Highlights
- •FERRY links mRNA to early endosomes in long-range transport of transcripts
- •Unique clamp-like architecture of the pentameric FERRY Rab5 effector complex
- •Complex RNA binding interface mainly involves flexible coiled-coil domains of Fy-2
- •Neurological disorder-related mutations impair Rab5 binding and FERRY assembly
Summary
The pentameric FERRY Rab5 effector complex is a molecular link between mRNA and early endosomes in mRNA intracellular distribution. Here, we determine the cryo-EM structure of human FERRY. It reveals a unique clamp-like architecture that bears no resemblance to any known structure of Rab effectors. A combination of functional and mutational studies reveals that while the Fy-2 C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils and Fy-5 concur to bind mRNA. Mutations causing truncations of Fy-2 in patients with neurological disorders impair Rab5 binding or FERRY complex assembly. Thus, Fy-2 serves as a binding hub connecting all five complex subunits and mediating the binding to mRNA and early endosomes via Rab5. Our study provides mechanistic insights into long-distance mRNA transport and demonstrates that the particular architecture of FERRY is closely linked to a previously undescribed mode of RNA binding, involving coiled-coil domains.
Introduction
Rab small GTPases are master regulators of intracellular transport that contribute to the structural and functional integrity of organelles. In the GTP-bound conformation, membrane-associated Rab proteins can recruit a plethora of diverse downstream effector proteins to accomplish membrane remodeling activities.
Rab5, one of the most extensively studied small GTPases, is mainly localized at the early endosome (EE) and regulates endocytosis and EE dynamics. The vast network of interaction partners of Rab5 includes GEFs, like Rabex-5 and RIN1, but also Rab5-specific GAPs such as RN-Tre and Rab-GAP5.
Prominent effectors like Rabaptin-5, Rabankyrin-5, Rabenosyn-5, EEA1, and APPL1/2 act downstream and can bind Rab5 via distinct domains such as zinc fingers. Co-structures of Rab effectors bound to their cognate GTPase, including Rab5-Rabaptin-5, and Rab4-Rabenosyn-5, have provided important insights in the functional interactions between Rab proteins and their effectors and regulators, demonstrating that binding is typically mediated by the switch and inter-switch regions of Rab proteins and either symmetric coiled-coils or α-helical bundles of effectors. In addition to individual proteins, Rab effectors comprise large multiprotein complexes, such as Exocyst and HOPS, that mediate crucial functions in the exocytic and endocytic pathways. These multiprotein complexes are non-symmetric, highly flexible and dynamic, and therefore challenging to analyze structurally. Hence, known structures are often limited to the core of the complexes, and it is difficult to reach high resolution.
Rab5 is also implicated in long-range endosomal motility. By harnessing the intracellular microtubule (MT) network, EEs can be actively transported via MT motor complexes to distal locations within the cell. Recent evidence suggests a role of endosomal motility also in RNA localization and expression. RNA transport and local translation serves as a prime example of how spatiotemporal control can influence the expression of genes underlying essential biological processes, such as embryonic development or neuronal plasticity. Local sites individually regulate gene expression, which is thus not limited to transcriptional control in the nucleus. The sophisticated mRNA localization pattern observed in polarized cells like neurons requires active transport of transcripts. Studies in a number of model systems, including yeast and Drosophila melanogaster, have identified components of the RNA transport machinery, both at the level of the mRNA and localization machinery. First, the pattern of localization depends on specific RNA elements, often positioned in the 3′UTR of the mRNA. Second, two distinct active transport pathways, both exploiting the cytoskeletal network in combination with motor proteins, have emerged: RNA is transported with the help of accessory proteins, termed trans-acting proteins, that bind RNA to form ribonucleoprotein particles (RNPs). These proteins can contain specific RNA binding domains. Alternatively, mRNA can be associated with endosomal compartments, both actively transported by co-opting cytoskeletal components.
In this case, the molecular interactions mediating mRNA binding to organelles are unknown. The role of late endosomes and lysosomes in mRNA localization has been subject of recent research, where Annexin 11A has been proposed to mediate the association between RNA and lysosomes.
While these initial insights are valuable, they are limited to a subclass of the endocytic system. In filamentous fungi, mRNA localization is mediated by the microtubule-based transport of vesicles, including EEs.
In neurons, long range transport of various types of cargo, including mRNA, requires active transport, which is mediated by endocytic organelles, particularly late endosomes.
However, mechanisms must ensure the localization of mRNA distant from the cell body, such as in dendrites and axons in neurons, as late endocytic organelles undergo bidirectional but primarily minus-end microtubule-directed motility. Candidate organelles for localizing mRNAs to the periphery of processes in neurons are also early endosomes. However, little is known whether and how mRNAs are transported via early endosomes to their target destination.
We have identified a human 5-subunit Rab5 effector complex termed five-subunit early endosomal Rab5 and RNA/ribosome intermediary (FERRY) complex, which interacts with mRNA and thus represents a prime candidate for early endosome-mediated mRNA transport. The FERRY complex is composed of Tbck (Fy-1), Ppp1r21 (Fy-2), C12orf4 (Fy-3), Cryzl1 (Fy-4), and Gatd1 (Fy-5), which have a molecular weight of 101, 88, 64, 39, and 23 kDa, respectively (Figure 1A). Here, we determined the cryoelectron microscopy (cryo-EM) structure of the FERRY complex at a resolution of 4 Å. Together with rotary-shadowing EM, hydrogen-deuterium exchange mass spectrometry (HDX-MS), crosslinking mass spectrometry (MS), electrophoretic mobility shift assay (EMSA) and mutational studies, the structure demonstrates that FERRY is an elongated complex with a clamp-like architecture at its center and protruding flexible coiled-coil structures at its periphery that mediate the interaction with the EE via Rab5 and mRNA. Moreover, the combination of biochemical and structural studies allowed us to delineate the complex RNA binding interface of FERRY, which is composed of the two-opposing coiled-coils of the central hub protein Fy-2 as well as Fy-5, shedding light on how FERRY links mRNA to early endosomes in long-range transport of transcripts…
