RNA-directed activation of cytoplasmic dynein-1 in reconstituted transport RNPs

Abstract

Polarised mRNA transport is a prevalent mechanism for spatial control of protein synthesis. However, the composition of transported ribonucleoprotein particles (RNPs) and the regulation of their movement are poorly understood. We have reconstituted microtubule minus end-directed transport of mRNAs using purified components. A Bicaudal-D (BicD) adaptor protein and the RNA-binding protein Egalitarian (Egl) are sufficient for long-distance mRNA transport by the dynein motor and its accessory complex dynactin, thus defining a minimal transport-competent RNP. Unexpectedly, the RNA is required for robust activation of dynein motility. We show that a cis-acting RNA localisation signal promotes the interaction of Egl with BicD, which licenses the latter protein to recruit dynein and dynactin. Our data support a model for BicD activation based on RNA-induced occupancy of two Egl-binding sites on the BicD dimer. Scaffolding of adaptor protein assemblies by cargoes is an attractive mechanism for regulating intracellular transport.

The Egalitarian binding partners Dynein light chain and Bicaudal-D act sequentially to link mRNA to the Dynein motor

“…2C). As expected given recent findings (McClintock et al, 2018;Sladewski et al, 2018), loss of RNA binding significantly compromised the Egl-BicD interaction (Fig. 2C).…”

“…Although the Egl/BicD/ Dynein complex can be reconstituted using purified components (McClintock et al, 2018;Sladewski et al, 2018), the existence of this complex in native ovarian lysates has been difficult to demonstrate. McClintock and colleagues were able to co-precipitate a small amount of Dynein heavy chain (Dhc, the motor subunit of Dynein; also known as Dhc64C) with Egl from ovarian lysates only upon addition of excess RNA cargo (McClintock et al, 2018). Because Egl_2pt and Egl_4e are deficient for binding RNA, a different strategy is needed to analyze the in vivo association of these mutants with Dynein.…”

“…Thus, in theory, both of the Egl binding partners are capable of linking mRNA-bound Egl with the Dynein motor. Recent in vitro studies have shown that a minimal complex consisting of RNA, Egl, BicD, Dynactin and Dynein is sufficient for transport (McClintock et al, 2018;Sladewski et al, 2018). Whether or not this complex is sufficient for mRNA localization in vivo, however, is not known.…”

Dynein activators and adaptors at a glance

“…BICD2 has also been implicated in nuclear positioning (Splinter et al, 2012;Hu et al, 2013). Furthermore, the C-terminal region of BICD proteins can bend back on itself to produce an autoinhibited state (Terawaki et al, 2015;Liu et al, 2013;Wharton and Struhl, 1989;Urnavicius et al, 2015), which can be relieved by cargo binding (Liu et al, 2013;Huynh and Vale, 2017;McClintock et al, 2018;Sladewski et al, 2018), suggesting an efficient mechanism to regulate dynein motility.…”

A reconstituted mammalian APC-kinesin complex selectively transports defined packages of mRNAs

“…Different cell types localise up to thousands of different mRNAs to specific domains which is required for a wide range of cellular functions [4][5][6][7] . New imaging techniques allowed to visualise individual-mRNA transport events in living neurons 8,9 , and reconstitutions revealed the first essential components of yeast and drosophila mRNA transport systems [10][11][12][13] . Still, as many building blocks of these reconstituted systems lack mammalian homologues, it remained unknown how any mammalian mRNA is loaded onto motor proteins, how copy number of transported mRNAs is controlled and what molecular mechanisms generate specificity to transport only certain mRNAs.…”