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
Source: PubMed


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
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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.
    Full-text · Article · May 2013 · RNA
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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.
    Preview · Article · Apr 2013 · PLoS ONE
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    • "In mammalian neurons, biochemical purification of Staufencontaining mRNPs revealed the presence of conventional kinesin (Mallardo et al., 2003). In oocytes of Xenopus laevis kinesin-1- type and kinesin-3-type motors coordinate mRNP transport to the vegetal cortex on subpopulations of microtubules (Messitt et al., 2008). In oocytes of D. melanogaster, oskar mRNPs move bidirectionally along a weakly polarised microtubule cytoskeleton, and conventional kinesin is responsible for most of the transport (Zimyanin et al., 2008). "
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    ABSTRACT: Long-distance transport of mRNAs is important in determining polarity in eukaryotes. Molecular motors shuttle large ribonucleoprotein complexes (mRNPs) containing RNA-binding proteins and associated factors along microtubules. However, precise mechanisms including the interplay of molecular motors and a potential connection to membrane trafficking remain elusive. Here, we solve the motor composition of transported mRNPs containing the RNA-binding protein Rrm4 of the pathogen Ustilago maydis. The underlying transport process determines the axis of polarity in infectious filaments. Plus-end-directed Kin3, a kinesin-3 type motor, mediates anterograde transport of mRNPs and is also present in transport units moving retrogradely. Split dynein Dyn1-Dyn2 functions in retrograde movement of mRNPs. Plus-end-directed conventional kinesin Kin1 is indirectly involved by transporting minus-end-directed dynein back to plus ends. Importantly, we additionally demonstrate that Rrm4-containing mRNPs colocalise with the t-SNARE Yup1 on shuttling endosomes and that functional endosomes are essential for mRNP movement. Either loss of Kin3 or removal of its lipid-binding pleckstrin-homology domain abolishes Rrm4-dependent movement without preventing colocalisation of Rrm4 and Yup1-positive endosomes. In summary, we uncovered the combination of motors required for mRNP shuttling along microtubules. Furthermore, intimately linked co-transport of endosomes and mRNPs suggests vesicle hitchhiking as mode of mRNP transport.
    Full-text · Article · Feb 2012 · Journal of Cell Science
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