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A′-form RNA helices are required for cytoplasmic mRNA transport in Drosophila

Abstract

Microtubule-based mRNA transport is widely used to restrict protein expression to specific regions in the cell and has important roles in defining cell polarity and axis determination as well as in neuronal function. However, the structural basis of recognition of cis-acting mRNA localization signals by motor complexes is poorly understood. We have used NMR spectroscopy to describe the first tertiary structure to our knowledge of an RNA element responsible for mRNA transport. The Drosophila melanogaster fs(1)K10 signal, which mediates transport by the dynein motor, forms a stem loop with two double-stranded RNA helices adopting an unusual A′-form conformation with widened major grooves reminiscent of those in B-form DNA. Structure determination of four mutant RNAs and extensive functional assays in Drosophila embryos indicate that the two spatially registered A′-form helices represent critical recognition sites for the transport machinery. Our study provides insights into the basis for RNA cargo recognition and reveals a key biological function encoded by A′-form RNA conformation.

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Figure 1: Solution structure of K10 TLS RNA.
Figure 2: Localization activity of WT and lower- and upper-stem mutant K10 RNAs.
Figure 3: Solution structure of mutant K10 TLS RNAs.
Figure 4: Localization activity of A′-form and bulge mutant K10 RNAs.

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Acknowledgements

We thank L. Easton for preparation of labeled NTPs and assistance with cloning of some constructs, J.-C. Yang for excellent help with NMR data collection and D. Neuhaus, J. Puglisi and L. Easton for the critical reading of the manuscript. This work was supported by the Medical Research Council (P.J.L. and S.L.B.) and Cancer Research UK (I.R. and D.I.-H.). S.L.B. is a Lister Institute Prize fellow.

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P.J.L. prepared isotope-labeled RNA samples, collected NMR data and determined the structures; P.J.L., I.R. and S.L.B. cloned mutant constructs for injection assays; S.L.B. and I.R. prepared fluorescently labeled RNAs and performed microinjections and microscopy; P.J.L., S.L.B. and D.I.H. prepared the manuscript.

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Correspondence to Peter J Lukavsky.

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Bullock, S., Ringel, I., Ish-Horowicz, D. et al. A′-form RNA helices are required for cytoplasmic mRNA transport in Drosophila. Nat Struct Mol Biol 17, 703–709 (2010). https://doi.org/10.1038/nsmb.1813

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