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Single-molecule assays reveal that RNA localization signals regulate dynein–dynactin copy number on individual transcript cargoes

Nature Cell Biology volume 14, pages 416–423 (2012)Cite this article

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Abstract

Subcellular localization of mRNAs by cytoskeletal motors plays critical roles in the spatial control of protein function1. However, optical limitations of studying mRNA transport in vivo mean that there is little mechanistic insight into how transcripts are packaged and linked to motors, and how the movement of mRNA–motor complexes on the cytoskeleton is orchestrated. Here, we have reconstituted transport of mRNPs containing specific RNAs in vitro. We show directly that mRNAs that are either apically localized or non-localized in Drosophila melanogaster embryos associate with the dynein motor and move bidirectionally on individual microtubules, with localizing mRNPs exhibiting a strong minus-end-directed bias. Single-molecule fluorescence measurements reveal that RNA localization signals increase the average number of dynein and dynactin components recruited to individual mRNPs. We find that, surprisingly, individual RNA molecules are present in motile mRNPs in vitro and provide evidence that this is also the case in vivo. Thus, RNA oligomerization is not obligatory for transport. Our findings lead to a model in which RNA localization signals produce highly polarized distributions of transcript populations through modest changes in motor copy number on single mRNA molecules.

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Figure 1: An assay for mRNP motility along single microtubules in vitro.
Figure 2: Differential motility of localizing and non-localizing mRNPs along polarity-marked microtubules in vitro.
Figure 3: Localizing mRNPs contain, on average, more copies of dynein and dynactin components than non-localizing mRNPs.
Figure 4: Single copies of mRNA molecules within most localizing mRNPs in vitro.
Figure 5: Single copies of an RNA species within most apically localized mRNPs in vivo.

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Acknowledgements

This work was financially supported by the UK Medical Research Council (project U105178790) and a Lister Institute prize fellowship (to S.L.B.). We are very grateful to A. Carter, A. Guichet, T. Hays, R. Lehmann, E. Miska, C. Navarro, I. Palacios, J. Raff, C. Samakovlis, B. Suter and R. Warrior for providing reagents, A. Paré for advice on in situ hybridization, N. Barry for advice on microscopy, A. Nicol and D. Zicha for help with mRNP tracking software, M. Dogterom for the Stepfinder algorithm, and members of the Bullock laboratory, D. St Johnston and A. Carter for their valuable insights.

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  1. Cell Biology Division, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK

    Mamta Amrute-Nayak & Simon L. Bullock

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  1. Mamta Amrute-Nayak
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Contributions

M.A-N. and S.L.B. conceived the project. M.A-N. developed the in vitro assays, carried out all in vitro experiments and analysed the data. S.L.B. carried out the embryo injections and in situ hybridizations and analysed the data. M.A-N. and S.L.B. interpreted the data. S.L.B. and M.A-N. wrote and edited the manuscript, respectively.

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Correspondence to Simon L. Bullock.

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The authors declare no competing financial interests.

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Amrute-Nayak, M., Bullock, S. Single-molecule assays reveal that RNA localization signals regulate dynein–dynactin copy number on individual transcript cargoes. Nat Cell Biol 14, 416–423 (2012). https://doi.org/10.1038/ncb2446

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