Receptor-specific interactome as a hub for rapid cue-induced selective translation in axons

Abstract

19During neuronal wiring, extrinsic cues trigger the local translation of specific mRNAs in axons 20 via cell surface receptors. The coupling of ribosomes to receptors has been proposed as a 21 mechanism linking signals to local translation but it is not known how broadly this mechanism 22 operates, nor whether it can selectively regulate mRNA translation. We report that receptor-23 ribosome coupling is employed by multiple guidance cue receptors and this interaction is 24 mRNA-dependent. We find that different receptors bind to distinct sets of mRNAs and RNA-25 binding proteins. Cue stimulation induces rapid dissociation of ribosomes from receptors and 26 the selective translation of receptor-specific mRNAs in retinal axon growth cones. Further, 27we show that receptor-ribosome dissociation and cue-induced selective translation are 28 inhibited by simultaneous exposure to translation-repressive cues, suggesting a novel mode 29 of signal integration. Our findings reveal receptor-specific interactomes and provide a general 30 model for the rapid, localized and selective control of cue-induced translation. 31 Lin and Holt, 2008). Unbiased detection of newly synthesized proteins in the axon 47 compartment has revealed further complexity showing that different guidance cues stimulate 48 the regulation of distinct signature sets of >100 axonal nascent proteins within just 5 mins, 49 many of which are not cytoskeletal-related , Yao et al., 2006, Wu et al., 50 2005. However, the mechanisms underlying the 51 localization and selectivity of translation are unclear. Several mechanisms are known to 52 control different aspects of axonal translation, including microRNA regulation (Bellon et al., 53 2017), mRNA modification (Yu et al., 2018), modulation of the phosphorylation of eukaryotic 54 initiation factors (Cagnetta et al., 2019), RNA-binding protein (RBP) phosphorylation (Sasaki 55 et al., 2010, Lepelletier et al., 2017, Huttelmaier et al., 2005) and receptor-ribosome coupling 56 (Tcherkezian et al. , 2010). The latter is a particularly direct and attractive mechanism to link 57 cue-specific signalling to differential mRNA translation. However, this mechanism has been 58 shown only for the Netrin-1 receptor, deleted in colorectal cancer (DCC), in commissural 59 axon growth cones and HEK293 cells (Tcherkezian et al., 2010) and it is unknown whether 60 receptor-ribosome coupling is a widespread mechanism used by different receptors and in 61 different cell types, and whether it regulates selective local translation. 62 63 Here, we show in retinal ganglion cell (RGC) axon growth cones that receptor-ribosome 64 coupling is used by several different axon guidance receptors (DCC, Neuropilin-1 and 65 Robo2), indicative of a common mechanism. Upon stimulation by specific cues, ribosomes 66 dissociate from their receptors within 2 minutes. Interestingly, the receptor-ribosome 67 interaction is mRNA-dependent, and immunoprecipitation (IP) reveals that distinct receptors 68 associate with specific RNA-binding proteins (RBPs) an...

Local Translation in Axons: When Membraneless RNP Granules Meet Membrane-Bound Organelles

“…As suggested by recent work, indeed, ribosomes of different compositions and functional properties may co-exist and exhibit preferential selectivity for subsets of mRNAs (Shi and Barna, 2015; Segev and Gerst, 2018). In axons, remarkable differences in the stoichiometry of defined ribosomal proteins were found when comparing the composition of ribosomes associated with distinct guidance molecule receptors (Koppers et al, 2019). Furthermore, on-site incorporation of axonally-synthesized ribosomal proteins and subsequent remodeling of ribosomes was observed (Shigeoka et al, 2019), thus opening the door for functional dissection of this additional layer of spatio-temporal regulation.…”

“…Analysis of mRNAs co-precipitating with COPIa, for example, revealed the presence of more than a thousand of mRNAs, 8 to 10% overlapping with the axonal transcriptome and about the same percentage encoding cytoskeletal components and/or regulators (Todd et al, 2013). Furthermore, RNP complexes with specific protein and RNA signatures were recently found in the immunoprecipitates of different guidance cue receptors in neuronal cells (Koppers et al, 2019). The RBP Staufen 1, for example, was found significantly associated with the Neuropilin receptor Nrp1, whereas hnRNPA2/B1 was found interacting with the Netrin-1 DCC receptor.…”

“…As demonstrated in Xenopus Retinal Ganglion neurons, translation of β -catenin ( ctnnb1 ) mRNA was for example activated in response to the DCC ligand Netrin-1, but not in response to Sema3A (Koppers et al, 2019). Translation activation correlated with a release of both RNAs and associated ribosomes from the receptor complex, suggesting (i) that mRNAs tethered to receptors are kept in a translationally repressed state and (ii) that cue-induced release may represent a rapid and direct means to trigger selective translation activation (Figure 1D; Tcherkezian et al, 2010; Koppers et al, 2019). How such release is achieved remains to be understood, but a possibility is that cue-induced signaling triggers the phosphorylation of RBPs associated with transmembrane receptors.…”

mRNA localization and local translation in neurons

“…Tethering components of the translation machinery to the cytoplasmic domain of TM receptors allows spatial and temporal regulation of protein expression in response to external cues. Furthermore, electron microscopy of developing Xenopus retinal ganglion cells axons demonstrated that receptor-ribosome coupling is used by guidance cues where guidance cues can induce dissociation of ribosomes followed by specific mRNA translation [42] .…”

Axonal mRNA translation in neurological disorders

“…Early scepticism sprang from concerns about sample (axonal) purity due to technical difficulties in obtaining axon-only material, and the paucity of ultrastructural evidence for the existence of ribosomes in axons. Technical advances in recent years have overcome these difficulties, enabling the collection of pure axons in vitro [6,7], the use of sophisticated RNA molecular analysis (transcriptomics and translatomics) [8][9][10] and the acquisition of ultrastructural evidence of ribosome localization in axons [9,11,12,13]. As a consequence, evidence now abounds that thousands of diverse sets of mRNAs reside and are translated in axons of both central nervous system (CNS) and peripheral nervous system (PNS) neurons.…”