Journal Article

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

Dix, Carly I, McClintock, Mark A, Johnson, Christopher M, Maizels, Rory J, Bullock, Simon L, Hoang, Ha Thi, McLaughlin, Stephen H

2018

Overview

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. In our cells, tiny molecular motors transport the components necessary for life’s biological processes from one location to another. They do so by loading their cargo, and burning up chemical fuel to carry it along pathways made of filaments. For example, one such motor, called dynein, can move molecules of messenger RNA (mRNA) to specific locations within the cell. There, the mRNA will be used as a template to create proteins, which will operate at exactly the right place. Transporting mRNA in this way is critical in processes such as embryonic development and the formation of memories; yet, this mechanism is still poorly understood. Previous work suggested that the mRNA is simply a passenger of the dynein motor, but McClintock et al. asked if this is really the case. Instead, could mRNA regulate its own sorting by controlling the activity of dynein? Studying mRNA trafficking within the complex molecular environment of a cell is challenging, so mRNA transporting machinery was recreated in the laboratory. Only the proteins necessary to build a working system were included in the experiments. In addition to the filaments, the components included dynein and a complex of proteins known as dynactin, which allows the motor to move together with a protein called BICD2. A protein named Egalitarian was used to link the mRNA to BICD2. By filming fluorescently labelled proteins and mRNAs, McClintock et al. discovered that mRNA strongly promotes the movement of the dynein motor. A structured section in the mRNA acts as a docking area for two copies of Egalitarian. This activates BICD2, which then binds to dynein and dynactin, thereby completing the transport machinery. According to these results, the mRNA directs the assembly of the system that will carry it within the cell. Viruses such as HIV and herpesvirus hijack dynein motors to have their genetic information moved around a cell in order to propagate infection. Understanding precisely how mRNA is transported may help to develop new strategies to fight these viruses.

• Publisher: eLife Sciences Publications Ltd,eLife Sciences Publications, Ltd
• Subject: Animals / Bicaudal-D / Binding Sites / Cell Biology / Cytoplasmic Dyneins - chemistry / Cytoplasmic Dyneins - genetics / Drosophila melanogaster - genetics / Drosophila Proteins - chemistry / Drosophila Proteins - genetics / Dynactin / Dynactin Complex - chemistry / Dynactin Complex - genetics / Dynein / Dyneins - chemistry / Dyneins - genetics / Egalitarian / in vitro reconstitution / Insects / Laboratories / Localization / microtubule / Molecular biology / Motility / mRNA / mRNA transport / Protein Binding - genetics / Protein biosynthesis / Protein composition / Protein Multimerization / Protein Transport - genetics / Proteins / Ribonucleoproteins - genetics / RNA transport / RNA Transport - genetics / RNA, Messenger - genetics / RNA-binding protein / RNA-Binding Proteins - chemistry / RNA-Binding Proteins - genetics / Velocity