Nijjar S and Woodland HR (2013), Localisation of RNAs into the germ plasm of vit...

XB-ART-46966

PLoS One 2013 Jan 01;84:e61847. doi: 10.1371/journal.pone.0061847.

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Localisation of RNAs into the germ plasm of vitellogenic Xenopus oocytes.

Nijjar S , Woodland HR .

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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.

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Species referenced: Xenopus laevis
Genes referenced: actl6a cat2 gdf1 mmut mtor nanos1 ocm pgat rbpms rbpms2 vegt velo1

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	Figure 1 Localisation of arly pathwaymRNAs and Hermes protein into the germ plasm of stage VI oocytes. 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 40oil-immersion lens. B. Full-length Cy5-labelled nanos1 and Xpat 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 nanos-1 and Xpat RNA 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-nanos1 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.
	Figure 4. Disruption of the tubulin and actin cytoskeletons does not prevent localisation to the germ plasm. Oocytes injected with Cy5-nanos1 and YFP-Hermes RNAs were treated with colcemid, cytochalasin D or both. After 48 h the vegetal cortex was examined by confocal microscopy. A. Control oocytes. Oocytes were treated with: B. Colcemid. C. Cytochalasin. D. Both inhibitors. (Nocodazole produced results similar to B, not shown).
	Figure 5. FRAP experiment showing that Hermes protein in germ plasm exchanges with a cytoplasmic pool YFP-Hermes. A. Confocal images, before during and after the bleaching step. B. Quantification of the bleached area and a small unbleached control region. C. Low power image to show that injected RNA diffuses more slowly than protein. Oocytes were co-injected at the equator with Cy5-nanos1 and YFP-Hermes RNAs (The latter does not have its own UTRs, so would not localise). After 48 h the vegetal pole was visualised and it is seen that the Cy5-nanos1 RNA has diffused more slowly from the injection point, at the top left, than Hermes protein.
	Figure 6. Localisation of RNAs in stage IV oocytes. The indicated RNAs were injected into stage IV oocytes and cultured in the presence of vitellogenin for 48 h. A show pattern I, with exclusively germ plasm localisation. A field of Cy3-nanos1 (A) and Cy5-VegT RNA (B) aggregates was seen at the vegetal pole at low magnification. C and D show higher power views of these oocytes. E and F show higher power views of oocytes showing Pattern II, a mixture of germ plasm localisation (RNP particles containing Hermes) and late pathway localisation (particles lacking Hermes). To illustrate our identification of structures formed by the activity of the two pathways of RNA localisation, in panels E and F examples of germ plasm are identified with arrows and late pathway particles with arrowheads. The former show coincident Cy5 and YFP signals, but the latter lack the YFP-Hermes.
	Figure 7. Localisation of nanos1 mutant transcripts in stage VI oocytes. 3′ and 5′ deletions used to create mutant transcripts are shown in schematic form (ORF, hatched; 5′ UTR, red; 3′ UTR, black). The map of the constructs is not drawn on a linear scale, but the residue numbers on the ruler correspond to the construct ends below. mut-nanos1 consisted of the full length transcript with its start codon mutated to TTG. Oocytes were injected with Cy5 labelled mutant transcripts and examined after 48 h using constant gain settings on the confocal microscope. The subsequent localisation of the injected RNA was scored as plus or minus. Examples of the different degrees of localisation are shown below.
	Figure 8. Co-operation between the mutant nanos1-3′Δ6 and the wild-type 3′UTR in germ plasm RNP localisation. Stage VI oocytes injected with Cy3-nanos1 3′Δ6 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 nanos1 3′UTR
	Figure 9. Localisation of non-germ plasm RNAs in stage V/VI oocytes. Cy5 or Cy3 labelled RNAs were injected into oocytes and examined 48 h later. In A and E vitellogenin was included in the culture medium. In panel F labelled Xvelo1 full length RNA was co-injected with RNA encoding YFP-Hermes protein. The localised RNA or protein detected is indicated in each panel. In panel F germ plasm aggregates are indicated with arrows and late pathway particles with arrowheads.
	Figure 2. YFP-Hermes localises into particles in islands containing concentrated mitochondria.A. Stage VI oocytes were injected with RNA encoding YFP-Hermes and after 18 h mitochondria were stained with TMRE [22], prior to visualisation of the vegetal cortex, as in Figure 1A. B Stage IV oocytes were injected with Cy5-nanos1 RNA and after 24 h the oocytes were stained with TMRE and analysed as in A.
	Figure 3. Disruption of micro- and intermediate filaments and its effect on germ plasm structure.Oocytes were cultured for 24 h with the inhibitors shown. They were fixed and Xpat protein particles were stained with purified antibodies [22]. Following Xpat staining oocytes were co-stained for microtubules, A, or cytokeratins, B.
	Figure 10. The behaviour of endogenous and exogenous germ plasm molecules during oocyte maturation.A–D. Fixed oocytes stained with antisera against Hermes (green) and Xpat (red). E–H. Live oocytes expressing YFP-Hermes (green) and injected Cy5-nanos1 (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-nanos1 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.
	Figure 1. Localisation of ‘early pathway’ mRNAs and Hermes protein into the germ plasm of stage VI oocytes.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 nanos1 and Xpat 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 nanos-1 and Xpat 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-nanos1 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.

References [+] :

Alarcón, RNA anchoring in the vegetal cortex of the Xenopus oocyte. 2001, Pubmed, Xenbase