D,E. In these islands Cy5-labelled <i>nanos-1</i> and <i>Xpat</i> RNA’s co-localise with YFP-Hermes protein, following co-injection of mRNA encoding YFP-Hermes. All the above oocytes were visualized live, as they are in later figures unless otherwise stated. F. Internal distribution of Cy5-<i>nanos1</i> RNA between the nucleus and the vegetal cortex of a fixed stage VI oocyte. Following injection of RNA, oocytes were cultured for 48 h in OCM, fixed and hemisected with a scalpel prior to visualization by confocal microscopy using a Z-stack.</p

Co-operation between the mutant nanos1-3′Δ6 and the wild-type 3′UTR in germ plasm RNP localisation.

<p>Stage VI oocytes injected with Cy3-<i>nanos1 3′Δ6</i> RNA display a very weak localisation pattern after 48 h in culture. Localization of this RNA was greatly improved when co-injected with Cy5-labelled RNA consisting of the full length <i>nanos1</i> 3′UTR.</p

Summary of localisation patterns displayed by injected RNA.

<p>In each table cell the number of oocytes showing a given localisation with respect to the total number of injected oocytes is shown. N = the number of experiments using oocytes from different females. Cy5 or Cy3-labelled RNAs were routinely injected, with or without RNA encoding YFP-Hermes. The YFP-Hermes alone denotes localisation by the translated protein following injection of the RNA. FL; full length.</p

The behaviour of endogenous and exogenous germ plasm molecules during oocyte maturation.

<p>A–D. Fixed oocytes stained with antisera against Hermes (green) and Xpat (red). E–H. Live oocytes expressing YFP-Hermes (green) and injected Cy5-<i>nanos1</i> (red). A. The wide field of large islands in a control oocyte; the overlay of Hermes and Xpat is shown. B. A similar view of newly fertilised eggs showing the dramatic reduction in fluorescent islands. C. Detail of islands in a control oocyte. D Detail of a fertilised egg. Note that Hermes and Xpat are in distinct particles. E. The wider field of YFP-Hermes coincident with Cy5-<i>nanos1</i> is similar to the endogenous molecules in A. F. When these oocytes were matured with progesterone the field was reduced, as in B, but less so; this is expected, since fertilised eggs have progressed further. G,H. Details of the fields in E and F respectively.</p