Multiple Kinesin Motors Coordinate Cytoplasmic RNA Transport on a Subpopulation of Microtubules in Xenopus Oocytes

Department of Molecular Biology, Cell Biology & Biochemistry, Brown University, Box G-L268, Providence, RI 02912, USA.
Developmental Cell (Impact Factor: 9.71). 10/2008; 15(3):426-36. DOI: 10.1016/j.devcel.2008.06.014
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RNA localization is a widely conserved mechanism for generating cellular asymmetry. In Xenopus oocytes, microtubule-dependent transport of RNAs to the vegetal cortex underlies germ layer patterning. Although kinesin motors have been implicated in this process, the apparent polarity of the microtubule cytoskeleton has pointed instead to roles for minus-end-directed motors. To resolve this issue, we have analyzed participation of kinesin motors in vegetal RNA transport and identified a direct role for Xenopus kinesin-1. Moreover, in vivo interference and biochemical experiments reveal a key function for multiple motors, specifically kinesin-1 and kinesin-2, and suggest that these motors may interact during transport. Critically, we have discovered a subpopulation of microtubules with plus ends at the vegetal cortex, supporting roles for these kinesin motors in vegetal RNA transport. These results provide a new mechanistic basis for understanding directed RNA transport within the cytoplasm.

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Available from: Young J Yoon, Oct 05, 2015
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    • "Staufen1 is associated with Vg1 and VegT mRNAs and is required for efficient localization (Yoon and Mowry 2004). Staufen1 also interacts with at least one of the two kinesin motors that direct transport of the Vg1 RNP complex, which tracks on a subpopulation of microtubules with their plus ends oriented toward the vegetal cortex (Yoon and Mowry 2004; Messitt et al. 2008). In light of its putative role as an adaptor to kinesin, we questioned whether Staufen1 plays a role in determining the direction of RNA movement and tested its association with other localized RNAs using immunoprecipitation assays (Fig. 4). "
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    ABSTRACT: The 3' untranslated region of mRNA encoding PHAX, a phosphoprotein required for nuclear export of U-type snRNAs, contains cis-acting sequence motifs E2 and VM1 that are required for localization of RNAs to the vegetal hemisphere of Xenopus oocytes. However, we have found that PHAX mRNA is transported to the opposite, animal, hemisphere. A set of proteins that cross-link to the localization elements of vegetally localized RNAs are also cross-linked to PHAX and An1 mRNAs, demonstrating that the composition of RNP complexes that form on these localization elements is highly conserved irrespective of the final destination of the RNA. The ability of RNAs to bind this core group of proteins is correlated with localization activity. Staufen1, which binds to Vg1 and VegT mRNAs, is not associated with RNAs localized to the animal hemisphere and may determine, at least in part, the direction of RNA movement in Xenopus oocytes.
    RNA 05/2013; 19(7). DOI:10.1261/rna.038232.113 · 4.94 Impact Factor
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    • "Routinely we injected 3 ng of Cy5-labelled RNA into each stage VI oocyte. In proportion to volume we injected about the same amount as did Messitt et al. into stage III oocytes [44], since stage VI oocytes are 50× larger by volume than those at stage III. In order to discount any effects on localisation by the amount of injected RNA we injected four times less than this and found that localisation was identical. "
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    ABSTRACT: 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.
    PLoS ONE 04/2013; 8(4):e61847. DOI:10.1371/journal.pone.0061847 · 3.23 Impact Factor
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    • "For example, a subpopulation of microtubules with plus ends oriented towards the vegetal cortex emerges prior to localization of Vg1 mRNA to the vegetal cortex in Xenopus oocytes (Messitt et al., 2008). Transport by plus end-directed kinesin motors is preceded by a dynein-dependent step in the vegetal RNA transport pathway (Gagnon et al., Submitted), and it is possible that the establishment of this subpopulation of microtubules triggers vegetal localization of Vg1 mRNA (Messitt et al., 2008). In addition, during the middle stages (7–10)of Drosophila oogenesis, microtubules are polarized with minus ends at the anterior and plus ends at the posterior (Clark et al., 1994), resulting in dynein-dependent transport of gurken RNA to the anterior pole (MacDougall et al., 2003). "
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    ABSTRACT: RNA localization, the enrichment of RNA in a specific subcellular region, is a mechanism for the establishment and maintenance of cellular polarity in a variety of systems. Ultimately, this results in a universal method for spatially restricting gene expression. Although the consequences of RNA localization are well-appreciated, many of the mechanisms that are responsible for carrying out polarized transport remain elusive. Several recent studies have illuminated the roles that molecular motor proteins play in the process of RNA localization. These studies have revealed complex mechanisms in which the coordinated action of one or more motor proteins can act at different points in the localization process to direct RNAs to their final destination. In this review, we discuss recent findings from several different systems in an effort to clarify pathways and mechanisms that control the directed movement of RNA.
    Critical Reviews in Biochemistry and Molecular Biology 06/2011; 46(3):229-39. DOI:10.3109/10409238.2011.572861 · 7.71 Impact Factor
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